5th EuropEan MolEcular IMagIng MEEtIng - ESMI

of the European network of excellence DiMI - Diagnostic Molecular Imaging, and is organised in collaboration with the 

ESR - European Society of Radiology, the European network of excellence CliniGene – Clinical Gene Transfer and Therapy, 

and is also supported through COST actions D38 and BM0607 – the European cooperation in science and technology. 

DAY 

0EMIM2010 is the 5thannual meeting of the European Society for Molecular Imaging - ESMI. The EMIM2010 is also the final meeting

WarSaW, poland May 26 – 29, 2010 

committees 

5 th EMIM EXECUTIVE COMMITTEE 

Andreas H. Jacobs, ESMI President 

Renata Mikołajczak, Local Organiser 

Clemens W.G.M. Löwik, ESMI Vice President 

Bertrand Tavitian, ESMI Past President 

Silvio Aime, ESMI Secretary 

Simone Mergui, ESMI Treasurer 

5 th EMIM SCIENTIFIC COMMITTEE 

Ludwig Aigner, Salzburg (A) 

Silvio Aime, Torino (I) 

Veerle Baekelandt, Leuven (B) 

Hervé Boutin, Manchester (Uk) 

Harald Carlsen, Oslo (N) 

John Clark, Edinburgh (Uk) 

Marion Dejong, Rotterdam (Nl) 

Silvana Del Vecchio, Naples (I) 

Frédéric Dollé, Orsay (F) 

Eugeniusz Dzuik, Warsaw (Pl) 

Cornel Fraefel, Zuerich (Ch) 

Jorgen Frokiaer, Aarhus (Dk) 

Nicolas Grenier, Bordeaux (F) 

Denis Guilloteau, Tours (F) 

Uwe Haberkorn, Heidelberg (D) 

Mathias Höhn, Cologne (D) 

Andreas H. Jacobs, Münster (D) 

Fabian Kiessling, Aachen (D) 

Izabela Kozlowicz-Gudzinska, Warsaw (P) 

Leszek Krolicki, Warsaw (Pl) 

Tony Lahoutte, Jette (B) 

Adriaan Lammertsma, Amsterdam (Nl) 

5 th EMIM Young Investigator Award SELECTION COMMITTEE 

Veerle Baekelandt 

Hervé Boutin 

Peter Brader 

John Clark (Chair) 

Frédéric Dollé 

Nicolas Grenier 

Fabian Kiessling 

Adriaan Lammertsma 

LOCAL ORGANIZING COMMITTEE 

Iliana Chwalinska, Warsaw (P) 

Eugeniusz Dzuik, Warsaw (P) 

Izabela Kozlowicz-Gudzinska, Warsaw (P) 

Leszek Krolicki, Warsaw (P) 

Renata Mikolajczak, Warsaw (P) 

Bengt Långstrom, (S) 

Clemens W.G.M. Löwik, Leiden (Nl) 

Helmut R. Maecke, Freiburg (Ch) 

Adriana Maggi, Milano (I) 

Serge Maitrejean, Paris (F) 

Renata Mikolajczak, Warsaw (P) 

Chrit Moonen, Bordeaux (F) 

Klaas Nicolay, Eindhoven (Nl) 

Vasilis Nziachristos,Munich (D) 

Sabina Pappata, Naples (I) 

David Parker, Durham (Uk) 

Bernd PIchler, Tübingen (D) 

Anna Planas, Barcelona (E) 

Juha Rinne, Turku (Fin) 

Jorge Ripoll, Heraklion (Gr) 

Markus Rudin, Zürich (Ch) 

Michael Schäfers, Münster (D) 

Markus Schwaiger, Munich (D) 

Bertrand Tavitian, Orsay (F) 

Eva Tóth, Orleans (F) 

Annemie Van Der Linden, Antwerp (B) 

David Wyper, Glasgow (Uk) 

Renata Mikolajczak 

Chrit Moonen 

Klaas Nicolay 

Markus Schwaiger 

Bertrand Tavitian (Chair) 

Eva Tóth 

David Wyper

content – per topic 

5th EuropEan MolEcular IMagIng MEEtIng – EMIM2010 

Day 0 - Wednesday 26,2010 6 

Overview oral presentations 6 

Welcome 8 

Venue 10 

Thank you! 11 

Programme at a Glance 12 

Programme Datails 14 

Inaugural Lecture by Christopher H. Contag 20 

Day 1 - Thursday May 27, 2010 22 

ESMI Plenary Lecture 1 by Violaine Sée 24 

Parallel Session 1: CANCER I - together with the ESR 26 

Parallel Session 2: NEUROSCIENCE I 32 

Parallel Session 3: TECHNOLOGY 38 

Parallel Session 4: PROBES - supported by COST actions 44 

ESMI Plenary Lecture 2 by Francesca Odoardi 50 

Plenary Session on Excellent and Late Breaking Abstracts 52 

Day 2 - Friday May 28, 2010 58 

ESMI Plenary Lecture 3 by Theodorus W.J. Gadella Jr. 60 

Parallel Session 5: NEUROSCIENCE II 62 

Parallel Session 6: CARDIOVASCULAR I - together with the ESR 68 

Parallel Session 7: PROBES II - together with the EANM 74 

Parallel Session 8: Gene and cell based therapies - together with CliniGene 80 

ESMI Plenary Lecture 4 by Hans-Jürgen Wester 86 

Plenary Session on Current Contribution of Imaging Technologies to 

Drug Development 88 

Day 3 - Friday May 29, 2010 92 

ESMI Plenary Lecture by Jagat Narula 94 

Parallel Session 9: CARDIOVASCULAR II 96 

Parallel Session 10: CANCER II - supported by COST actions 102 

Poster Session, guided poster walks, topics, and titles 108 

Index 224 

EuropEan SocIEty for MolEcular IMagIng – ESMI

6 


overview oral presentations – per title 

Inaugural Lecture by Christopher H. Contag 

imaging life 

Day 0 - Wednesday May 26, 2010 

Point-of-Care Microscopy: Molecular Imaging with Cellular Resolution 20 

Day 1 - Thursday May 27, 2010 

ESMI Plenary Lecture 1 by Violaine Sée 

Transcription factors dynamics: from discrete pulses to oscillatory pattern to control gene expression 24 

Parallel Session 1: CANCER I - together with the ESR 

Multimodal imaging approaches for cell-cell interaction 26 

Molecular imaging for monitoring treatment with protein kinase inhibitors 27 

Potentials of imaging biomarkers in patients 28 

Pre-clinical screening of anti - HER2 nanobodies for molecular imaging of breast cancer 29 

[18F]-FDG/[18F]-FLT-PET and bioluminescence imaging of therapy response in B-cell lymphoma mice treated with 

cytotoxic and antiproliferative agents 30 

TF-Pimonidazole: an hypoxia marker suitable for in vivo 19 F MRS imaging 31 

Parallel Session 2: NEUROSCIENCE I 

[11C](R)-PK11195- a tricky tracer. Do we really need it to image microglial activation? 32 

Preclinical imaging of the type 1 cannabinoid receptor in neurodegenerative diseases 

Longitudinal non-invasive detection of amyloid plaques by combined molecular, functional and morphological imaging 

33 

in transgenic mouse models of Alzheimer‘s disease 34 

Serotonergic neurotransmission in early Alzheimer’s disease 

Qualitative and quantitative assessment of florbetapir F-18 PET ( 

35 

18F-AV45) amyloid deposition PET imaging - validation 

by pathology. Preliminary report 36 

In-vivo imaging of a mouse traumatic brain injury model using dual-isotope quantitative spect/ct 37 

Parallel Session 3: TECHNOLOGY 

Molecular analysis of adaptive immune function through microscopic and mesoscopic imaging 38 

Magnetic resonance imaging – from structure to molecular interactions 39 

Imaging modalities: PET and SPECT 40 

In vivo fluorescence kinetic imaging for improved contrast and studies of temporal and quantitative biodistribution 41 

Cerenkov radiation imaging of a xenograft murine model of mammary carcinoma 42 

A system for 4D (3D+Kinetics) molecular imaging in bioluminescence and fluorescence 43 

Parallel Session 4: PROBES - supported by COST actions 

The alignment of target-specific lipoCEST MRI contrast agents 44 

Lanthanide-based in vivo luminescence imaging 45 

New perspectives for imaging molecules and metabolism in vivo 46 

In vivo biodistribution of radiolabeled matrix metalloproteinase-2 activatable cell penetrating peptides 47 

Bioresponsive MRI contrast agents based on self-assembling β-cyclodextrin nanocapsules 48 

Photochemical activation of endosomal escape of MRI-Gd-agents in tumor cells 49 

ESMI Plenary Lecture 2 by Francesca Odoardi 

Immune surveillance and autoimmunity: how encephalitogenic T cells enther their target organ 50 

Plenary Session on Excellent and Late Breaking Abstracts 

Exendin-4 derivatives labeled with radiometals for the detection of insulinoma: a “from bench to bed approach” 52 

Assessment of HIF transcriptional activity in a mouse tumor model using GPI anchored avidin– a novel protein 

reporter for in vivo imaging 53 

A leukocyte ligand of vascular adhesion protein-1 as an imaging tool in PET 54 

Clinical translation of ex vivo sentinel lymph node mapping for colorectal cancer using invisible near-infrared 

fluorescence light 55 

Development of strategies for in vivo MRI and Optical Imaging of neural stem cells distribution for the treatment of 

traumatic spinal cord injury 56 

Validation of bone marrow-derived stromal cell graft position by colocalisation of histology, bioluminescence and 

magnetic resonance imaging 57 

Day 2 - Friday May 28, 2010 

ESMI Plenary Lecture 3 by Theodorus W.J. Gadella 

New probe-based strategies for quantitative microscopy of signaling dynamics in single cells 60

Parallel Session 5: NEUROSCIENCE II 


Potential to use transgenic animals for imaging of neurological diseases 62 

Specific cell labeling with imaging reporters in vivo by viral vectors: the challenges 63 

Optimization of an MRI method to measure microvessel density: application to monitor angiogenesis after stroke 64 

MRI study of intra-arterial bone marrow-derived macrophage administration after acute focal ischemic stroke in rats 65 

Inactivated paramagnetic tissue plasminogen activator predicts thrombolysis outcome following stroke 66 

Matrix metalloproteinase-9 – a possible therapeutic target in intractable epilepsy 67 

Parallel Session 6: CARDIOVASCULAR I - together with the ESR 

Potentials of new contrast agents for vascular molecular imaging in patients 68 

Protease specific nanosensors in atherosclerosis 69 

Imaging of inflamed carotid artery atherosclerotic plaques with the use of 99mTc-HYNIC-IL-2 scintigraphy in the 

end-stage renal disease patients 70 

Uptake of 68 Ga-Chloride in Atherosclerotic Plaques of LDLR -/- ApoB 100/100 Mice 71 

Hybrid imaging using dual energy µCT and FMT for characterization of atherosclerotic plaques in ApoE -/- mice 72 

Long circulating emulsion based ct contrast agents 73 

Parallel Session 7: PROBES II - together with the EANM 

Application of microfluidics to the ultra-rapid preparation of fluorine-18 and carbon-11 labelled compounds 74 

Radioligand for in vivo measuring neurotransmitters release 75 

18f-tracers for amyloid plaques 76 

Chelators for radiocopper: biological/pharmacokinetic differences of [Tyr3 ,Thr 8 ]octreotide conjugates 77 

[ 11C]SOMADAM: potential sert ligand for pet studies and comparison with [ 11C]MADAM 78 

MR imaging of extracellular redox by a thiolsensitive gd(iii)-do3a derivative 79 

Parallel Session 8: Gene and cell based therapies - together with CliniGene 

Everything but not cell replacement with somatic neural stem cell transplantation 80 

Mesenchymal stem cell transplantation: an option to promote remyelination? 81 

Viral vector-mediated transcriptional targeting of dendritic cells for antigen-specific tolerance induction in EAE/MS. 82 

Delivery of a bioluminescent transgene to a tumor via bone marrow engraftment and local control of gene expression 

by non invasive local hyperthermia 

Dendritic cell labelling with paramagnetic nanoparticles and 

83 

111In-oxine for in vivo magnetic resonance imaging and 

scintigraphic imaging 84 

The type 2 cannabinoid receptor as a new PET reporter gene for the brain 85 

ESMI Plenary Lecture 4 by Hans-Jürgen Wester 

Molecular imaging of CXCR4 receptors 86 

Plenary Session on Current Contribution of Imaging Technologies to Drug Development 

Evaluation of the temporal window for drug delivery following ultrasound mediated membrane 

permeability enhancement 88 

Integrisense: a novel near-infrared fluorescent probe for α β integrin and its applications in drug discovery v 3 88 

Harnessing the power of bioluminescence to cross the in vitro–in vivo divide 90 

In vivo imaging in drug discovery: the example of application in the development of novel estrogenic compounds 91 

Day 3 - Saturday May 29, 2010 

ESMI Plenary Lecture by Jagat Narula 

Molecular imaging of unstable coronary plaques 94 

Parallel Session 9: CARDIOVASCULAR II 

Advances in contrast-enhanced MRI of the mouse heart 96 

Molecular imaging of α β integrin expression with v 3 18F-galacto-RGD after experimental myocardial infarction: 

comparison with left ventricular remodeling and function 97 

Existing and emerging animal models mimicking cardiovascular disease and their relevance for molecular imaging 

Imaging of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques with Multispectral Optoacoustic 

98 

Tomography 99 

c-Jun N-terminal kinase promotes inflammation at atherosclerosis-prone sites by enhancing expression and activity 

of NF-κB transcription factors 100 

Absolute Quantification in Small Animal Pinhole Gated Myocardial Perfusion SPECT 101 

Parallel Session 10: CANCER II - supported by COST actions 

Cancer imaging 102 

Controlled drug delivery under image guidance 103 

Comparative biodistribution of twelve gastrin/CCK2 receptor targeting peptides 104 

Targeting cancer stem cells using radiolabeled Sonic Hedgehog 105 

Evaluation of the photosensitizer Bremachlorin for photodynamic treatment of breast cancer bone metastasis. 106 

In vivo targeting of HEK-hsst Xenografts by 2/3/5 111in-labeled [(DOTA)Ser 1 ,Leu 8 ,trp22 ,Tyr25 ]-SS-28 in SCID mice 107 

EuropEan SocIEty for MolEcular IMagIng – ESMI 

overview oral presentations

8 


Welcome to the 5 th EMIM in Warsaw, Poland 

Dear Colleagues, 

on behalf of the ESMI as well as the other organising and contributing societies and networks it is our great pleasure 

to welcome you to Warsaw, Poland, for the 5 th European Molecular Imaging Meeting. EMIM 2010 brings together top 

European scientists from 3 societies, 2 European Networks of Excellence (NoEs) and 2 COST actions. With great honour 

we may state that your fellow attendees represent not only colleagues from all over Europe, but also Asia and the US. 

The societies and networks with primary responsibility for organising the meeting are: 

• European Society for Molecular Imaging – ESMI 

• NoE Diagnostic Molecular Imaging – DiMI 

• European Cooperation in Science and Technology – COST Actions D38 and BM0607 

• European Society of Radiology – ESR 

• We also gratefully acknowledge the contribution from the following participating organisations: 

• European Association of Nuclear Medicine – EANM 

• NoE Clinical Gene Transfer and Therapy – Clinigene 

All these organisations were working together, with input from a scientifically complementary scientific steering committee, 

to develop a strong scientific programme which integrates developments in imaging technologies and molecular 

imaging agents with applications for drug development, basic science investigations, and clinical translation with special 

emphasis in the main disease burdens of our society such as cancer, cardiovascular disease and neurodegeneration. 

We developed the 5 th EMIM scientific programme primarily on the outstanding strength of submitted abstracts. 

About 150 abstracts were submitted, reviewed and scored. Then, each of the concurrent session co-chairs reviewed 

the scored abstracts which were considered relevant to their particular sessions. Session co-chairs selected abstracts 

for oral presentation based on average abstract score, session content and structure. Attention was also paid to diversity 

of disciplines and geographical distribution. From this effort 39 abstracts were integrated into the Parallel as 

well as Plenary Sessions that bring attendees from different disciplines together for a comprehensive examination of 

the role of molecular imaging in particular biomedical problems. 21 of these oral abstract presentations will be held 

by Young Investigator Award (YIA) applicants. The YIA selection committee is going to select the best 3 YIA presentations 

to be held again during the final Plenary Session on Saturday where then THE Young Investigator Awardee 

will be announced. Furthermore, special attention has been put on seven guided poster walks which have been 

scheduled on Thursday and Friday afternoon with no other competing sessions in order to enhance the knowledge 

exchange of experienced and young researchers. Each of the guided poster walks will be chaired by two chairs and will 

result in one poster award. The seven Poster Awards will also be offered during the Closing Ceremony on Saturday. 

We are especially proud and thankful that Prof. Dr. Christopher Contag will give the inaugural lecture, 

entitled, “Point-of-care microscopy: molecular imaging with cellular resolution”. In addition, we have planned 5 exciting 

plenary lectures, covering topics on probe design, systems biology, cancer, neuroinflammation, and atherosclerosis. 

Furthermore, we have dedicated one Plenary Session each to Excellent, Late Breaking, and YIA Abstracts as 

well as to Current Contribution of Imaging Technologies to Drug Development. 

We would like to take this opportunity to acknowledge the support of this year’s sponsors of the conference, especially 

recognizing the continued interest and support from DG Research of the European Commission. Moreover 

we would like to thank YOU for participating in this year’s EMIM and making it possible by your attendance. We 

hope you will enjoy the conference. If this is not your first time attending an EMIM conference then welcome back 

and we hope you will establish new contacts and continue to expand your network of scientific excellence. Please 

help first-time attendees to meet others and generally make people feel at home. Regardless whether this is your 

first time with us or not we will endeavour to have you come back again in the future. 

As in previous years our intention is that this high-level meeting will foster the coherence of a sustainable European 

Molecular Imaging Community with the common goal to translate fundamental research discoveries into medical 

application and health benefit for the European Society. 

Sincerely, 

The 5th Andreas H. Jacobs, ESMI President 

Renata Mikołajczak, Local Organiser 

Clemens W.G.M. Löwik, ESMI Vice President 

Bertrand Tavitian, ESMI Past President 

Silvio Aime, ESMI Secretary 

European Molecular Imaging Meeting Executive Committee 

Simone Mergui, ESMI Treasurer 

imaging life

Dear Colleagues, 


it is a pleasure for me to welcome you to the 5 th European Molecular Imaging Meeting in Warszawa. With an impressive 

history of previous successful meetings, this time the European Society of Molecular Imaging decided 

in favour for Poland, the country belonging to East as well as to Central Europe. 

In some way this in-between-situation is also characteristic for the field of MI: the discipline MI is not defined by 

a profession, no one is automatically a MI professional. Molecular Imaging is multidisciplinary. This makes it on 

the one hand difficult: we have to define ourselves and the direction of our research; it forces us to look beyond 

our bench and own disciplines. On the other hand this is also the advantage of MI: it means at any time to be in 

process, be in cross-communication and stay open-minded. 

Me and my colleagues involved in the organisation of this meeting believe that it will strengthen Molecular Imaging 

research activities within Eastern and Central Europe, as well as between the various disciplines working 

in the broad field of MI. 

The efforts of the ESMI to provide a platform for exchange have started to lead into the formation of a European 

MI community. This is just the beginning of our common efforts and with our activities and willingness for 

cross-interaction we can further establish this development. 

We hope that the familiar arrangement of the meeting venue will create a friendly atmosphere and provide a 

maximum of opportunities for you to meet friends and exchange views. Enjoy the interesting scientific programme 

of invited talks, oral and poster presentations as well as the technical exhibition. I also want to say 

thanks to the companies who are supporting this meeting and the MI community; especially to those who come 

to Warszawa. 

Do not forget that Warszawa is a lively city, with a number of historical sites, museums and parks. We are also in 

the year of Chopin; you can find a lot of information about his music and places he used to live. Take some time 

to immerse yourself in the history of the city. 

On behalf of local organizing committee I wish your visit to Warszawa will be informative, interesting and enjoyable. 

Sincerely, Renata Mikołajczak, Local Organiser 

Dear Ladies and Gentlemen, 

It is my great pleasure to welcome you to the 5 th annual meeting of the European Society for Molecular Imaging. 

In my capacity as president of the European Society of Radiology I would like to thank the organisers of this important 

event that has been organised in close cooperation with the ESR for their kind invitation. 

It is an honour to be here today as the ESR has supported the creation of the European Society for Molecular Imaging 

from the very beginning and many ESR representatives have been and are still represented in this organisation. 

We are looking forward to enhancing the successful cooperation between the European Society for Molecular Imaging 

and the European Society of Radiology in the future. 

Realising the importance of molecular imaging, latest research discoveries in this field and possible translations 

into medical practice, I will reinforce the topic of molecular imaging within the European Society of Radiology during 

my presidency and make sure that the work of all ESR bodies is coordinated with a special focus on this issue. 

I would also like to take this opportunity to invite you all to attend ECR 2011 from March 3 to 7 in Vienna, Austria. 

The ESR would be most happy to welcome you at its annual meeting. 

I thank you for having taken the time to be with us in Warsaw this week and I wish you a most successful meeting. 

Sincerely, Professor Maximilian F. Reiser, ESR President 


Welcome

10 


Why Warsaw? 

Warsaw, the capital of Poland, a captivating city with 

a distinctive atmosphere and definitely worth visiting. 

Warsaw is a perfect embodiment of changes that 

have taken place in Poland in the past 20 years. It is a 

city of many faces - a contrasting blend of historical 

and socrealist buildings neighbouring post-modern 

skyscrapers, of cosy cafes and vibrant clubs, of historical 

Żoliborz and artistic Praga district, but most of all 

it is a thriving European capital. Whether you come 

to Warsaw on a business trip, for a conference, or as a 

tourist, Warsaw will definitely exceed your expectations. 

Novotel Warsawa Centrum 

Marszałkowska 94/98, 00-510 Warszawa 

Tel: +48 22 596 25 48 | Fax: +48 22 596 01 22 

The Novotel Warsawa Centrum hotel provides excellent 

conference facilities. Close to the iconic Palace of Culture 

and Science and 500 m from the railway station with a 

convenient connection to the airport. The plenary lectures 

will take place in ROZA and IRYS, the parallel sessions will 

take place in ROZA or IRYS. Visit the exhibition in LILIA, 

FREZJA as well as in the FOYER; and get in communication! 

The poster sessions will take place in PROMENADA. You are 

mostly welcome to view the posters also besides of the 

“official” poster sessions. 

Floor Plan / Exhibition 

1 

2 

3 

coffee break 

exhibition and coffee 

7 8 9 

exhibition and coffee 

14 16 

15 

13 

coffee 

12 

4 

5 

6 

internal meetings 

internal meetings 

imaging life 

11 

19 

10 

lectures 

exhibition space 3x2m 

20 

office 

17 

How to get there 

From OKECIE Airport, take bus route No.175 to Centrum. From 

Warszawa Centralna railway station take tram No.7, 8, 9, 22, 24 

or 25 to Centrum (one stop). Via the E30, E77 or E67 highways, 

head in the direction of Centrum. By metro, alight at Centrum. 

Airport : WARSZAWA OKECIE AIRPORT 

Railway Station : WARSZAWA CENTRALNA STATION 

The Gala dinner will take place on Friday May 28, 2010 at 

Zamojski Palace Warsaw 2, Foksal street. Zamojski Palace is 

within walking distance. Looking forward to meeting you there. 

Floor plan 

poster session 

lectures 

18 

1. GEHC 

2. ART 

3. BIOSCAN/PHILIPS 

4. ZINSSER ANALYTIC 

5. CaliperLS 

6. MR solutions 

7. Carestream 

8. BRUKER 

9. VISUALSONICS 

10. SIEMENS 

11. BIOSPACE LAB 

12. LOT-Oriel 

13. CT Imaging 

14. SEDECAL 

15. VisEn 

16. LI-COR 

17. SKYSCAN/MILabs 

18. Polatom 

19. information


This meeting would not have been possible without your support. 


THANK YOU!

WMIC 2010 Speakers 

Keynote Address by Shizuo Akira, MD, PhD 

Professor of Department of Host Defense, Research Institute for Microbial 

Diseases and Director of the WPI Immunology Frontier Research Center 

(WPI-IFReC), Osaka University 

Featured Plenary Speakers 

Kevin Brindle, PhD 

Professor, Dept of Biochemistry 

University of Cambridge 

Robert J. Gropler, MD 

Chief, Cardiovascular Imaging Center, 

Mallinckrodt Institute of Radiology 

Gregory Lanza, MD, PhD 

Professor of Medicine 

Div. of Cardiovascular Diseases 

Washington University School of Medicine 

Herman P. Spaink, PhD 

Professor of Molecular Cell Biology 

Leiden University 

Jun Takahashi, MD, PhD 

Associate Professor 

Center for iPS Cell Research and Application (iCeMS) 

Kyoto University, Japan 

September 8-11, 2010 

Kyoto, Japan 

Scientific Sessions 

Preliminary List. For full list, visit our website. 

In Vivo Studies 

* Clinical Studies 

* Translational Studies 

* Animal Models 

Imaging Instrumentation and Methodology 

* Hybrid & Multimodality Imaging 

* Magnetic Resonance Imaging 

* Optical and Opto-acoustic Imaging 

* PET/SPECT/CT Imaging 

* Ultrasonic Imaging & Drug Delivery 

Development/Novel Use of Imaging Probes 

* CT 

* MRI 

* Multi-modality 

* Optical and Photoacoustic 

* PET/SPECT 

* Ultrasound 

Imaging Disease/Organ Processes 

* Cancer 

* Cardiovascular System 

* Central Nervous System 

* Immune System 

Drug and/or Radiation Therapy 

* Image Guided Therapy 

* Monitoring Therapy 

* Novel Therapy Development 

REGISTER NOW 

September 8-11, 2010 

Kyoto, Japan 

www.WMICmeeting.org


Wednesday - 26 May 2010 Thursday - 27 May 2010 Friday - 28 May 2010 Saturday - 29 May 2010 

room ROZA IRYS ROZA IRYS ROZA IRYS ROZA IRYS 

08:00-09:00 Registration Registration 

09:00-10:00 

ESMI Plenary Lecture 1 by Violaine Sée 

(Liverpool, UK) 

ESMI Plenary Lecture 3 by Theodorus W.J.Gadella 

(Amsterdam, The Netherlands) 

10:00-10:30 Coffee break & Exhibition Coffee break & Exhibition 

10:30-12:00 

PS 1: Cancer I - 

together with ESR 

PS 2: Neuroscience I PS 5: Neuroscience II 

PS 6: Cardiovascular I - 

together with ESR 

ESMI Plenary Lecture 5 by Klaas Nicolay 

(Eindhoven, The Netherlands) 

PS 9: Cardiovascular II 

12:00-13:00 Lunch break & Exhibition Break 

13:00-14:30 PS 3: Technology 

14:30-16:00 

16:00-17:00 

17:00-18:00 

Registration 

18:00-19:30 

Open Ceremony and Inaugural Lecture by 

19:00 

Christopher H. Contag (Stanford, USA) 

19:30 Opening Reception and Exhibition 

PS 4: Probes I - 

together with COST 

PS 7: Probes II 

PS 8: Gene and Cell 

Based Therapies - 

together with CliniGene 

Guided Poster Session 1 with Coffee break Guided Poster Session 2 with Coffee break 

ESMI Plenary Lecture 2 by Francesca Odoardi 

(Goettingen, Germany) 

ESMI Plenary Session on Excellent, Late Breaking 

and YIA Abstracts 

Sightseeing Tour 

ESMI Plenary Lecture 4 by Hans-Jürgen Wester 

(Munich, Germany) 

ESMI Plenary Session on Current Contribution of 

Imaging Technologies to Drug Development 

ESMI Executive Committee meets Industry 

Gala Dinner at Zamojski Palace 

Warsaw 2, Foksal street 

Programme at a Glance 

PS 10: Cancer II - 

together with COST 

12:30-13:30 

ESMI Plenary Session and Closing Ceremony 

5th EuropEan MolEcular IMagIng MEEtIng – EMIM2010


ROZA IRYS GERBERA 

09:00-12:00 Exhibitor’s set up 

14:00-18:00 Registration 

13:00-15:00 

15:00-17:00 

imaging life 

Posters set up 

Speaker’s documents set up 

18:00-19:30 Opening Ceremony and Inaugural Lecture 

Mariusz Klubczuk plays Chopin: 

Nocturne B flat Minor Op.9 No 1; Mazurka C Major Op.24 No 2 

Welcome – Andreas H. Jacobs (ESMI President) 

Welcome – Renata Mikolajczak (EMIM Local Organiser) 

Welcome – Andrzey Siemaszko (NCP in Poland for EU research) 

Welcome – Maximilian Reiser (ESR President) 

Announcement of ESMI Award 2010, John Clark (ESMI Award Winner 2009) 

Mariusz Klubczuk plays Chopin: 

Etude C sharp Minor Op. 25 No 7; Etude A Minor Op. 25 No 12 

Inaugural lecture by Christopher H. Contag (Stanford, USA) 

Point-of-Care Microscopy: Molecular Imaging with Cellular Resolution 

19:30-21:00 Opening Reception and Exhibition 

DiMI WP Meetings 

(Closed Meetings) 

ESMI Executive Committee 

(Closed Meeting) 

ESMI Council Meeting 



08:00-08:45 DiMI SMB (Closed Meeting) 


09:00-10:00 

ESMI Plenary Lecture 1 by Violaine Sée (Liverpool, UK) 

Transcription factors dynamics: from discrete pulses to oscillatory pattern to control gene expression 

Co-Chairs: Clemens WGM Löwik (Leiden, The Netherlands), Leszek Krolicki (Warsaw, Poland) 

10:00-10:30 Coffee break & Exhibition 

10:30-12:00 

Parallel Session 1: Cancer I (together with ESR) 

Co-Chairs: Markus Schwaiger (Munich, Germany), 

Peter Brader (Vienna, Austria) 

10:30-10:45 Multimodal imaging approaches for cell-cell interaction 

Steffen Maßberg, (Munich, Germany) 

10:45-11:00 Molecular imaging for monitoring treatment with 

protein kinase inhibitors 

Wolfgang Weber (Freiburg, Germany) 

11:00-11:15 Potentials of imaging biomarkers inpatients 

Bernard Van Beers (Paris, France) 

11:15-11:30 YIA Applicant’s Presentation: Pre-clinical screening of anti - her2 

nanobodies for molecular imaging of breast cancer 

Ilse Vaneycken (Brussels, Belgium) 

11:30-11:45 YIA Applicant’s Presentation: [18f]-fdg/[18f]-flt-pet and 

bioluminescence imaging of therapy response in b-cell lymphoma 

mice treated with cytotoxic and antiproliferative agents, 

Marijke De Saint-Hubert (Leuven, Belgium) 

11:45-12:00 tf-pimonidazole: an hypoxia marker suitable for in vivo 19f mrs 

imaging 

Arend Heerschap (Nijmegen, The Netherlands) 

12:00-13:00 Lunch break 

Day 0 - Wednesday May 26, 2010 


YIA Selection Committee 


Parallel Session 2: Neuroscience I 

Co-Chairs: Annemie van der Linden (Antwerp, Belgium), 

David Wyper (Glasgow, UK), Hervé Boutin (Manchester, UK) 

[11C](R)-PK11195- a tricky tracer. Do we really need it to image 

microglial activation? 

Alex Gerhard (Manchester, UK) 

YIA Applicant’s Presentation: Preclinical imaging of the type 1 

cannabinoid receptor in neurodegenerative diseases 

Cindy Casteels (Leuven, Belgium) 

YIA Applicant’s Presentation: Longitudinal non-invasive detection of 

amyloid plaques by combined molecular, functional and morphological 

imaging in transgenic mouse models of alzheimers disease, 

Florian C. Maier (Tübingen, Germany) 

Serotonergic neurotransmission in early Alzheimer’s disease, 

Steen G. Hasselbalch (Copenhagen, Denmark) 

Qualitative and quantitative assessment of florbetapir f-18 pet (18fav45) 

amyloid deposition PET imaging - validation by pathology. 

preliminary report 

Grzegorz Romanowicz (Gdańsk, Poland) 

YIA Applicant’s Presentation: In-vivo imaging of a mouse traumatic 

brain injury model using dual-isotope quantitative SPECT/CT 

Domokos Máthé (Budapest, Hungary) 

TOPIM2011 committee 

(Closed meeting)


Parallel Session 3: Technology 

13:00-14:30 Co-Chairs: Jorge Ripoll (Heraklion, Greece), 

Markus Rudin (Zürich, Switzerland) 

13:00-13:15 Molecular analysis of adaptive immune function through 

microscopic and mesoscopic imaging 

Jens Stein (Bern, Switzerland) 

13:15-13:30 Magnetic resonance imaging – from structure to molecular interactions 

Markus Rudin (Zürich, Switzerland) 

13:30-13:45 Imaging modalities: PET/SPECT 

Sibylle Ziegler (Munich, Germany) 

13:45-14:00 In vivo fluorescence kinetic imaging for improved contrast and 

studies of temporal and quantitative biodistribution 

James Mansfield (Cambridge, UK) 

14:00-14:15 Cerenkov radiation imaging of a xenograft murine model of 

mammary carcinoma 

Federico Boschi (Verona, Italy) 

14:15-14:30 A system for 4d (3d+kinetics) molecular imaging in 

bioluminescence and fluorescence 

Serge Maitrejean (Paris, France) 

14:30-16:00 

16:00-17:00 

17:00-18:00 


Parallel Session 4: Probes I (together with COST action D38) 

Co-Chairs: Eva Jakab-Tóth (Orléans, France), 

Silvio Aime (Torino, Italy) 

The alignment of target-specific lipoCEST MRI contrast agents 

Sander Langereis (Eindhoven, The Netherlands) 

Lanthanide-based in vivo luminescence imaging 

Stephane Petoud (Orléans, France and Pittsburgh, US) 

New perspectives for imaging molecules and metabolism in vivo 

Rolf Gruetter (Lausanne, Switzerland) 

YIA Applicant’s Presentation: In vivo biodistribution of radiolabeled 

matrix metalloproteinase-2 activatable cell penetrating peptides 

Sander Van Duijnhoven (Eindhoven, The Netherlands) 

YIA Applicant’s Presentation: Bioresponsive mri contrast agents 

based on self-assembling beta-cyclodextrin nanocapsules 

Jonathan Martinelli (Alessandria, Italy) 

Photochemical activation of endosomal escape of MRI-gd-agents in 

tumor cells 

Eliana Gianolio (Torino, Italy) 

Guided Poster Session 1 with Coffee break (see page 110) 

Poster Walk 1: Imaging Cancer Biology (P001-P012) & (P105-P106) 

Co-Chairs: Fabian Kiessling (Aachen, Germany), Markus Rudin (Zürich, Switzerland) 

Poster Walk 2: Technology & Data Analysis Methods (P070-P078) & (P097-P104) 

Co-Chairs: Serge Maitrejean (Paris, France), Adriaan Lammertsma (Amsterdam, The Netherlands) 

Poster Walk 3: Molecular Neuroimaging (P034-P047) & (P107) 

Co-Chairs: Sabina Pappata (Naples, Italy), Andreas H. Jacobs (Münster, Germany) 

Poster Walk 4: Probe Design (P058-P069) 

Co-Chairs: Frédéric Dollé (Orsay, France), Helmut Maecke (Freiburg, Germany) 

ESMI Plenary Lecture 2 by Francesca Odoardi (Göttingen, Germany) 

Immune surveillance and autoimmunity: how encephalitogenic T cells enter their target organ 

Co-Chairs: Renata Mikolajczak (Warsaw, Poland), Chrit Moonen (Bordeaux, France) 

Plenary Session on Excellent, Late Breaking, and Young Investigator Award Abstracts 

Co-Chairs: Renata Mikolajczak (Warsaw, Poland), Chrit Moonen (Bordeaux, France) 

17:00-17:10 Exendin-4 derivatives labeled with radiometals for the detection of insulinoma: a “from bench to bed approach” 

Damian Wild (Freiburg, Germany) 

17:10-17:20 YIA Applicant’s Presentation: 

Assessment of hif transcriptional activity in a mouse tumor model using gpi anchored avidin- 

a novel protein reporter for in vivo imaging 

Steffi Lehmann (Zürich, Switzerland) 


A leukocyte ligand of vascular adhesion protein-1 as an imaging tool in PET 

Anu Autio (Turku, Finland) 


Clinical translation of ex vivo sentinel lymph node mapping for colorectal cancer using invisible near-infrared fluorescence light 

Merlijn Huttemann (Leiden, The Netherlands) 


Development of strategies for in vivo MRI and optical imaging of neural stem cells distribution for the treatment of traumatic 

spinal cord injury 

Ramona Lui (Milano, Italy) 

17:50-18:00 Validation of bone marrow-derived stromal cell graft position by colocalisation of histology, 

bioluminescence and magnetic resonance imaging 

Nathalie de Vocht (Antwerp, Belgium) 

18:15-18:45 

19:00-20:00 

DiMI General Assembly 


DiMI WP Meeting 





08:00-08:45 


imaging life 





09:00-10:00 

ESMI Plenary Lecture 3 by Theodorus W.J. Gadella Jr. (Amsterdam, Netherlands) 

New probe-based strategies for quantitative microscopy of signaling dynamics in single cells 

Co-Chairs: Vasilis Nziachristos (Munich, Germany), Bengt Långström (Uppsala, Sweden) 

10:00-10:30 Coffee break & Exhibition 

10:30-12:00 

Parallel Session 5: Neuroscience II 

Co-Chairs: Markus Rudin (Zürich, Switzerland), 

Veerle Baekelandt (Leuven, Belgium), 

Mathias Hoehn (Cologne, Germany) 

10:30-10:45 Potential to use transgenic animals for imaging of neurological 

diseases 

Ludwig Aigner (Salzburg, Austria) 

10:45-11:00 Specific cell labeling with imaging reporters in vivo by viral vectors: 

the challenges 

Veerle Baekelandt (Leuven, Belgium) 

11:00-11:15 YIA Applicant’s Presentation: Optimization of an MRI method to 

measure microvessel density: application to monitor 

angiogenesis after stroke 

Philipp Boehm-Sturm (Cologne, Germany) 

11:15-11:30 MRI study of intra-arterial bone marrow-derived macrophage 

administration after acute focal ischemic stroke in rats 

Adrien Riou (Lyon, France) 

11:30-11:45 YIA Applicant’s Presentation: Inactivated paramagnetic tissue 

plasminogen activator predicts thrombolysis 

outcome following stroke 

Maxime Gauberti (Caen, France) 

11:45-12:00 Matrix metalloproteinase-9 – a possible therapeutic target 

in intractable epilepsy 

Grzegorz Wilczynski (Warsaw, Poland) 

12:00-13:00 Lunch Break 

13:00-14:30 

Parallel Session 7: Probes II 

(together with EANM) 

Co-Chairs: Frédéric Dollé (Orsay, France) , 

Denis Guilloteau (Tours, France) 

13:00-13:15 Application of microfluidics to the ultra-rapid preparation of 

fluorine-18 and carbon-11 labelled compounds 

Philip Miller (London, UK) 

13:15-13:30 Radioligand for in vivo measuring neurotransmitters release, 

Christer Halldin (Stockholm, Sweden) 

13:30-13:45 18F-tracers for Amyloid plaques 

Denis Guilloteau (Tours, France) 

13:45-14:00 YIA Applicant’s Presentation: Chelators for radiocopper: biological/ 

pharmacokinetic differences of [tyr3,thr8]octreotide conjugates, 

Abiraj Keelara (Basel, Switzerland) 

14:00-14:15 [11c]SOMADAM: potential sert ligand for PET studies 

and comparison with [11c]MADAM 

Fabienne Gourand (Caen, France) 

14:15-14:30 MR imaging of extracellular redox by a thiolsensitive 

gd(iii)-do3a derivative 

Giuseppe Digilio (Torino, Italy) 

Day 2 - Friday May 28, 2010 

ESMI Exec. Com. & ESMI Council 

Meeting (Closed Meeting) 

together with Treasurer 

Parallel Session 6: Cardiovascular I (together with ESR) 

Co-Chairs: Tony Lahoutte (Mons, Belgium), 

Nicolas Grenier (Bordeaux, France) 

Potentials of new contrast agents for vascular molecular imaging in 

patients 

Philippe Douek (Lyon, France) 

Protease specific nanosensors in atherosclerosis 

Eyk Schellenberger (Berlin, Germany) 

YIA Applicant’s Presentation: Imaging of inflamed carotid artery 

atherosclerotic plaques with the use of 99mtc-hynic-il-2 scintigraphy 

in the end-stage renal disease patients 

Marta Opalinska (Krakow, Poland) 

Uptake of 68ga-chloride in atherosclerotic plaques of ldlr-/apob100/100mice 

Johanna Silvola (Turku, Finland) 

YIA Applicant’s Presentation: Hybrid imaging using dual energy µct 

and fmt for characterization of atherosclerotic 

plaques in apoe -/- mice 

Felix Gremse (Aachen, Germany) 

YIA Applicant’s Presentation: Long circulating emulsion 

based CT contrast agents 

Anke De Vries (Eindhoven, The Netherlands) 

ESMI Training Committee 


Parallel Session 8: Gene and Cell Based Therapies 

(together with Clinigene) 

Co-Chairs: Ludwig Aigner (Salzburg, Austria), 

Cornel Fraefel (Zürich, Switzerland) 

Everything but not cell replacement with neural 

stem cell transplants 

Stefano Pluchino (Milano, Italy) 

Mesenchymal Stem Cell Transplantation: An Option to Promote 

Remyelination? Ludwig Aigner (Salzburg, Austria) 

Viral vector-mediated transcriptional targeting of dendritic cells for 

antigen-specific tolerance induction in EAE/MS 

Christiane Dresch (Zürich, Switzerland) 

Delivery of a bioluminescent transgene to a tumor via bone marrow 

engraftment and local control of gene 

expression by non invasive local hyperthermia 

Pierre-Yves Fortin (Bordeaux, France) 

YIA Applicant’s Presentation: Dendritic cell labelling with 

paramagnetic nanoparticles and 111in-oxine for in vivo magnetic 

resonance imagingand scintigraphic imaging 

Cristina Martelli (Milan, Italy) 

YIA Applicant’s Presentation: The type 2 cannabinoid receptor 

as a new PET reporter gene for the brain 

Caroline Vandeputte (Leuven, Belgium)

14:30-16:00 

16:00-17:00 

17:00-18:00 



Guided Poster Session 2 with Coffee break (see page 110) 

Poster Walk 5: Imaging in Cancer & Drug Development (P013-P030) 

Co-Chairs: Peter Brader (Graz, Austria), Nicolas Grenier (Bordeaux, France) 

Poster Walk 6: Imaging in Other Diseases (P031-P033) & (P079-P091) 

Co-Chairs: Bertrand Tavitian (Orsay, France), John Clark (Edingburgh, UK) 

Poster Walk 7: Imaging-guided Gene and Cell based and Targeted Therapies (P048-P057) & (P092-P096) 

Co-Chairs: Ludwig Aigner (Salzburg, Austria), Adriana Maggi (Milano, Italy) 

ESMI Plenary Lecture 4 by Hans-Jürgen Wester (Munich, Germany) 

Molecular Imaging of CXCR4 Receptors 

Co-Chairs: Adriana Maggi (Milano, Italy), Adriaan Lammertsma (Amsterdam, The Netherlands) 

Plenary Session on Current Contribution of Imaging Technologies to Drug Development 

Co-Chairs: Adriana Maggi (Milano, Italy), Adriaan Lammertsma (Amsterdam, The Netherlands) 

17:00-17:15 Evaluation of the temporal window for drug delivery following ultrasound mediated membrane permeability enhancement 

Matthieu Lepetit-Coiffe (Bordeaux, France) 

17:15-17:30 Integrisense: a novel near-infrared fluorescent probe for a v b 3 integrin and its applications in drug discovery 

Cyrille Sur, Merck Research Laboratories (Westpoint, USA) 

17:30-17:45 Harnessing the power of bioluminescence to cross the in vitro–in vivo divide 

John Watson, PROMEGA (Madison, Wisconsin, USA) 

17:45-18:00 In vivo imaging in drug discovery: the example of application in the development of novel estrogenic compounds 

Andrea Biserni, TOP Srl (Milano, Italy) 

18:00-19:00 

19:30 Gala Dinner at Zamojski Palace 

08:00-08:45 ESMI General Assembly (Closed Meeting) 

09:00-10:00 

ESMI Executive Committee 

meets Industry 



ESMI Plenary Lecture 5 by Klaas Nicolay (Eindhoven, The Netherlands) 

Multi-modality molecular imaging of atherosclerosis, usingtargeted contrast agents 

Co-Chairs: Uwe Haberkorn (Heidelberg, Germany), Helmut Maecke (Freiburg, Germany) 

10:00-10:30 Coffee break & Exhibition YIA Selection Committee 

10:30-12:00 

Parallel Session 9: Cardiovascular II 

Co-Chairs: Klaas Nicolay (Eindhoven, The Netherlands), 

Michael Schäfers (Münster, Germany) 

10:30-10:45 Advances in contrast-enhanced MRI of the mouse heart 

Gustav Strijkers (Eindhoven, The Netherlands) 

10:45-11:00 Molecular imaging of α v β 3 integrin expression with 18F-galacto- 

RGD after experimental myocardial infarction: comparison with left 

ventricular remodeling and function 

Antti Saraste (Turku, Finland/Munich, Germany) 

11:00-11:15 Existing and emerging animal models mimicking cardiovascular 

disease and their relevance for molecular imaging 

Michael Schäfers (Münster, Germany) 

11:15-11:30 Imaging of matrix metalloproteinase activity in vulnerable human 

carotid plaques with multispectral optoacoustic tomography 

Daniel Razansky (Munich, Germany) 

11:30-11:45 C-jun n-terminal kinase promotes inflammation at atherosclerosisprone 

sites by enhancing expression and activity 

of nf-κb transcription factor 

Paul Evans (London, UK) 

11:45-12:00 YIA Applicant’s Presentation: Absolute quantification in small 

animal pinhole gated myocardial perfusion SPECT 

Lode Goethals (Brussels, Belgium) 

Parallel Session 10: Cancer II(with COST action BM0607) 

Co-Chairs: Marion de Jong (Rotterdam, The Netherlands), 

Fabian Kiessling (Aachen, Germany) 

Cancer Imaging 

Uwe Haberkorn (Heidelberg, Germany) 

Controlled Drug Delivery under Image guidance 

Holger Grüll (Eindhoven, The Netherlands) 

Targeted radionuclide imaging and therapy (COST Action BM0607), 

Marion de Jong (Rotterdam, The Netherlands) 

Targeting cancer stem cells using radiolabeled sonic hedgehog, 

Izabela Tworowska (Houston, USA) 

YIA Applicant’s Presentation: Evaluation of the photosensitizer 

bremachlorin for photodynamic treatment of 

breast cancer bone metastasis 

Pieter Van Driel (Leiden, The Netherlands) 

In vivo targeting of hek-hsst2/3/5 xenografts by 111in-labeled 

[(dota)ser1,leu8,trp22,tyr25]-ss-28 in scid mice 

Theodosia Maina (Athens, Greece) 

12:00-12:30 Break YIA Selection Committee 

12:30-13:30 

ESMI Plenary Session and Closing Ceremony and Young Investigator Award Presentations 

Young Investigator Awards & Poster Awards Ceremony 

Co-Chairs: John Clark (Edinburgh, UK), Bertrand Tavitian (Orsay, France) 

Day 3 - Saturday May 29, 2010 



Do not forget: 

Join the next ESMI Winter Conference – TOPIM2011: 

further information/pre-registration 

send mail to office@ e-smi.eu 

WWW.E-SMI.Eu 

HOT TOPIC 2011: Emerging image-guided methods in Medicine 

Jan. 16-21, 2011 in Les Houches, France & 


MY NOTES fl

20 


Dr. Contag is an Associate Professor of Pediatrics in the Division of Neonatal and Developmental Medicine, 

and a member of the BioX faculty at Stanford University. He is the Director of the Stanford Infrared Optical 

Science and Photomedicine Program, director of Stanford’s Center for Innovation in In Vivo Imaging (SCI3) 

and co-director of the Molecular Imaging Program at Stanford (MIPS). Dr. Contag received his B.S. in 

Biology from the University of Minnesota, St. Paul in 1982; and earned his Ph.D. in Microbiology from the 

University of Minnesota, Minneapolis in 1988. He was a postdoctoral fellow at Stanford University from 

1990-1994, and joined the faculty in Pediatrics at Stanford in 1995 with a joint appointment in Microbiology 

and Immunology and a courtesy appointment in Radiology. 

Dr. Contag is a pioneer in the emerging field of molecular imaging and is developing imaging approaches 

aimed at revealing molecular processes in living subjects and advancing therapeutic strategies through 

imaging. His laboratory develops macroscopic and microscopic optical imaging tools and uses imaging to 

assess tissue responses to stress, reveal immune cell migration patterns, understand stem cell biology and 

advance biological therapies. He is a founding member, and a past president, of the Society for Molecular 

Imaging, and for his fundamental contributions in imaging, is a recipient of the Achievement Award from 

the Society for Molecular Imaging. Dr. Contag is a scientific founder of Xenogen Corp. – now Caliper 

LifeSciences. He is also a founder of ConcentRx Corp. 

Selected References 

• Cao, Y-A, Stevenson, DKS, Weissman, I, Contag, CH. 2008. Heme Oxygenase-1 Deficiency leads to disrupted response 

to acute stress in stem cells and progenitors. Blood. 112: 4494-4502 

• Banaszynski, LA, Sellmyer, MA, Thorne, SH, Contag, CH, Wandless, TJ. 2008. Chemical control of protein stability and 

function in living mice. Nature Med. 14 (10):1123-7. 

• Wender, PA, Goun, EA, Jones, LR, Pillow, TH, Rothbard, JB, Shinde, R, Contag, CH. 2007. Real-time analysis of uptake 

and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice. Proc Nat Acad Sci, USA. 

104(25):10340-5. 

• Thorne SH, Negrin, RS, Contag, CH. 2006. Synergistic antitumor effects of immune cell-viral biotherapy. Science. 311:1780-1784. 

• Cao, Y-A, Wagers, A, Beilhack, A, Dusich, J, Bachmann, MH, Negrin, RS, Weissman, IL, Contag, CH. 2004. Shifting foci of 

hematopoeisis during reconstitution from single stem cells. Proc. Natl. Acad. Sci. USA. 101(1):221-226. 

• Hardy, JK, Chu, P, Gibbs, K, Contag, CH. 2004. Extracellular replication of Listeria monocytogenes in the gall bladder. 

Science. 303(5659): 851-853 

• Contag, CH and Bachmann, MH. 2004. The writing is on the vessel wall. Nature. 429:618-620. 

• Shachaf, CM, Kopelman, A, Arvanitis, C, Beer, S, Mandl, S, Bachmann, MH, Borowsky, AD, Ruebner, B, Cardiff, RD, Yang, 

Q, Bishop, JM, Contag, CH and Felsher, DW. 2004. MYC inactivation uncovers stem cell properties and induces the state 

of tumor dormancy in hepatocellular cancer. Nature, 431:1112-1117. 

• Contag, PR, Olomu, IN, Stevenson, DK, Contag, CH. 1998. Bioluminescent indicators in living mammals. Nature Med. 

4(2):245-247. 

imaging life 

Christopher H. Contag


Point-of-Care Microscopy: Molecular Imaging with Cellular Resolution 

Contag C.H. . 

Division of Neonatal and Developmental Medicine, and a member of the BioX faculty at Stanford University. 

ccontag@stanford.edu 

Micro-optical designs are enabling the development 

of miniaturized microscopes that can reach inside 

the body to interrogate disease states microscopically. 

This is leading to an emerging field of in vivo 

pathology that is changing the diagnostic paradigm 

from biopsy 

and conventional 

histopathology to 

one of point-ofcare 

histopathology 

coupled with telepathology. 

These 

advances are closing 

the gap between 

micro–mirror 

the patient and the 

pathologist and 

have the potential of accelerating diagnosis and 

guiding therapy. While recent advances in this 

field have been significant, many issues must be 

resolved before this clinical transformation may be 

fully realized. There are technological and translational 

advances that are driving this field and are 

leading towards in vivo microscopy becoming a standard 

clinical tool. By removing the spatiotemporal 

separation between the pathologist and patient, we 

can accelerate clinical diagnosis and advance clinical 

care for patients with a wide variety of diseases. 

References 

1. Liu, JTC, Mandella, MJ, Loewke, NO, Haeberle, H, Ra, H, 

Piyawattanametha, W, Solgaard, O, Kino, GS, Contag, 

CH (2010) Micromirror-scanned dual-axis confocal 

microscope utilizing a gradient-index relay lens for 

image guidance during brain surgery. JBO. In Press. 

2. Piyawattabanetha, W, Ra, H, Mandella, MJ, Loewke, 

Wang, TD, Kino, GS, Solgaard, O, Contag, CH (2009) 3-D 

near-infrared fluorescence imaging using a MEMSbased 

miniatuire dual axis confocal microscope. IEEE J. 

Sel. Topics Quantum Electronics. 15(5): 1344-1350. 

3. Mackanos, MA, Hargrove, J, Wolters, R, Du, CB, 

Friedland, S, Soetikno, RM, Contag, CH, Arroyo, MR, 

Crawford, JM, Wang, TD (2009) Use of an endoscopecompatible 

probe to detect colonic dysplasia with 

Fourier transform infrared spectroscopy. J Biomed 

Optics 14, 044006. PMID: 19725718 

4. Liu, JT, Mandella, MJ, Crawford, JM, Contag, CH, Wang, 

TD, Kino, GS. (2008) Efficient rejection of scattered light 

enables deep optical sectioning in turbid media with 

low-numerical-aperture optics in a dual-axis confocal 

architecture. J Biomed Opt. 13(3):034020. 

5. Hsiung, P-L, Hardy, JW, Friedland, S, Soetikno, R, Du, CB, 

Wu, APW, Sahbaie, P, Crawford, JM, Lowe, AW, Contag, 

CH, Wang, TD. (2008) Detection of colonic dysplasia in 

vivo using a targeted fluorescent septapeptide and 

confocal microendoscopy. Nat. Med. 14(4): 454-8. 

6. Wang, TD, Triadafilopoulos, G, Crawford, JM, Dixon, LR, 

Bhandari, T, Sahbaie, P, Friedland, S, Soetikno, R, Contag, 

CH. (2007) Detection of Endogenous Biomolecules 

in Barrett’s Esophagus by Fourier Transform Infrared 

Spectroscopy. Proc. Natl. Sci, USA. 104(40): 15864-9. 


day0 

Inaugural Lecture by Christopher H. Contag

DAY 

1


• ESMI Plenary Lecture 1: Violaine Sée (Liverpool, UK) 

Transcription factors dynamics: from discrete pulses to oscillatory pattern to control gene expression 

Chairs: Clemens W.G.M. Löwik (Leiden, The Netherlands) , Leszek Krolicki (Warsaw, Poland) 

• Parallel Session 1: Cancer I – together with ESR 

Chairs: Markus Schwaiger (Munich, Germany), Peter Brader (Vienna, Austria) 

• Parallel Session 2: Neuroscience I 

Chairs: Annemie van der Linden (Antwerp, Belgium), David Wyper (Glasgow, UK), 

Hervé Boutin (Manchester, UK) 

• Parallel Session 3: Technology 

Chairs: Jorge Ripoll (Heraklion, Greece) , Bernd Pichler (Tuebingen, Germany) 

• Parallel Session 4: Probes I – together with COST 

Chairs: Eva Jakab-Tóth (Orléans, France) , Silvio Aime (Torino, Italy) 

• ESMI Plenary Lecture 2: Francesca Odoardi (Göttingen, Germany) 

Immune surveillance and autoimmunity: how encephalitogenic T cells enter their target organ 

Chairs: Renata Mikolajczak (Warsaw, Poland), Chrit Moonen (Bordeaux, France) 

• Plenary Session on Excellent and Late Breaking Abstracts 

Chairs: Renata Mikolajczak (Warsaw, Poland), Chrit Moonen (Bordeaux, France) 



day1 

Day 1 - Thursday May 27, 2010

24 


Violaine Sée is a BBSRC David Phillips Research Fellow at the University of Liverpool. She graduated in 

Chemistry and Molecular and Cellular Biology at the University Louis Pasteur in Strasbourg (France). After a 

Master in Pharmacology, in 2001 she obtained her PhD in Pharmacology and Neurobiology at the University 

Louis Pasteur. She was then assistant lecturer and subsequently moved to the University of Liverpool as a 

Post-doctoral Research Fellow. In 2005, she obtained a prestigious David Phillips Fellowship, to develop 

her work on intracellular signalling dynamics. She is focusing on the imaging of single living cells in order 

to understand regulation of gene transcription and cell fate. She has recently been interested in using new 

techniques for single molecule imaging in live cells based on the use of gold nanoparticles. 

Selected References 

• Nelson, D. E., Ihekwaba, A. E., Elliott, M., Johnson, J. R., Gibney, C. A., Foreman, B. E., Nelson, G., See, V., Horton, C. A., 

Spiller, D. G., Edwards, S. W., McDowell, H. P., Unitt, J. F., Sullivan, E., Grimley, R., Benson, N., Broomhead, D., Kell, D. B., 

and White, M. R. (2004) Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306, 

704-708 

• See, V., Rajala, N. K., Spiller, D. G., and White, M. R. (2004) Calcium-dependent regulation of the cell cycle via a novel 

MAPK--NF-kappaB pathway in Swiss 3T3 cells. The Journal of cell biology 166, 661-672 

• Nelson, D. E., See, V., Nelson, G., and White, M. R. (2004) Oscillations in transcription factor dynamics: a new way to 

control gene expression. Biochemical Society transactions 32, 1090-1092 

• Ashall, L., Horton, C. A., Nelson, D. E., Paszek, P., Harper, C. V., Sillitoe, K., Ryan, S., Spiller, D. G., Unitt, J. F., Broomhead, D. 

S., Kell, D. B., Rand, D. A., See, V., and White, M. R. (2009) Pulsatile stimulation determines timing and specificity of NFkappaB-dependent 

transcription. Science 324, 242-246 

• See, V., Free, P., Cesbron, Y., Nativo, P., Shaheen, U., Rigden, D. J., Spiller, D. G., Fernig, D. G., White, M. R., Prior, I. A., Brust, 

M., Lounis, B., and Levy, R. (2009) Cathepsin L digestion of nanobioconjugates upon endocytosis. ACS nano 3, 2461- 

2468 

• Meley, D., Spiller, D.G., White, M.R., McDowell, H., Pizer, B.L., and See, V. (2010) p53-mediated delayed NF-kB activity 

enhances etoposide-induced cell death in medulloblastoma. Cell Death and Disease 1, In press 

imaging life 

Violaine Sée


Transcription factors dynamics: from discrete pulses to oscillatory pattern to control gene 

expression 

Sée V. 

Centre for Cell Imaging, University of Liverpool 

violaine@liverpool.ac.uk 

At a given time-point, cells in a population are 

heterogeneous in their functions and fate and it is 

therefore vital to develop and apply methods that 

allow the measurement of dynamic molecular processes 

in single cells. We have shown, using single 

cell imaging, the critical role of nucleo-cytoplasmic 

localisation oscillations of the NF-κB transcription 

factor to control downstream pattern of gene transcription 

(Nelson et al, Science 2004; Ashall et al, 

Science 2009). NF-κB regulates cellular stress and 

immune responses to infection. In most cases, oscillations 

had previously been masked in population 

level studies by cellular heterogeneity. We developed 

a protocol based on cell treatment by repeated short 

pulses of TNFα at various intervals to mimic pulsatile 

inflammatory signals. This allowed obtaining 

synchronous cycles of NF-κB nuclear translocation. 

We have also observed cell to cell heterogeneity in 

other signalling systems such as in cellular response 

to low oxygen environment (hypoxia). In both inflammatory 

and hypoxic signalling systems one 

source of heterogeneity is due to the presence of extrinsic 

dynamic processes that are functionally coupled 

and that are occurring over different time scales. 

We identified the cell cycle as one source of variability 

as cells must coordinate and prioritise their 

response to the environment depending on their 

cell cycle status. We apply mathematical modelling 

using the quantitative data generated by imaging 

experiments to predict the role of the negative feedback, 

to unravel new network motifs and to characterise 

the cross-talk between the signalling systems. 


day1 

ESMI Plenary Lecture 1 by Violaine Sée

26 Cell-cell 


Multimodal imaging approaches for cell-cell interaction 

Massberg S. 

Deutsches Herzzentrum München, Technische Universität München 

massberg@idi.harvard.edu 

interactions play a pivotal role in the control 

of critical cellular functions, such as cell adhesion, 

cell migration as well as cell differentiation and 

proliferation. The scientific focus of our lab is on 

the mechanisms that regulate cell-cell interactions 

during scenarios, such as arterial or venous thrombosis, 

stem cell homing and differentiation, during 

inflammation as well as in cancer development and 

progression. To evaluate the interaction between 

different cell subsets in their physiological (micro-) 

environment, we have established novel imaging approaches 

that allow dissecting all the steps involved 

both in reductionist in vitro assays and in vivo. 

imaging life 

This includes the used of gene-targeted mice, in 

which distinct cellular lineages are genetically 

marked in combination with innovative imaging 

techniques, including intravital multi-photon microscopy. 

The in vivo approaches are complemented 

by sophisticated in vitro assays allowing investigation 

of cell-cell interactions on a subcellular and 

molecular level. The multimodal imaging approaches 

will contribute to a better understanding of the 

molecular mechanisms and the kinetic aspects of 

the dynamic process of cell-cell interactions under 

physiological conditions and in the disease states.


Molecular imaging for monitoring treatment with protein kinase inhibitors 

Weber W. 

University Freiburg, Germany 

wolfgang.weber@uniklinik-freiburg.de 

Targeted drugs that modulate the function of specific 

molecules in diseased tissues hold great promise for 

the treatment of many diseases including malignant 

tumors. However, there are several challenges for 

the efficient evaluation of these drugs in clinical 

trials as well as for the use in clinical practice. 

These include 

(i) the selection of patients likely to benefit from 

treatment with a specific targeted drug, 

(ii) finding the right dose and dose schedule, 

(iii) monitoring target inhibition, and 

(iv) assessing tumor response to therapy. 

Standard anatomic imaging continues to play an 

important role for addressing these challenges, 

but molecular imaging provides several new 

opportunities to make the use of targeted drugs more 

efficient. Using molecular imaging the expression 

of drug targets can be assessed non-invasively, the 

concentration of drugs can be measured in the 

tumor tissue, target inhibition can be monitored and 

tumor response to therapy can be evaluated earlier 

than with anatomic imaging techniques. 

Therefore it is expected that molecular imaging 

will play an increasing role for guiding molecularly 

defined therapeutic interventions. 


day1 

Parallel Session 1: CANCER I - together with the ESR

28 Introduction: 


Potentials of imaging biomarkers in patients 

Van Beers B.E. 

Department of Radiology and INSERM U 773, Beaujon University Hospital, University Paris Diderot, France. 

bernard.van-beers@bjn.aphp.fr 

There is an increasing need for imaging 

biomarkers to objectively assess the prognosis and 

response to treatment in cancer. 

Methods: Quantitative anatomical, molecular, and 

functional characteristics can be used as imaging 

biomarkers. 

Results: The RECIST criteria are anatomical imaging 

biomarkers that are accepted surrogate endpoints 

in cancer treatment. However, they are not early 

biomarkers and are limited in the assessment of 

some tumors and targeted treatments. 

The use of molecular imaging biomarkers is 

restricted by their narrow target specificity and 

by the complexity of the biology in tumors. In a 

given tumor type, several different subgroups often 

exist with overexpression of different proteins and 

signaling pathways. 

Therefore, it is often preferred to assess downstream 

changes of molecular pathways with functional 

imaging life 

imaging. The rationale of this approach is that 

most cancers have acquired the same functional 

capabilities including upregulated glycolysis, limitless 

proliferation, extensive angiogenesis, resistance to 

apoptosis and metastasis formation. These capabilities 

are assessed with functional biomarkers using PET, 

dynamic contrast-enhanced CT and MRI, diffusionweighted 

MRI, MR elastography and spectroscopy. 

Conclusions: Functional biomarkers have an 

important potential to help in selecting the patients 

and assessing the response to new treatments, 

especially in phase 1 and 2 clinical trials. 

However, important efforts of validation and 

standardization remain to be done before the wide 

use of functional biomarkers and their acceptance 

as surrogate endpoints that can replace clinical 

endpoints such as survival or time to progression. 

References: 

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 

2000;100(1):57-70. 

2. Rudin M. Imaging readouts as biomarkers or surrogate 

parameters for the assessment of therapeutic 

interventions. Eur Radiol. 2007 (10):2441-57. 

3. Padhani AR, Liu G, Koh DM, Chenevert TL, et al. 

Diffusion-weighted magnetic resonance imaging as a 

cancer biomarker. Neoplasia. 2009;11(2):102-25. 

4. Van Beers BE, Vilgrain V. Biomarkers in abdominal 

imaging. Abdom Imaging. 2009;34(6):663-7.


Pre-clinical screening of anti - HER2 nanobodies for molecular imaging of breast cancer 

Vaneycken I. (1) , Devoogdt N. (1) , Peleman C. (1) , Xavier C. (1) , Lahoutte T. (1) , Caveliers V. (1) . 

Vrije Universiteit Brussel, Belgium 

ilse.vaneycken@gmail.com 

Introduction: Nanobodies are small antigen-binding 

fragments of camelid heavy-chain antibodies. Besides 

therapeutic agents, they are also effective as a 

diagnostic tool for targeted imaging when labeled 

with a suitable radionuclide[1]. We produced Nanobodies 

for molecular imaging of Human Epidermal 

Growth Factor Receptor 2 (HER2) expression in 

breast cancer patients[2] for a phase I clinical trial. 

After production, a number of candidate binders 

are obtained. we designed a standardized selection 

procedure based on production yield, in vitro and 

in vivo criteria to identify the lead anti-HER2 Nanobody 

with the highest potential as a tracer for clinical 

translation. 

Methods: A camel was immunized with HER2 recombinant 

protein and serum IgG2 and IgG3 subclasses 

with heavy-chain-only antibodies were 

separated. Eleven different anti-HER2 Nanobodies 

were produced in E. coli, purified and labeled with 

99mTc(I)-tricarbonyl. In vitro saturation binding 

studies were performed on recombinant HER2 and 

HER2 positive SKOV3 cells with the native and radiolabeled 

anti-HER2 Nanobodies. Competition 

studies allowed assessing whether the anti-HER2 

99mTc-Nanobodies competed with the therapeutic 

anti-HER2 antibodies Trastuzumab and Pertuzumab. 

In vivo, the biodistribution and tumor targeting 

potential of all 99mTc-Nanobodies was evaluated 

using pinhole SPECT/micro-CT in nude mice bearing 

HER2 positive SKOV3 xenografts. The Nanobody 

showing the most favourable in vivo characteristics 

was additionaly evaluated in nude mice 

bearing HER2 positive LS174T and HER2 negative 

MDAMB4357d xenografts. 

Results: Nanobodies were labeled with 99mTc and 

purified to a final radiochemical purity of ≥99%. 

Saturation binding studies showed that 99mTc labeling 

was not associated with a significant reduction of 

immunoreactivity. The Nanobodies appeared to be 

not or only weakly competitive with the commercial 

therapeutic antibodies. In vivo biodistribution demonstrated 

that tumor accumulation varied between 

0,78 and 4,44 percent injected activity per gram 

(%IA/g). Of the eleven 99mTc-Nanobodies tested in 

SKOV3 xenografts, three presented a tumor uptake 

above 4 %IA/g. One was selected and also showed 

high tumor uptake (3,76±0,82 %IA/g) in LS174T xenografts, 

but low uptake in MDAMB435d xenografts 

(0,71±0,07 %IA/g). In addition, all 99mTc-Nanobodies 

displayed high renal uptake but low non-specific 

accumulation in liver, muscle and blood, resulting 

in high tumor-to-background ratios. 

Conclusions: Using a standardized selection procedure 

for identificiation of high affinity Nanobodies 

for molecular imaging of cancer, we identified one 

Nanobody as the lead compound for a phase I clinical 

trial. This Nanobody meets with all criteria that 

characterize a good diagnostic tracer. 

Acknowledgement: The research at ICMI is funded 

by the Interuniversity Attraction Poles Program– 

Belgian State–Belgian Science Policy. Tony Lahoutte 

is a Senior Clinical Investigator of the Research 

Foundation–Flanders (Belgium) (FWO). 

References: 

1. Gainkam LO, Huang L, Caveliers V, et al. Comparison of 

the biodistribution and tumor targeting of two 99mTclabeled 

anti-EGFR nanobodies in mice, using pinhole 

SPECT/micro-CT. J Nucl Med. 2008;49(5):788-795. 

2. Hicks,D.G. et al (2008). HER2+ breast cancer: review of 

biologic relevance and optimal use of diagnostic tools. 

American Journal of Clinical Pathology, 129, 263-273. 


YIA applicant 

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YIA applicant 



[18F]-FDG/[18F]-FLT-PET and bioluminescence imaging of therapy response in B-cell 

lymphoma mice treated with cytotoxic and antiproliferative agents 

De Saint-Hubert M. (1) , Brepoels L. (1) , Devos E. (1) , Degroot T. (1) , Ibrahimi A. (1) , Tousseyn T. (1) , Verbruggen A. (1) , 

Mortelmans L. (1) , Mottaghy F. (2) . 

(1) KULeuven, Belgium 

(2) RWTH Aachen. 

marijke.desainthubert@med.kuleuven.be 

Early and reliable evaluation of therapy 

outcome is essential for optimal medical care 

in oncology. Shortly after treatment the influx of 

inflammatory cells can interfere with [18F]-FDG 

uptake while [18F]-FLT is less hampered by this 

phenomenon. The aim of this study was to image 

[18F]-FDG and [18F]-FLT response to either cytotoxic 

or antiproliferative therapy and to correlate the 

response to the amount of viable cells assessed with 

Bioluminescence Imaging (BLI). 

Methods: Daudi cells (Burkitt lymphoma) were 

transduced with a lentiviral vector (blasticidin selection 

marker and firefly luciferase reporter gene) and 

inoculated in SCID-mice (n=57). When the tumors 

reached a diameter of 15mm2 animals were treated 

with either cyclophosphamide (125mg/kg, n=25) 

or temsirolimus (50mg/kg, n=25). [18F]-FDG and 

[18F]-FLT small-animal PET was performed on 

day0 (d0, before treatment) day (d) 2, 4, 7, 9 and 14. 

On these days we also imaged the tumor cells with 

BLI. At each time point, 2 mice of each treatment 

condition were sacrificed and tumors were excised 

for histopathology (H&E, Ki-67, CD20, TUNEL). 

Quantitative imaging data were expressed as % signal 

of the baseline signal measured on d0 and expressed 

as mean ± standard error of the mean (SEM). 

Results: [18F]-FDG uptake decreased immediately 

(-38% on d2) after cyclophosphamide treatment 

without a significant reduction in [18F]-FLT uptake 

which could be due to DNA repair as DNA FACS 

showed an increased amount of cells in the S-fase 

early after cyclophosphamide. From d7 [18F]-FLT 

uptake decreased significanlty corresponding to histology 

data (ki-67). [18F]-FDG uptake stabilized between 

d7 and d9 (-57% and -59% respectively) likely 

due to a high influx of inflammatory cells observed 

in histology (±40% on d7-d9). Caliper measurements 

showed tumor shrinkage from d7. Surprisingly the 

BLI measured an increased signal early after therapy 

(d2-d4) while only late after therapy a reduction in 

the signal was observed (d9-d14). 

Temsirolimus treatment reduced both the [18F]- 

FDG and [18F]-FLT uptake immediately (d2) after 

therapy. [18F]-FDG stabilized between d9 and d14 

imaging life 

maybe due to inflammation which was only observed 

on d14 but this might as well be due to regrowth. The 

early [18F]-FLT decrease corresponded well to ki-67 

stainings and to DNA FACS where an increase of the 

G0/G1 fase is observed. Tumor size reduced already 

from d4 after temsirolimus treatment. BLI showed 

the same signal increase as cyclophosphamide early 

after therapy and only in a late stage after therapy 

we observed a reduced amount of viable cells. This 

early increase might be due to an upregualtion of the 

promotor after therapy but further evalution of this 

phenomenon is on the way. 

Conclusions: [18F]-FDG reduced early after therapy 

but stabilized due to a temporary rise in inflammatory 

cells. [18F]-FLT-PET was able to detect a 

reduced proliferation after therapy. However, after 

cytotoxic therapy [18F]-FLT did not change probably 

due to DNA repair. Therefore caution should be 

made when imaging [18F]-FLT response immediately 

after cytotoxic therapy. Bioluminescence imaging 

showed an early increase which may have important 

implication for BLI monitoring of therapy. 

Acknowledgement: This work was financially supported 

by the Center of Excellence ‘MoSAIC’ of the 

K.U.Leuven.


TF-Pimonidazole: an hypoxia marker suitable for in vivo 19 F MRS imaging 

Heerschap, A. . 

Centre for Molecular Life Science, University Nijmegen, The Netherlands 

a.heerschap@rad.umcn.nl 

Introduction: Hypoxia in tumours is associated 

with enhanced progression, increased aggressiveness 

and metastatic potential and poor prognosis. 

Moreover, hypoxic tumour cells are resistant to 

radiotherapy and some forms of chemotherapy. 

Over the years several approaches to assess hypoxia 

in vivo have been explored, ranging from 

needles measuring local pO 2 invasively to a range 

of non-invasive (imaging) methods using oxygen 

sensitive agents. Unfortunately until today none 

of these methods or agents has entered widespread 

clinical practice. Here we demonstrate a 

novel hypoxia marker completely analogous to 

the commonly used histological hypoxia marker 

Pimonidazole (PIMO) and labelled with fluorine 

for in vivo 19 F MRS imaging. 

Methods:1-(2-nitro-1H-imidazol-1-yl)-3-[4- 

(trifluoromethyl)piperidin-1-yl]propan-2-ol 

(TF-PIMO) was synthesized in a similar way as 

described in [1]. C57BL/6 mice carrying a C38 

colon carcinoma on the upper leg (size approx. 

250 mm 3 ) were given either 80 or 200 mg / kg TF- 

PIMO intraperitoneal (IP) at least 3 hours before 

MR investigations. The mice were anesthetized 

using a single urethane IP injection. This avoids 

any spectral interference by fluorinated inhalation 

anaesthetics. Experiments were performed 

on a 7 T horizontal bore MR system. A homemade 

14 mm solenoid coil was used for transmit/receive 

of 19 F and 1 H. After initial localization and basic 

1 H MR imaging (T2*w GRE) an unlocalized pulse 

acquire sequence (FID) on 19 F was used to detect 

TF PIMO validating correct injection of the compound. 

Subsequently a series of 3D 19 F chemical 

shift imaging (CSI) FID experiments using an 

ultrashort adiabatic half passage pulse was used 

for TF-PIMO 19 F imaging. Further settings of the 

MRSI sequence: FOV 32 × 32 ×32 mm, matrix 

8 × 8 × 8, TR 597 ms, acquisition time 58 m 02 

s, 256 averages, weighted phase encoding scheme. 

After MR, tumours were removed immediately 

and stored in liquid nitrogen. Frozen tumour sections 

of 5 μm thickness were cut for staining and 

further analysis. The tumor sections were subsequently 

stained and scanned for Hoechst and rabbit 

anti-pimonidazole. 

Results: Figure 1 shows a heat map generated from 

the 3D 19 F MRSI representing the in vivo TF-PIMO 

accumulation in a hypoxic C38 colon carcinoma as 

was validated by subsequent immunohistochemical 

analysis of the tumour tissue. 

Figure 1: 1H MRI of a C38 murine colon carcinoma on a mouse leg 

with a heat map generated from a 3D 19F MRSI representing the in 

vivo TF-PIMO accumulation over-layed. 

Conclusions: TF-PIMO was synthesized and shown 

to be a potential non-invasive marker to image tissue 

hypoxia in tumours by 19F MRSI. Background 

free functional images are obtained that can be coregistered 

with conventional MRI. In addition this 

marker has the advantage that it can be stained with 

the same anti-body as used to detect the common 

clinical used marker Pimonidazole and thus allows 

for easy matching of hypoxia by histology and by 

MR on the same tumour sample. 

Acknowledgement: This work is supported in part 

by EMIL (LSHC-CT-2004-503569) and NWO (VIS- 

TA and INV911-06-021) 

References: 

1. Raleigh, J. A. et al; Magn Reson Med 22:451-466 (1991) 


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32 Detailed 


[11C](R)-PK11195- a tricky tracer. Do we really need it to image microglial activation? 

Gerhard A. . 

Wolfson Molecular Imaging Centre Manchester, UK 

alex.gerhard@manchester.ac.uk 

experimental and human post mortem 

studies have shown that activation of microglia, the 

brain’s resident tissue macrophages, has a particularly 

close association with active brain pathology. One 

of the molecules expressed de novo during the 

‘activation’ of microglia is the translocator protein 

(18kDa), TSPO.PK11195 is a selective ligand for the 

TSPO. 

In vivo and in the absence of invading blood-borne 

cells the de novo expression of TSPO occurs primarily 

in activated microglia. Based on this relative cellular 

selectivity [11C]PK11195 PET has been used to 

image microglial activation in vivo for about 20 years 

now and it has been possible to demonstrate patterns 

of increased signal distribution in the brain that 

correspond well with the localisation of pathological 

changes in a number of neurological disorders (1). 

Recently [11C](R)- PK11195 PET has even 

successfully been used to show in vivo the effect of a 

pharmacological intervention in a neurodegenerative 

disorder on microglial activation (2). 

imaging life 

Nevertheless there are methodological challenges 

when using [11C]PK11195 particularly an 

unfavourable signal to noise ratio. Therefore 

numerous new ligands for the TSPO have been 

evaluated at different stages over the last years 

with some of them having also been assessed in 

diseases known to be accompanied by microglial 

activation (3). 

The talk will give a brief overview of relevant studies 

with PK11195, as well as newer TSPO tracers, and 

will try to highlight their potential advantages and 

problems. 

References: 

1. Venneti S, Lopresti BJ, Wiley CA. The peripheral 

benzodiazepine receptor (Translocator protein 

18kDa) in microglia: from pathology to imaging. Prog 

Neurobiol. 2006;80(6):308-322. Epub 2006 Dec 2006. 

2. Dodel R, Spottke A, Gerhard A, et al. Minocycline 1-year 

therapy in multiple-system-atrophy: Effect on clinical 

symptoms and [(11)C] (R)-PK11195 PET (MEMSA-trial). 

Mov Disord. 2010;25(1):11. 

3. Chauveau F, Boutin H, Van Camp N, Dolle F, Tavitian 

B. Nuclear imaging of neuroinflammation: a 

comprehensive review of [(11)C]PK11195 challengers. 

Eur J Nucl Med Mol Imaging. 2008;1:1.


Preclinical imaging of the type 1 cannabinoid receptor in neurodegenerative diseases 

Casteels C. (1) , Van Laere K. (1) . 

University Leuven, Division of Nuclear Medicine, Belgium 

cindy.casteels@med.kuleuven.be 

The endocannabinoid system (ECS) is an important 

modulatory system in the brain. It consists of a family 

of naturally occurring lipids, the endocannabinoids, 

of transport and degradation proteins, and of 

cannabinoid receptors. Type 1 cannabinoid (CB1) 

receptors are abundantly expressed in all brain areas, 

especially those involved in the control of motor 

function. CB1 receptor stimulation modulates 

GABA, glutamate and dopamine neurotransmitter 

release in a dynamic activity manner.1 

Dysregulation of cannabinoid-mediated control 

of basal ganglia function have been suggested to 

play a critical role in the pathogenesis and symptom 

development of Parkinson’s disease (PD) and 

Huntington’s disease (HD), providing rationale for 

potential ECS-targeted therapy in these diseases.2 

However, at present, limited clinical pilot trials have 

been inconclusive. As only in vitro, ex vivo and 

post mortem data on the ECS existed until recently, 

functional in vivo imaging may play an important 

role in the further evaluation of the neurobiological 

and clinical impact of the ECS in PD and HD. 

Here, we present the in vivo characterization of CB1 

receptor alterations in preclinical models of PD and 

HD using PET and [18F]MK-94703. Both genetic 

and toxin-based experimental models will be presented. 

Their relevance to mimic the human condition 

will be discussed as well. 

Acknowledgement: Financial support of the Research 

Council of the Katholieke Universiteit Leuven 

(OT/05/58), the Fund for Scientific Research, 

Flanders, Belgium (FWO/G.0548.06), and the Institute 

for the Promotion of Innovation by Science and 

Technology in Flanders (SBO50151) is gratefully 

acknowledged. This work is performed under European 

Commission FP6-project DiMI,LSHB-CT- 

2005-512146 

References: 

1. Katona et al., Nat. Med. 2008; 

2. Maccarrone et al., Prog. Neurobiol. 2007; 

3. Burns et al., Proc. Natl. Acad. Sci. U.S.A. 2007. 


YIA applicant 

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Parallel Session 2: NEUROSCIENCE I

YIA applicant 

34 


Longitudinal non-invasive detection of amyloid plaques by combined molecular, functional 

and morphological imaging in transgenic mouse models of Alzheimers Disease 

Maier F. C. (1) , Wehrl H. F. (1) , Schmid A. (1) , Odenthal J. (2) , Reischl G. (3) , Wiehr S. (1) , Mannheim J. (1) , Stiller D. (4) , Jucker M. (2) , 

Pichler B. (1) . 

(1) Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, 

(2) Hertie Institute for Clinical Brain Research, 

(3) University Hospital Tuebingen, Radiopharmacy, 

(4) Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG. 

florian.maier@med.uni-tuebingen.de 

Introduction: Current small animal imaging 

instrumentation provides powerful tools to study 

disease characteristics and progression. However, 

biomarkers for imaging amyloid plaque deposition 

and associated physiological changes are not yet 

fully understood – impeding diagnosis in daily 

clinics. Thus our aims were to assess the ongoing 

plaque deposition in an animal model of Alzheimers 

Disease longitudinally using [11C]PIB, to compare 

the binding properties in different transgenic 

mouse models and to monitor perfusion differences 

with [15O]H2O and Arterial Spin Labeling (ASL) – 

reflecting disease induced changes in physiology. 

Methods: APPPS1 tg mice and Tg2576 mice 

with respective littermate controls were injected 

intravenously with 7.6±2.8 MBq [11C]PIB (specific 

activity > 50GBq/µmol) and with 28.5±3 MBq 

[15O]H2O for measurement of cerebral perfusion. 

Dynamic small animal PET scans were performed 

for 1h p.i. and the mice were anesthetised with 

1.5% isofluran in 100% oxygen. In addition, 3D MR 

images and ASL were acquired for each mouse. The 

PET images were analysed using cortical regions 

as target and the cerebellum as internal reference. 

The obtained time activity curves were processed 

with the Logan Plot and the simplified reference 

tissue model (SRTM) by Lammertsma. ASL-MRI 

data were analyzed with an inhouse programmed 

Matlab routine using a simplified version of the 

Bloch equation. Furthermore, the animals were 

analysed histopathologically. 

Results: Both analysis methods revealed 

significant differences in the binding potential 

(BP) of [11C]PIB in cortical regions of transgenic 

APPPS1 mice (Logan 0.28±0.10; SRTM 0.32±0.18) 

in comparison to littermate controls (Logan 

0.13±0.07; SRTM 0.05±0.07, n=8, p


Serotonergic neurotransmission in early Alzheimer’s disease 

Hasselbalch S. G. (1) , Marner L. (1) , Madsen K. (1) , Lehel S. (1) , Barré W. (1) , Knudsen G. M. (1) . 

(1) Copenhagen University, Denmark 

sgh@nru.dk 

Introduction: Post mortem studies suggest involvement 

of the serotonin system in Alzheimer’s disease 

(AD) and serotonin 2A (5-HT2A) receptors are 

globally reduced early in the course of the disease 

(1). However, few studies have investigated other 

aspects of the serotonergic system, including presynaptic 

markers (serotonin transporters, SERT) as 

a measure of serotonergic degeneration (2). Further, 

recent evidence points to a disease-modifying effect 

of 5-HT4 agonism through a decrease in beta-amyloid 

load (3). Therefore, we have investigated SERT 

and 5-HT4 in early and compared these measures to 

the reduction in 5-HT2A receptors. 

Methods: For the SERT/5-HT2A comparison, we 

included 12 patients (mean age 73.7 ±7.6 years, 8 

males) with AD (average MMSE of 24, range 19-26) 

and 11 healthy age-matched subjects (mean age 72.5 

±6.8 years, 6 males). Subjects were investigated with 

a 90 min dynamic [11C]DASB-PET recording to 

measure SERT (4) and a 40 min steady-state [18F] 

altanserin-PET recording to measure 5-HT2A receptors 

(5). In a separate study of 5-HT4 receptors, 

the novel radioligand [11C]SB207145 was used in 

twelve healthy individuals (mean age 67.2 y, 6 males) 

and eleven newly diagnosed AD patients (mean age 

70.6 y, 6 males, mean MMSE 24, range 19-27) using 

the simplified reference tissue model. Volumes of 

interest (VOIs) were delineated automatically on coregistered 

3T MRIs, and partial volume correction 

was applied to correct for differences in atrophy. 

Results: The 5-HT2A receptors were markedly reduced 

(25-66%) in AD patients in all regions but 

the striatum. Inc contrast, we found a reduction of 

SERT binding by 34% (p=0.0003) in the hippocampus, 

while most other regions were unaffected. 

Midbrain showed no change in binding (p=0.30). 

No statistically significant differences in 5-HT4 receptor 

binding between healthy individuals and AD patients 

were found in any of the included brain regions: 

Hippocampus (p = 0.55), posterior cingulate gyrus 

(p = 0.22), amygdala (p = 0.77), parietal cortex (p = 

0.44), temporal cortex (p = 0.74) and prefrontal cortex 

(p = 0.43). 

Conclusions: We showed a marked decrease of 

5-HT2A binding in patients with mild AD, consistent 

with previous findings in MCI (1). The SERT 

binding was unaffected by the disease in almost all 

cortical regions and in midbrain, suggesting that 

the serotonergic innervations and the neuron bodies 

in dorsal nucleus raphe are intact, at least early 

in the disease. We interpret the SERT reduction in 

hippocampus in patients as a decreased serotonergic 

innervation, which could be secondary to the neuronal 

degeneration taking place in hippocampus of 

AD patients. The marked reduction in 5-HT2A may 

be related to beta-amyloid accumulation. In contrast 

to the 5-HT2A receptor subtype, the 5-HT4 receptor 

levels seemed to be unaffected in AD. However, 

5-HT4 binding in relation to cognitive function, 

neuropsychiatric symptoms and beta-amyloid load 

should be further investigated. 

Acknowledgement: Supported by The Lundbeck 

Foundation, Rigshospitalet, and the Danish Medical 

Research Council. The John and Birthe Meyer Foundation 

is gratefully acknowledged for the donation 

of the Cyclotron and PET-scanner. These studies 

were funded in part by the EC - FP6-project DiMI, 

LSHB-CT-2005-512146. 

References: 

1. S. G. Hasselbalch et al., Neurobiol. Aging. 29, 1830 

(2008). 

2. K. Nielsen et al., Synapse. 59, 270 (2006). 

3. S.J. Robert, et al., Neurodegener Dis 5(3-4):163 (2008). 

4. M. Ichise et al., J Cereb. Blood Flow Metab. 23, 1096 

(2003). L. H. Pinborg et al., J. Cereb. Blood Flow Metab. 

23, 985 (2003). 


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Qualitative and quantitative assessment of florbetapir F-18 PET ( 18 F-AV45) amyloid 

deposition PET imaging - validation by pathology. Preliminary report. 

Romanowicz G. (1) , Saha K. (2) , Krautkramer M. (2) , Joshi A. (2) , Pontecorvo M. (2) , Clark C. (2) , Carpenter A. (2) , Skovronsky D. (2) . 

(1) Gdański Uniwersytet Medyczny, Poland 

(2) Avid Radiopharmaceuticals Inc.. 

greg@amg.gda.pl 

Various methods for analyzing amyloid 

brain PET scans have been proposed and tested. 

The lack of reliable truth standards has complicates 

the validation and implementation of these methods, 

and investigators have relied on theoretical modeling 

and clinical diagnoses as surrogates for true 

pathology. We applied three methods for assessing 

florbetapir F18 PET (18F-AV45) amyloid images: a 

semiquantitative visual read, a mean SUVr quantitation 

based on VOIs, and a novel automated histogram 

based quantitative approach. Each method 

was validated by comparison with amyloid burden 

measured at post-mortem. 

Methods: 6 subjects (3 men; 3 women; mean age at 

imaging 76y (47-86); diagnosis at enrollment, 1 MCI, 

4 AD, 1 PDD) with a life expectancy of < 6 months 

had a 10 minute PET scan 50 minutes after IV injection 

of 10 mCi of florbetapir F 18. Images were reconstructed 

by iterative technique with 4i/16s and 3 

mm FWHM post-reconstruction Gaussian filter. For 

visual assessment (VR) an experienced reader rated 

florbetapir cortical uptake intensity (VR score) on a 

5 point scale ranging from 0, (no difference in uptake 

between cortex and cerebellum), to 4 (uptake 

in all cortical brain regions significantly higher vs. 

both cerebellum and white matter). For the SUVr 

technique, PET images were spatially normalized by 

12 parameter affine registration to Talairach/MNI 

space and counts extracted from six regional VOIs 

(frontal, temporal, precuneus, parietal, anterior 

cingulated and posterior cingulated). Average standardized 

uptake value ratios (SUVr) were calculated 

with cerebellum as reference. The values for the six 

cortical regions were averaged to generate a global 

SUVr. For the histogram based analysis image data 

were processed automatically to generate a128 bin 

count histogram. The first order derivative Gaussian 

(FWHM= 3mm) convolved histogram curve was 

searched in the intensity direction to estimate separation 

of high/low intensity areas. The high/low intensity 

curve area ratio (CAR) was calculated to estimate 

specific amyloid binding. After the patient’s 

death (mean interval from imaging 43 days (range 

12-158)) their brain was evaluated for of β-amyloid 

deposition using immunohistochemistry (IHC) and 

by neuritic plaque rating (modified CERAD method). 

imaging life 

CAR score, VR score and SUVr were compared with 

IHC/CERAD using Spearman’s (ρ) or Pearson’s correlation 

coefficient (r) as appropriate. 

Results: A strong correlation was obtained between 

each measure of PET amyloid binding and both 

measures of amyloid deposition at autopsy (VR and 

IHC (ρ=0.88), VR and CERAD (ρ =0.92); SUVr and 

IHC (r=0.89), SUVr and CERAD (ρ=0.93); CAR and 

IHC (r=0.97), CAR and CERAD (ρ =0.64)). 

Conclusions: The results confirm that florbetapir F 

18 PET imaging provides relevant in vivo estimate of 

brain amyloid pathology irrespectively of the choice 

of image assessment method. The novel histogram 

based method is particularly interesting because it 

is quantitative and does not require fitting images 

to a template or reorientation of images, kinetic data 

acquisition, input functions or drawing / applying 

regions of interest. 

Acknowledgement: This work is supported by AVID 

Radiopharmaceuticals Inc. We would also like to 

thank A.S. Fleisher, J.A. Schneider, T.G. Beach, B.J. 

Bedell, S.P. Zehntner and M. Mintun for their contribution 

to the work.


In-vivo imaging of a mouse traumatic brain injury model using dual-isotope quantitative 

spect/ct 

Máthé D. (1) , Szigeti K. (2) , Fekete K. (3) , Horváth I. (2) , Benyó Z. (3) . 

(1) CROmed Ltd, 

(2) Nanobiotechnology and In Vivo Imaging Centre Semmelweis University Faculty of Medicine, 

(3) Semmelweis University Faculty of Medicine. 

domokos.mathe@gmail.com 

Introduction: Using a standardized mouse neurotrauma 

model we aimed at determining the role 

of high-resolution SPECT/CT in detecting and 

quantifying the volumetric and functional extent of 

blood-brain barrier disruption. We chose the freezing 

trauma model because of its reproducibly standard 

damage. 

Methods: Mice were subjected to a standardized 

freezing method transcranially using a cooled 

needle tip. In the presented initial studies, we compared 

the imaging pattern of the animals at equally 5 

hours post the freezing trauma. We used a dedicated, 

quantitative multiplexed multipinhole SPECT imaging 

system for laboratory animals. X-ray CT of the 

structures was also performed together with SPECT 

data acquisition. 

We correlated BBB disruption and blood perfusion 

with quantitative imaging of the decrease in glial 

and neuronal potassium uptake. We applied the validated 

intravenous tracer 99mTc-DTPA to detect the 

disruption of the BBB. Blood perfusion of the brain 

was assessed using the also validated tracer 99mTc- 

HMPAO. A radioactive isotopic potassium analogue, 

201Tl was used to track K+ uptake changes quantitatively. 

201Tl ions were passed through the BBB by 

means of diethyl-dithiocarbamate (DDC) complex 

that redistributes to ionic 201Tl after being taken 

up in brain tissues. 

Uptake volumes (in mm3) and total brain as well as 

lesion radioactivity values (in kBq) were evaluated 

on 3D reconstructed brain images of treated mice 

(n=5 per group) and a non-treated group. Besides 

imaging of single tracers (3 groups), simultaneous 

multi-channel imaging was used in two other groups 

of mice having received 99mTc-DTPA plus 201Tl- 

DDC or, 99mTc-HMPAO plus 201Tl-DDC. 

After imaging, histological control of the brain 

trauma size and localization was performed in the 

formaldehyde-fixed brains of all animals. 

Results: The uptake of 99mTc-DTPA correlated well 

with the size and localization of the lesion whereas 

the cold spot of the lesion at the perfusion image 

was evident in all animals having received 99mTc- 

HMPAO. However the cold spot was surrounded by 

an area of increased perfusion as compared to the 

same area in control animals. When comparing with 

201Tl-DDC images, the penumbra effect became evident, 

especially in the animals imaged with 99mTc- 

HMPAO and 201Tl-DDC in the same time. The 

area of non-active glial and neuronal potassium uptake 

was significantly larger than the non-perfused 

area and the hyper-perfused area co-localized with 

the edge of the disappeared neural potassium uptake 

volumes in 4 animals out of 5. SPECT/CT identified 

the dislocation of brain due to increased intracranial 

pressure in all treated animals. 

Conclusions: In vivo whole-animal imaging using 

quantitative SPECT is a very promising method to 

dissect different activation patterns and regulation 

of different mechanisms behind the events following 

neurotrauma (and consequently stroke too). Using 

a unique dual-isotopic approach to detect multiple 

events in the same time in the same animals, the size 

and presence of a penumbral region where perfusion 

is elevated but neural (both glial and neuronal) K+ 

utilization is decreased, could be identified. 

Acknowledgement: We are grateful for Dr. Jürgen 

Goldschmidt (Magdeburg) and Roberto Pasqualini 

(Orsay) for valuable advice and kind hints in 201Tl- 

DDC application and preparation. 

References: 

1. Goldschmidt et al. NeuroImage 49 (2010) 303–315 


YIA applicant 

day1 




Molecular analysis of adaptive immune function through microscopic and mesoscopic 

imaging 

Stein J. . 

University of Bern, Switzerland 

jstein@tki.unibe.ch 

Cell migration is of central importance 

during development, cancer metastasis and the 

functioning of the immune system. A powerful 

approach to dissect molecular mechanisms during 

cell trafficking on a single cell level requires the 

adaptation of microscopy techniques to anesthetized 

animals, usually mice. 

Twophoton microscopy (2PM) was developed to 

examine the molecular mechanisms governing 

transmigration through endothelium and within 

tissue. Here, a brief overview is given over the 

techniques and potential applications of 2PM in the 

field of lymphocyte trafficking. 

imaging life 

The overall internal organization of organ 

microenvironments, such as B cell follicles and 

blood microvessels in lymph nodes, has thus far 

been mainly determined by two-dimensional tissue 

sectioning, whereby three-dimensional information 

is lost. Optical Projection Tomography (OPT) and 

selective plane illumination (SPIM) are recently 

developed mesoscopic imaging approaches to dissect 

the overall organization of specimen of 1-15 mm 

diameter. Their applications in lymphoid structure 

analysis will be discussed.


Magnetic resonance imaging – from structure to molecular interactions 

Rudin M. . 

ETH and University of Zürich, Switzerland 

rudin@biomed.ee.ethz.ch 

Magnetic resonance imaging (MRI) yields images 

characterized by high spatial resolution and 

unsurpassed soft-tissue contrast. The latter is 

the consequence of the fact that the MRI signals 

depends on multiple parameters such a various 

relaxation rates, water diffusivity, proton exchange 

reactions, which are tissue specific. By choice of 

experimental parameters contrast can be varied to 

highlight structures of interest. Paramagnetic or 

superparamagnetic contrast agents can be used to 

further enhance specific structures. It is therefore 

not surprising that today MRI has become one of the 

most important modalities for structural imaging. 

Yet MRI provides information beyond mere anatomy. 

The analysis of dynamic signal changes - e.g. during 

administration of a contrast agent - yields relevant 

quantitative information on physiological processes 

such as blood flow, vascular permeability, or renal 

function. Probably the most important physiological 

MRI application is functional MRI (fMRI), which 

has become an indispensable tool for studying 

brain function, or more precisely the hemodynamic 

response to neuronal activity, under normal and 

pathological conditions. fMRI measures local 

hemodynamic changes in the CNS that are linked to 

neural activity through neurovascular coupling. 

More recently, MRI has tapped into the field of 

molecular imaging. By coupling the MRI contrast 

agent to a targeting moiety, molecular information 

can be derived at high spatial resolution. Yet the 

MRI molecular imaging approach suffers from two 

limitations. MRI is insensitive due to the small 

quantum energy involved in the process, typically 

concentrations have to be in the micromolar range 

to induced chances detectable by MRI. This can be 

in part be accounted for by increasing the relaxivity 

of MRI reporters by increase of the payload or by 

using physiological amplification. The second 

limitation is that MRI contrast agents are in 

general bulky, which renders target accessibility 

difficult. Straightforward molecular targets that 

can be reached by MRI contrast agents are those 

expressed on the endovascular side. On the other 

hand the efficiency of MRI probes, similar to that 

of fluorescent probes, can be modulated through 

the molecular interaction with the target. This 

potentially allows discriminating target-bound from 

free floating probe, thereby enhancing the contrastto-background 

ratio. An attractive application of 

targeted MRI imaging are studies of cell trafficking. 

As many cells are rather tolerant to the amount of 

contrast agent they can carry, sensitivity appears 

to be less an issue. In fact there are some reports, 

that under favorable conditions single cells might be 

detected. 

The lecture will discuss the various aspects of MRI with 

focus on molecular and cellular imaging applications. 


day1 

Parallel Session 3: TECHNOLOGY

40 


Imaging modalities: PET and SPECT 

Ziegler S. . 

TU Munich, Department of Nuclear Medicine, Germany 

sibylle.ziegler@tum.de 

Nuclear medical imaging methods, positron emission 

tomography (PET) and single photon emission 

computed tomography (SPECT), utilize the detection 

of gamma rays leaving the body after a radioactive 

tracer has been administered in vivo. The choice 

of tracer substance as well as radio-nuclide for labelling 

depends on the biological process of interest 

and the organ which is imaged. While electronic 

collimation is used in PET, mechanical collimators 

made of high-Z materials need to be employed in 

SPECT imaging. Depending on the imaging situation, 

these collimators can be designed for parallel, 

imaging life 

diverging, or converging projection. Gamma ray detection 

in either modality is accomplished by scintillation 

detectors or semiconductor detectors, as in 

more recent developments. Raw data are projections 

of the activity distribution, which is reconstructed 

using analytical or statistical algorithms. Sensitivity 

of PET allows detecting pico-molar tracer amounts 

in vivo and current technology offers mm (PET) or 

sub-mm (SPECT) spatial resolution. Quantitative 

measurement of activity concentration in vivo is the 

basis for more advanced dynamic studies including 

biokinetic modelling.


in vivo fluorescence kinetic imaging for improved contrast and studies of temporal and 

quantitative biodistribution 

Mansfield J. (1) , Agarwal A. (2) , Curtis A. (2) , Krucker T. (2) . 

(1) Cambridge Research & Instrumentation, 

(2) Novartis Institutes for BioMedical Research. 

jmansfield@cri-inc.com 

Introduction: In vivo fluorescence imaging has 

added an easy and economical modality to the rapidly 

growing field of molecular imaging. It offers 

the possibility to perform experiments analogous 

to bioluminescence imaging through fluorescent 

proteins, but at longer and more efficient wavelengths. 

A further distinction is the translational 

and research aspects enabling the imaging of fluorophore-labelled 

biologics (e.g., antibodies, peptides, 

siRNA) and activatable reagents. One unique 

aspect limiting the sensitivity of in vivo fluorescence 

methodologies is the confounding effect of 

tissue autofluorescence, which can be addressed 

through the proper use of spectral imaging [1,2]. 

However, like other molecular imaging modalities, 

reagent-based in vivo fluorescence imaging also 

has to contend with sensitivity and contrast problems 

due to non-specific signals and long wash-out 

times, both limiting the detection of specifically 

bound reagent and preventing accurate determination 

of uptake rates. 

Methods: A kinetic imaging modality, Dynamic 

Contrast Enhancement, or DyCETM, that combines 

rapid imaging (up to 15 frames/sec monochrome 

or 10 sec/frame multispectrally) with an advanced 

data processing methodology was developed. When 

combined with analysis software, these allow determination 

of (1) the rates of change of the intensity 

of the fluorophore in each pixel of the image and 

(2) the rate of uptake and wash-out in the animal. 

Mice were injected with a near-infrared fluorescent 

agent (IgG antibody) that was taken up in the liver 

and imaged for 80 minutes following injection at 

a rate of 1 multispectral dataset per minute. Each 

timepoint’s data were unmixed and kinetic data 

and “movies” assembled from these. 

Results: By utilizing the uptake and wash-out rate 

information, a much higher contrast image of the 

accumulating fluorophore was obtained in a much 

shorter period of time (minutes and hours vs. days). 

In addition, body compartment data determined 

from the kinetic data can provide information on 

the temporal distributions of a fluorescent agent 

during the experiment and can act as inputs for 

rate-of-change or body compartment models. 

Conclusions: This broadly applicable temporalbiodistribution 

methodology can be used, for 

example, to differentiate between the rate of liver 

uptake vs. the rate of tumor uptake and quantitate 

each signal relative to general body and/or bladder 

distribution. This information can then be 

combined with multispectral imaging and used to 

quantitate the biodistribution of multiple fluorophores 

simultaneously, each without interference 

from autofluorescence. When combined with 

models of peripheral, tumor, blood and wash-out 

(bladder) distributions, this kinetic imaging data 

can be transformed into a physiologically based 

pharmacokinetic model of agent distribution that 

provides an estimate of the pK values between the 

various compartments. Potentially such a method 

can also be used to establish optimal drug treatment 

schedules aiding drug discovery and development. 

References: 

1. Levenson, Mansfield, Cytometry A. 2006 Aug; 

69(8):748-58. 

2. Tam et al., Mol Imaging. 2007 Oct-Dec;6(4):269-76. 


day1 




Cerenkov radiation imaging of a xenograft murine model of mammary carcinoma 

Boschi F. (1) , Calderan L. (1) , D’ambrosio D. (2) , Marengo M. (2) , Fenzi A. (1) , Calandrino R. (3) , Sbarbati A. (1) , Spinelli A. E. (3) . 

(1) University of Verona, 

(2) S. Orsola – Malpighi University Hospital, 

(3) S. Raffaele Scientific Institute. 

federico@anatomy.univr.it 

2-[18F]fluoro-2-deoxy-D-glucose 

( 18 F-FDG) is a well known radiotracer for the in 

vivo studies of several diseases. In a previous paper 

[1] we showed that the positrons emitted by 18 F-FDG, 

travelling into tissues faster than the speed of light 

in the same medium, are responsible of Cerenkov 

Radiation (CR) emission which is prevalently in the 

visible range. The purpose of this work was to show 

that Cerenkov radiation escaping from tumour tissues 

of small living animals injected with 18 F-FDG 

can be detected with optical imaging (OI) techniques 

using a commercial optical instrument equipped 

with charged coupled detectors. 

Methods: In order to demonstrate as a proof of 

principle the possibility of imaging tumours using 

CR we studied an experimental model of mammary 

carcinoma named BB1. BB1 tumours were obtained 

by subcutaneous injection of BB1 cells, which are 

epithelial cells, from spontaneous mammary carcinomas 

of FVB transgenic mice for HER-2/neuT 

oncogene. The BB1 carcinomas exhibit histopatological 

and vascular features very similar to those 

of the parent spontaneous tumours. The BB1 mouse 

tumour model was well explained by Galiè and coworkers 

[2] and will not be described here. 

The images show a comparison between images acquired pre injection (left image) and 1 hour after 18F- 

FDG injection for two mice. White arrows indicate the position of the tumour masses. 

imaging life 

Mice injected with 18 F-FDG or saline solution underwent 

dynamic OI acquisition and a comparison 

between images were performed. Multispectral analysis 

of the radiation was used to estimate the deepness 

of the source of Cerenkov light. Small animal 

PET images were also acquired in order to compare 

the 18 F-FDG bio-distribution measured using OI 

and PET scanner. 

Results: The first Cerenkov in vivo whole body images 

of tumour bearing mouse and the measurements 

of the emission spectrum (560-660 nm range) were 

presented. Brain, kidneys and tumour were identified 

as a source of visible light in the animal body: 

the tissue time activity curves reflected the physiological 

accumulation of 18 F-FDG in these organs. 

The identification is confirmed by the comparison 

between CR and 18 F-FDG images. 

Conclusions: These results will allow the use of 

conventional optical imaging devices for the in 

vivo study of the cancer glucose metabolism and 

the assessment, for example, of anti-cancer drugs. 

Moreover this demonstrates that 18 F-FDG can be 

employed as it is as bimodal tracer for PET and OI 

techniques. 

References: 

1. Spinelli A E et al; Phys. 

Med. Biol. 55(2):483- 

495 (2010) 

2. Galiè M et al; 

C a r c i n o g e n e s i s . 

26:1868-1878 (2005)


A system for 4D (3D+Kinetics) molecular imaging in bioluminescence and fluorescence 

Maitrejean S. (1) , Kyrgyzov I. (1) , Bonzom S. (1) , Levrey O. (1) , Le Masne Q. (1) , Hernandez L. (1) , Raphael B. (2) . 

(1) Biospace Lab, 

(2) CEA/DSV/ I2BM / SHFJ / LIME. 

smaitrejean@biospacelab.com 

Introduction: Several technologies and systems are 

now capable of delivering tri-dimensional data in Bioluminescence 

and Fluorescence imaging. However, all 

are based on sequential acquisitions, and therefore any 

of them allow the production of real kinetic data in 

3D. In this work we demonstrate a new device which 

is able to acquire Bioluminescence and Fluorescence 

data from several views simultaneously, allowing the 

reconstruction of tri-dimensional images while keeping 

all kinetic properties of the acquisition. 

Methods: Multiple views of Bioluminescence and Fluorescence 

data are acquired simultaneously using a photon 

counting device (Photon ImagerTM) in which a 

specific add-on with four mirrors is installed. This device 

(named 4Views) allows the concurrent acquisition 

of the ventral, dorsal, right, and left views of the animal 

in a single image. The size and intensity of the four subimages 

(i.e. the four views) are corrected by taking the 

true optical path into account. The corrected photon 

counting data are recorded in a list mode file with time 

information (43 frame/s) as can be seen in Fig1. 

In a second step a micro video projector is used to 

project a moving spot on the animal. For each position 

of the spot, the 

height of the animal 

is calculated 

by triangulation 

and a map of the 

surface of the animal 

is produced. 

As the chosen 

reconstruction 

Fig1: simultaneous acquisition of four views 

in bioluminescence imaging. 

method is based 

on the finite elements 

method, 

the volume of the animal is represented by a tetrahedral 

mesh (about 30 000 tetrahedrons) using the Delaunay 

algorithm, while the surface is approximated 

by triangles, using a marching cube method. Then, the 

forward problem is solved for given light sources that 

are placed at the nodes of tetrahedrons and the light 

intensities on the surface of the triangles are computed. 

The forward problem is based in this first version on a 

diffusion model with average constant absorption and 

diffusion parameters. In a next version, a registration of 

the surface of the animal with an anatomical atlas will 

allow to use optical parameters associated with each 

organ. In the following step measured data is extracted 

from the list mode file and mapped on the triangulated 

surface. Since the detected events are stored separately, 

any time interval from 22 ms (one frame) up to the 

total scan duration can be used to create the data. In 

the last step of the process, the inner light sources are 

estimated as the result of the inverse problem using a 

least square criterion with a Tikhonoff regularization 

term. This regularisation term is chosen as an entropic 

term in order to favour connected solution. The two 

last steps can be performed using any time interval of 

the list mode file and therefore 3D kinetic data can be 

computed for times scales larger than 22 ms. 

Results: The validity of the method has been first tested 

using light beads (Microtek) inside a half cylinder of a 

scattering material (delrin). It has been demonstrated 

that two light beads as close as three millimetres could 

be separated, at a depth of one centimetre. A second 

set of tests has been realised on an animal (nude mice) 

by placing the light beads in the rectum of the animal. 

On the second image (Fig 2) , two beads 9 mm far from 

each other were placed in the rectum. It was possible to 

reconstruct the two positions of the light sources. The 

distance between the two reconstructed sources is 9.77 

mm in good agreement. 

Fig2: Reconstruction of two light sources based 

on the four views data of fig1. 

Conclusions: 3D optical reconstruction is known to 

be approximate, but can give useful and satisfactory 

results in a large number of applications. We have 

demonstrated that this four view approach is able to 

provide 3D kinetic molecular imaging data with a 

spatial accuracy similar to other methods. 

Acknowledgement: This work was supported in part 

by the DIMI and ENCITE networks. 


day1 


44 Magnetic 


The alignment of target-specific lipoCEST MRI contrast agents 

Langereis S. (1) , Burdinski D. (1) , Pikkemaat J. (1) , Gruell H. (1) , Huskens J. (2) . 

(1) Philips Research Europe, 

(2) University of Twente. 

sander.langereis@philips.com 

resonance imaging (MRI) offers a unique 

combination of advantages such as the recording 

of contrast enhanced and anatomical images with 

a high spatial resolution, while avoiding the use of 

ionizing radiation. MRI for imaging sparse molecular 

epitopes on diseased cells is hampered by its low 

sensitivity, which can potentially be overcome with 

liposomal chemical exchange saturation transfer (lipoCEST) 

contrast agents (CAs).1-3 In this case, MR 

detection is based on the saturation of the intraliposomal 

water signal with a selective radiofrequency 

pulse. Water exchange across the liposomal membrane 

causes partial saturation of the bulk water 

signal and, as a consequence, negative contrast enhancement 

in the MR image. For in vivo applications, 

it is crucial to achieve large intraliposomal chemical 

shifts of the water protons, as larger shifts allow for 

better lipoCEST contrast enhancement, reduce the 

interference with background magnetization transfer 

effects, and allow for frequency-based multiplexing. 

Large chemical shift differences have been 

obtained upon aspherical deformation of liposomes 

encapsulating a chemical shift agent in response to 

osmotic shrinkage, and the additional incorporation 

of amphiphilic, paramagnetic lanthanide complexes 

within the liposomal bilayer. 

The direction of the chemical shift is governed by 

the alignment of the aspherical liposomes in the external 

magnetic field, which in turn is dictated by 

the sign of the magnetic anisotropy of the incorporated 

amphiphilic lanthanide complex. The use of 

lipoCEST CAs as targeted probes for molecular MRI 

applications entails their specific binding and immobilization 

at the target site, for example, the surface 

of a biological structure or a cell. To maximize the 

imaging life 

attractive interactions, such aspherical liposomes 

will tend to align with the target structure (Scheme 

1).4 This enforced orientation may, however, be 

different from the magnetic alignment. As a consequence, 

the chemical shift of the intraliposomal 

water of the bound lipoCEST CA may differ from 

that of the unbound CA. It is therefore essential 

to understand the interplay between the preferred 

magnetic and the enforced mechanical alignment of 

such aspherical lipoCEST CAs. Herein, we address 

the alignment change of such aspherical liposomes 

upon multivalent binding to a target surface, which 

was studied by using routine CEST MR methods.4 

References: 

Fig1.: Reorientation of aspherical 

lipoCEST contrast agents upon 

binding to a target surface with 

respect to an external magnetic 

field (B0).4 

1. Aime, S.; Delli Castelli, D.; Terreno, E. Angew. Chem. Int. 

Ed. 2005, 44, 5513-5515. 

2. Terreno, E.; Cabella, C.; Carrera, C.; Delli Castelli, D.; 

Mazzon, R.; Rollet, S.; Stancanello, J.; Visigalli, M.; Aime, 

S. Angew. Chem. Int. Ed. 2007, 46, 966-968. 

3. Langereis, S.; Keupp, J.; van Velthoven, J. L. J.; de Roos, 

I. H. C.; Burdinski, D.; Pikkemaat, J. A.; Grüll, H. J. Am. 

Chem. Soc. 2009, 131, 1380-1381. 

4. Burdinski, D.; Pikkemaat, J. A.; Emrullahoglu, M.; 

Costantini, F.; Verboom, W.; Langereis, S.; Grüll, H.; 

Huskens, J. Angew. Chem. Int. Ed. 2010, 49, 2227-2229.


Lanthanide-based in vivo luminescence imaging 

Petoud S. . 

CNRS, France 

spetoud@pitt.edu 

Introduction: Fluorescence and luminescence 

are detection techniques that possess important 

advantages for bioanalytical applications and 

biologic imaging: high sensitivity, versatility and 

low costs of instrumentation. A common characteristic 

of biologic analytes is their presence in 

small quantities among complex matrices such as 

blood, cells, tissue and organs. These matrices 

emit significant background fluorescence (autofluorescence), 

limiting detection sensitivity. 

The luminescence of lanthanide cations has several 

complementary advantages over the fluorescence 

of organic fluorophores and semiconductor 

nanocrystals, such as sharp emission bands for 

spectral discrimination from background emission, 

long luminescence lifetimes for temporal 

discrimination and strong resistance to photobleaching. 

In addition, several lanthanides emit 

near-infrared (NIR) photons that can cross deeply 

into tissues for non-invasive investigations and 

that result in improved detection sensitivity due 

to the absence of native NIR luminescence from 

tissues and cells. The main requirement to obtain 

lanthanide emission is to sensitize them with an 

appropriate chromophore. 

Methods: An innovative concept for such sensitization 

of lanthanide cations is proposed herein; 

the current limitation of low quantum yields 

experienced by most mononuclear lanthanide 

complexes is compensated for by using larger 

numbers of lanthanide cations and by maximizing 

the absorption of each discrete molecule, 

thereby increasing the number of emitted photons 

per unit of volume and the overall sensitivity 

of the measurement. To apply this concept, we 

are developing a family of dendrimer-naphthalimide 

ligands that are able to incorporate several 

lanthanide cations. Polyamidoamine (PAMAM) 

dendrimers have been chosen as a basis for these 

complexes because the oxygen atoms of the amido 

groups located along their branches can bind 

and protect the lanthanide cations inside the 

dendrimer core.1,2 Derivatives of naphthalimide 

groups, required for the sensitization of the lanthanide 

cations, are located at the branch termini. 

Our synthetic approach allows facile modification 

of the dendrimer complex for control over 

photophysical properties and solubility. It also 

provides for the attachment of different types 

of targeting agents such as peptides, oligonucleotides 

or proteins, as well as other sensing agents, 

to provide functionality to these compounds in a 

broad range of applications. 

Results: In this paper, we will describe several 

examples of luminescent polymetallic lanthanide 

complexes based on dendrimers. We will also 

present examples of their applications as reporters 

and sensors for biologic imaging in living cells 

and small animals. 

References: 

1. J. P. Cross, M. Lauz, P. D. Badger, S. Petoud, Journal of 

the American Chemical Society, 2004, 126, 16278. 

2. D. R. Kauffman, C. M. Shade, H. Uh, A. Star and S. Petoud, 

Nature Chemistry 2009, 1, 500. 


day1 

Parallel Session 4: PROBES - supported by COST

46 


New perspectives for imaging molecules and metabolism in vivo 

Gruetter R. . 

University of Lausanne and Geneva, Switzerland 

rolf.gruetter@epfl.ch 

Magnetic resonance imaging is subjected to continuous 

advances and discoveries, much of which are lead 

by either improved data acquisition techniques and/ 

or higher magnetic fields or the detection of novel 

principles/contrast mechanisms altogether. This presentation 

will highlight on one hand the possibility 

to detect contrast agents at very low concentrations 

using a new approach to MR, based on hyperpolarized 

media using dissolution DNP. The potential 

will be demonstrated especially for long-lived nuclei 

with long T1, such as Lithium-6, Yttrium-89 and 

N-15, but also C-13 will be illustrated. The ability to 

detect low-concentration nuclei or rare spin nuclei 

imaging life 

opens new possibilities for molecular imaging. On 

the other side, for imaging molecules (metabolism) 

recent advances have allowed to attain in rodent 

brain a spatial resolution of below 1umol at 14.1 

Tesla that matches or even exceeds that possible by 

nuclear imaging modalities, in particular PET. The 

quantitative nature of the MR spectroscopic imaging 

approach allows insights into cellular biochemistry 

and metabolism that complements the capabilities 

of PET and SPECT and opens new windows on 

characterizing disease evolution and treatment opportunities, 

as will be illustrated with the example of 

ischemia and cancer, among others.


In vivo biodistribution of radiolabeled matrix metalloproteinase-2 activatable cell 

penetrating peptides 

Van Duijnhoven S. (1) , Robillard M. (2) , Nicolaij K. (1) , Gruell H. (1) . 

(1) University of Technology Eindhoven, The Netherlands 

(2) Philips Research Laboratories. The Netherlands 

s.m.j.v.duijnhoven@tue.nl 

Introduction: Activatable cell penetrating peptides 

(ACPPs) are a new class of promising molecular 

imaging probes for the visualization of proteolytic 

activity in vivo [1-3] . The cell penetrating function of 

a polycationic peptide is efficiently blocked by intramolecular 

electrostatic interactions with a polyanionic 

peptide. Proteolysis of a cleavable linker 

present between the polycationic cell penetrating 

peptide and the polyanionic peptide affords dissociation 

of both domains and enables the activated 

cell penetrating peptide to enter cells. Fluorescently 

labeled ACPPs cleavable by matrix metalloproteinase-2 

(MMP-2) have been reported as specific 

probes for MMP-2 expressing tumors in mice [1-3] . 

Here, we developed MMP-2 activatable dual isotope 

radiolabeled ACPPs and assessed their in vivo 

biodistribution in HT-1080 tumor-bearing mice. 

These probes enabled us to discriminate activated 

from intact ACPP. 

Methods: MMP-2 activatable and non-activatable 

cell penetrating peptides (ACPP and non-ACPP, respectively) 

containing an n-terminal tyrosine were 

prepared by Fmoc solid-phase peptide synthesis, 

site specifically conjugated with DOTA chelate succinimidyl 

ester at the c-terminus, purified by HPLC, 

and characterized by LC-MS. Furthermore, a DOTAconjugated 

cell penetrating peptide (CPP) was synthesized 

as positive control. Nude mice were injected 

subcutaneously with approximately 3.0 x 10 6 MMP-2 

positive HT-1080 fibrosarcoma cells. When the 

tumors reached a size of 8-50 mm 3 , the mice (n=6 

per probe) were used for in vivo studies. Therefore, 

the cell penetrating peptide and polyanionic peptide 

of ACPP or non-ACPP (60 nmol) were labeled 

with 177 Lu and 125 I, respectively, analyzed by iTLC 

and HPLC, injected into the tail vein, and in vivo 

biodistribution was determined 24h post-injection. 

Results: Radiolabeled ACPP (>98% radiochemical 

purity for both 177 Lu and 125 I) showed a significant 

~3-fold increase in tumor uptake relative to a negative 

control peptide with a scrambled linker (t-test, 

p

YIA applicant 



Bioresponsive MRI contrast agents based on self-assembling β-cyclodextrin nanocapsules 

Martinelli J. (1) , Fekete M. (1) , Tei L. (1) , Botta M. (1) . 

Università del Piemonte Orientale “A. Avogadro”, Italy 

jonathan.martinelli@unipmn.it 

One of the most exciting research area 

in the development of MRI contrast agents is the design 

of responsive or “smart” probes, whose performance 

is modulated by changes in physiological environment 

such as pH, partial oxygen pressure, metal 

ion concentration, enzyme activity etc. We have designed 

and synthesized a new type of nanosized Gdbased 

MRI probe containing disulfide bonds, that 

can be activated (i.e. switched on or off) under reducing 

conditions like those where specific enzymes 

or high radical concentrations are associated with a 

disease state (e.g. tumors, strokes etc.). The activation 

of the agent is signaled by a large change in the 

relaxation properties. 

Methods: The macromolecular architecture of the 

agent is based on nanocapsules prepared from perthiolated 

β-cyclodextrins via oxidation of the thiol 

groups to form S-S bridges.1 The assembly was 

carried out in the presence of either an equimolar 

amount or an excess of Gd-benzyl-AAZTA2 as the 

MRI-active paramagnetic complex, able to form 

inclusion adducts with the cyclodextrin buildingblocks 

and thus being “trapped” inside the capsules 

during their formation. After purification of the 

nanomaterials and removal of the external Gd-complexes 

by prolonged dialysis, the macromolecular 

systems were characterized by analytical and NMR 

relaxometric techniques. Reduction kinetic experiments 

were carried on by monitoring the variation 

in the longitudinal relaxation rate vs. time upon addition 

of tris(2-carboxyethyl)phosphine (TCEP)3 as 

a reducing agent able to cleave the disulfide bonds 

between the CD units. 

Results: High relaxivity nanoparticles made up of several 

β-CD units were formed which include in the inner 

core a relatively large number of Gd-chelates. The 

ICP analyses and NMRD profiles confirmed that a 

significant amount of complex was caged within the 

macromolecular structures. The relaxivity is as high 

as 22 mM-1 s-1 at 20 MHz and 25 °C for the system 

prepared with a 5:1 complex/CD ratio. Dynamic light 

scattering measurements also showed that the size of 

the nanocapsules prepared in the presence of the Gdcomplex 

is sensibly bigger than in its absence (120- 

200 nm vs. 30 nm). Following addition of TCEP and 

imaging life 

cleavage of the S-S bridges, a significant decrease (ca. 

30%) in the water proton relaxation rate was observed. 

Conclusions: The high relaxivity values measured for 

the Gd-loaded nanocapsules imply a high permeability 

of water through the cyclodextrin units of the surface 

shell. Upon disruption of the nanoparticles following 

the cleavage of the disulfide bonds between 

the CD units by the reducing agent TCEP, the complexes 

are released and originate an equilibrium between 

the free form and their adduct with the monomeric 

CDs. This is signaled by a remarkable decrease 

in the relaxivity as a consequence of the shortening 

of the reorientational correlation time τR. 

According to these data, our β-cyclodextrin nanocapsules 

containing Gd-complexes appear to be 

promising as MRI contrast agents responsive to 

reducing biological environments. 

Acknowledgement: This work is supported by the 

Regione Piemonte (Italy) as part of the NanoIGT 

project. 

References: 

1. Jones, L. C.; Lackowski, W. M.; Vasilyeva, Y.; Wilson, K.; 

Chechik, V. Chem. Commun. 2009, 1377-1379. 

2. Aime, S.; Calabi, L.; Cavallotti, C.; Gianolio, E.; Giovenzana, 

G. B.; Losi, P.; Maiocchi, A.; Palmisano, G.; Sisti, M. Inorg. 

Chem. 2004, 43, 7588-7590. 

3. Burns, J. A.; Butler, J. C.; Moran, J.; Whitesides, G. M. J. 

Org. Chem. 1991, 56, 2648-2650.


Photochemical activation of endosomal escape of MRI-Gd-agents in tumor cells 

Gianolio E. (1) , Arena F. (1) , Hogset A. (2) , Aime S. (3) . 

(1) Centro di Imaging Molecolare, 

(2) PCI Biotech AS Laboratories, 

(3) Molecular Imaging Center, Torino Italy. 

eliana.gianolio@unito.it 

Introduction: The cellular uptake of xenobiotics often 

procedes through the entrapment into endosomes. As 

recently reported1,2, in the case of cellular labeling with 

Gd-based complexes, the confinment into endosomal 

vesicles negatively affects the attainable relaxation enhancement 

of water protons. In fact it has been shown 

that, upon increasing the number of Gd(III) per cell, a 

quenching effect on the observed relaxivity takes place. 

It is the consequence of the fact that the term |R1end 

– R1cyt| is > than the water exhange rate between the vesicular 

and cytosolic compartments. In this communication 

we report an efficient method for endosomal escape 

of paramagnetic Gd(III) chelates that yields to a marked 

improvement of the efficiency in cellular labeling. 

Methods: The Photosensitizer TPPS2a (LumiTrans®) 

and the Lumisource® lamp were provided by PCI Biotech 

AS laboratories (Oslo, Norway). For cellular labeling, 

ca. 3-4×106 HTC,K562, NEURO2A and C6 cells 

were incubated for 18 hours at 37°C with different concentrations 

(5-100mM) of Gd-HPDO3A in the absence 

and in the presence of the Photosentisizer (2µl/ml). After 

this incubation time cells were washed three times 

and incubated for additional 4 hours at 37°C. Then the 

cells containing the Photosentisizer were exposed to LumiSource 

light for 5 minutes. Then cells were detached, 

washed and transferred into glass capillaries for registraion 

of MR-images on a Bruker Avance300 spectrometer 

operating at 7.1T. 

K562 cells 

B 

K562 cells 

A 

C 

D 

E 

F 

G 

R1obs (s -1 ) 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0,0 5,0x10 9 

1,0x10 10 

Number of Gd 3+ / cell 

1,5x10 10 

Fig.1: A) T 1 -weighted spin echo images of K562 cells labeled with Gd-HPDO3A. Phantoms E, F 

and G contain cells treated with the PCI technology while phantoms B, C, and D contain cells 

simply labeled by pynocitotic uptake. B) Observed relaxation rates of cells (HTC stars; NEURO-2a 

circles; C6 triangles) labelled with Gd-HPDO3A with endosomic (black symbols) distribution as 

a function of the number of Gd(III) found in each cell. 

Results: The cellular labeling has been pursued 

by pynocitosis (18h at 37°C) at different concentration 

of Gd-HPDO3A in the presence and in 

the absence of PCI treatment. As shown in the 

figure 1A, a marked enhancement in the labeling 

efficiency has been observed upon application 

of photochemical stimulus to cells entrapping 

Gd-HPDO3A and TPPS2a. This considerable 

gain in signal intensity achieved when cells are 

processed with PCI tehnology is due to the release 

of Gd-units from endosomes to cytosol. As 

shown in Fig. 1B, the “quenching” effect on the 

relaxivity of cells processed with PCI technology 

is reached when the number of Gd-HPDO3A per 

cell is ca. One order of magnitude higher than the 

number causing the same effect when the paramagnetic 

complexes are confined into endosomes. 

Thus, on going from the endosome-entrapped to 

cytoplasm-entrapped Gd(III), the paramagnetic 

loading can be several times higher thus allowing 

a marked improvement in the MRI detection of 

labelled cells. 

Conclusions: The PCI methodology appears an 

excellent route to pursue the endosomal escape of 

Gd-HPDO3A molecules entrapped by pynocitosis 

in different types of cells. It as been shown that 

it allows to exploit high payload of Gd-HPDO3A 

before the quenching effect becomes detectable. 

Fig. 1: A) T1-weighted spin echo 

Neuro 

images of K562 cells Neuro_lumi labeled with 

Gd-HPDO3A. Phantoms Htc E, F and G 

contain cells treated Htc_lumi with the PCI 

C6 

technology while phantoms C6_Lumi B,C and 

D contain cells simply labeled by 

pynocitotic uptake. B) Observed 

relaxation rates of cells (HTC stars; 

NEURO-2a circles; C6 triangles) 

labelled with Gd-HPDO3A with 

endosomic (black symbols) or 

cytoplasmatic (red symbols) 

distribution as a function of the 

number of Gd(III) found in each cell. 

References: 

1. Terreno, E et al., Magnetic 

Resonance in Medicine, 

2006, 55, 491-497. 

2. Strijkers G.J. et al. 2009, 

61, 1049-58. 


day1 

Parallel Session 4: PROBES - supported by COST

50 Education 


and Research Experience: 

Degree in Medicine and Surgery at the Catholic University of Rome (Italy). 

Membership of the College of Physicians and Surgeons of Rome (Italy). 

Specialization in Neurology at the Catholic University of Rome (UCSC). 

Post-graduate research at the laboratory of muscular disease of the neurological department at the Catholic 

University of Rome (UCSC). 

PhD in Neuroscience at the Neuroscience Department of the Catholic University of Rome (UCSC) within a 

joint collaboration with the department of Neuroimmunology of the Max Planck Institute for Neurobiology 

(Martinsried, Germany). 

Post doctoral position at the Max Planck Institute of Neurobiology, department of Neuroimmunology 

(Martinsried, Germany). 

Group leader position at the Institute for Multiple Slerosis Research (IMSF) of the Georg-August Universität 

of Göttingen. 

Dissertation: Visualization of antigen presentation and T cell activation after treatment with high doses of 

soluble antigen. 

Selected references: 

• I. Bartholomäus, N. Kawakami, F. Odoardi, C. Schläger, D. Miljkovic, J.W. Ellwart., W.E.F Klinkert., C. Flügel-Koch, T.B. 

Issekutz, H. Wekerle, A. Flügel. Effector T cell interactions with meningeal vascular structures in nascent autoimmune 

lesions, Nature. 2009 Nov 5; 462 (7269):94-8. 

• W. Dammermann+, B. Zhang+, M. Nebel+, C. Cordiglieri+, F. Odoardi, T. Kirchberger, N. Kawakami, J. Dowden, F. 

Schmid, K. Dornmair, M. Hohenegger, A. Flügel*, A.H. Guse*, B.V.L. Potter* (2009) NAADP mediated Ca2+ signaling via 

type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist, PNAS, 2009 Jun 30; 106 (26): 10678-83. 

+,* equal contribution. 

• Odoardi F, Kawakami N, Klinkert WE, Wekerle H, Flügel A. Blood-borne soluble protein antigen intensifies T cell activation 

in autoimmune CNS lesions and exacerbates clinical disease. Proc Natl Acad Sci U S A. 2007 Nov 20;104(47):18625-30. 

• Flügel A, Odoardi F, Nosov M, Kawakami N. Autoaggressive effector T cells in the course of experimental autoimmune 

encephalomyelitis visualized in the light of two-photon microscopy. J Neuroimmunol. 2007 Nov;191(1-2):86-97. 

• Odoardi F, Kawakami N, Li Z, Cordiglieri C, Streyl K, Nosov M, Klinkert WE, Ellwart JW, Bauer J, Lassmann H, Wekerle H, 

Flügel A. Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of 

autoimmune encephalomyelitis. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):920-5. 

• Kawakami N, Nägerl UV, Odoardi F, Bonhoeffer T, Wekerle H, Flügel A. Live imaging of effector cell trafficking 

and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med. 2005 Jun 

6;201(11):1805-14. 

imaging life 

Francesca Odoardi


Immune surveillance and autoimmunity: how encephalitogenic T cells enther their target organ 

Odoardi F. . 

Max Planck Institute of Neurobiology, Germany 

odoardi@neuro.mpg.de 

Introduced in the immunological field in 2002 

two-photon microscopy is the method of choice 

for visualizing living cells in their anatomical 

compartment because it allows to image deep 

in the tissue with negligible phototoxicity and 

photobleaching. We applied this technique in order 

to visualize in living Lewis rat the fate of genetically 

labeled MBP specific effector T cells in the menigeal 

vessels during the initial phase of experimental 

autoimmune encephalomyelitis, a classical model of 

multiple sclerosis. We observed that the incoming 

cells remained in close association with pial blood 

vessels, crawling on surfaces within the outline of 

the vessels. This behavior was specific to the CNS: 

in peripheral organs, for example in peripheral 

nerves, muscle or subcutaneous tissue, MBP T 

cells mainly rolled along the inner surface of the 

vessels. The crawling was completely abolished by 

the treatment with anti VLA-4 and LFA-1 antibodies. 

After diapedesis, the cells continued their scan on 

the abluminal vascular surface and the underlying 

leptomeningeal (pial) membrane. Here they 

established contact with local meningeal phagocytes 

and these interactions were crucial to induce the 

production of proinflammatory and to trigger 

tissue invasion. 


day1 

ESMI Plenary Lecture 2 by Francesca Odoardi



Exendin-4 derivatives labeled with radiometals for the detection of insulinoma: a “from 

bench to bed approach” 

Wild D. (1) , Wicki A. (1) , Mansi R. (1) , Béhé M. (1) , Keil B. (1) , Bernhardt P. (1) , Christofori G. (1) , Ell P. J. (1) , Reubi J. C. (1) , Maecke H. (1) . 

(1) University of Freiburg, Department of Nuclear Medicine 

damian.wild@uniklinik-freiburg.de 

Strong overexpression of glucagonlike 

peptide-1 (GLP-1) receptors in human insulinoma 

provides an attractive target for imaging. 

We have shown that GLP-1 receptor SPECT/CT 

using a Lys(Ahx-DOTA)NH2 C-terminal extended 

Exendin-4 derivative labeled with 111In shows 

high sensitivity in the detection of hardly detectable 

insulinoma [1]. For obvious reasons 111In 

is not an ideal nuclear imaging probe label. We 

therefore aimed at the development of 99mTc- 

(SPECT/CT) and 68Ga-, 64Cu-(PET/CT) labeled 

peptides. In addition we extended our clinical 

studies with a DTPA-modified peptide for higher 

specific activity labeling with 111In. 

Methods: Internalisation, biodistribution and 

imaging studies were performed in the Rip1Tag2 

mouse model and the corresponding cell line. 

The Results were compared with the “gold standard” 

Lys40(Ahx-DOTA-111In)NH2-Exendin-4. 

Kidney blocking was studied with poly-glutamic 

acid and Gelofusine. Clinical data were obtained 

with the 111In-labeled DTPA-analog. 

Results: The tumor uptake of Lys40(Ahx-DOTA- 

68Ga)NH2-Exendin-4 was very high, at 205±59 

%IA/g (1h pi) and was very similar to Lys40(Ahx- 

DOTA-111In) NH2-Exendin-4. > 90% of this 

uptake could be blocked by the preinjection of 

cold peptide. Normal organs showed much lower 

uptake resulting in a high lesion-to-background 

ratio. Kidney uptake was high and comparable 

to the tumor uptake. Lys40(Ahx-HYNIC-99mTc/ 

EDDA)NH2-Exendin-4 shows distinctly lower 

tumor but also normal tissue uptake. Tumors 

with 1-3.2 mm in size could be visualised easily 

by SPECT and PET. 

The kidney uptake could be reduced by 49-78% 

using poly-glutamic acid, Gelofusine or a combination 

of both. 

Clinical studies using SPECT/CT with Lys40(Ahx- 

DTPA-111In)NH2-Exendin-4showed 100% sensitivity 

in the localisation of benign insulinoma 

of the first consecutive 13 patients. Some of the 

localisations such as an ectopic insulinoma could 

imaging life 

only be localised with SPECT/CT. Due to the long 

residence time in the tumor intraoperative localisation 

with an endoscopic gamma probe was possible 

even 2 weeks i.p.. 

Conclusions: These very promising pharmacokinetic 

data show that the two new imaging probes 

are good candidates for clinical translation: Especially 

the 99mTc-labeled peptide may increase 

the availability and distribution of the method 

whereas the 68Ga derivative may even allow to 

image and localise very small lesions 

Acknowledgement: Oncosuisse grant No OSC- 

01778-08-2005 and grants from the Novartis 

Foundation and Swiss National Science Foundation 

are gratefully achnowledged. 

References: 

1. Wild D, Macke H, Christ E, Gloor B, Reubi JC. Glucagonlike 

peptide 1-receptor scans to localize occult 

insulinomas. N Engl J Med. 2008;359(7):766-768.


Assessment of HIF transcriptional activity in a mouse tumor model using GPI anchored avidin– 

a novel protein reporter for in vivo imaging 

Lehmann S. (1) , Garcia E. (2) , Blanc A. (2) , Schibli R. (2) , Keist R. (1) , Rudin M. (1) . 

(1) Animal imaging center, 

(2) Paul Scherrer Institute, Villigen, Switzerland. 

lehmann@biomed.ee.ethz.ch 

Introduction: With the emergence of multimodal 

imaging approaches genetic reporters, which can be 

flexibly combined with multiple imaging methods, 

are highly attractive. Here we present the feasibility 

of using glycosylphosphatidylinositol anchored avidin 

(av-GPI) (1) as a novel reporter for multimodal 

in vivo imaging. Expressed on the extracelluar side 

of cell membranes, av-GPI can be targeted with 

biotinylated imaging probes. In this study, we employed 

av-GPI to read out on the activity of hypoxia 

inducible factors (HIFs) in tumors. Induced by tumor 

hypoxia to mediate the adaptation of cells to 

low oxygen tensions these transcription factors play 

an important role in cancer progression. Typically, 

the expression of HIF in human cancer patients is 

associated with more aggressive tumor phenotypes 

and poor patient prognosis. Imaging HIF activity 

in live tumors hence provides an important tool to 

study the mechanisms leading to its activation in 

cancer. 

Methods: Mouse C51 cells were stably transfected 

with pH3SVG, a reporter construct driving the 

expression of avidin-GPI from a minimal SV40 

promoter and 3 hypoxia response element (HRE), 

bound by HIF, from the human transferrin gene 

(2). To monitor HIF activity in vivo, pH3SVG transfected 

C51 cells were subcutanouesly implanted into 

Balb/C nude mice. 10 days after tumor inoculation, 

mice received an i.v. injection of alexa-594-biocytin 

and were imaged using fluorescence reflectance 

imaging. 

Results: Fluorescence stainings of av-GPI expressing 

cells demonstrated that this protein is specifically expressed 

on the extracellular side of cell membranes. 

Moreover, upon pharmacological activation of HIF, 

we observed a shift in fluorescence indicative of 

an increased expression of av-GPI in fluorescent 

activated cell sorting (FACS) experiments involving 

pH3SVG transfected cells. In vivo fluorescence 

imaging showed a specific uptake of a biotinylated 

dye (alexa-594-biocytin) in the tumor from 60 minutes 

after contrast injection, whilst there was no 

accumulation of an unbiotinylated control probe 

(alexa-594-cadaverine). On ex vivo tissue sections 

alexa-594 biocytin was found to co-localize with 

zones positive for pimonidazole, a commonly used 

hypoxia marker (3) but also showed staining in regions 

devoid of pimonidazole uptake. In additional 

experiments, biotinylated 67Ga-DOTA was shown 

to specifically label avidin expressing cells in vitro. 

Conclusions: Overall, we demonstrate the utility 

of av-GPI as a reporter for in vivo imaging of HIF 

transcriptional activity in an optical, fluorescence 

reflectance approach. In vitro binding studies with 

67Ga-DOTA showed a high specificity of the probe 

in targeting av-GPI in cells, which implies that 

this reporter can indeed be combined with different 

imaging modalities. Its application in SPECT is 

currently being tested. 

References: 

1. Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) 

Bioactivation and Cell Targeting of Semiconductor 

CdSe/ZnS Nanocrystals with Phytochelatin-Related 

Peptides. Journal of the American Chemical Society 

126: 6115-6123 

2. Wanner, R. M., et al. (2000) Epolones induce 

erythropoietin expression via hypoxia-inducible 

factor-1 alpha activation. Blood 96: 1558-65 

3. Raleigh, J. A., et al. (1998) Hypoxia and vascular 

endothelial growth factor expression in human 

squamous cell carcinomas using pimonidazole as a 

hypoxia marker. Cancer Res 58: 3765-8 


YIA applicant 

day1 

Plenary Session on Excellent and Late Breaking Abstracts

YIA applicant 



A leukocyte ligand of vascular adhesion protein-1 as an imaging tool in PET 

Autio A. (1) , Saanijoki T. (1) , Sipilä H. (1) , Jalkanen S. (2) , Roivainen A. (3) . 

(1) Turku PET Centre, 

(2) MediCity Research Laboratory, National Public Health Institute, 

(3) Turku PET Centre, Turku Centre for Disease Modeling. 

akauti@utu.fi 

Vascular adhesion protein-1 (VAP-1) 

is both an endothelial glycoprotein and a semicarbazide-sensitive 

amine oxidase (SSAO) enzyme playing 

a critical role in leukocyte trafficking to the sites of 

inflammation. Although VAP-1 was identified more 

than 15 years ago, the leukocyte ligand has remained 

unknown until very recently. Last year it was shown 

that Siglec-10 (sialic acid-binding immunoglobulinlike 

lectin) expressed on a subpopulation of lymphocytes 

can bind to VAP-1 and serve as its substrate [1]. 

According to phage display screening and structural 

modeling also Siglec-9 expressed on granulocytes 

and monocytes interacts with VAP-1. In this study, 

we investigated a Siglec-9 peptide as a potential imaging 

tool in positron emission tomography (PET). 

Methods: A cyclic peptide binding to recombinant 

human VAP-1 was conjugated with DOTA-chelator 

through PEG-linker and 68Ga-labeled for PET 

studies as previously described [2]. The interaction 

between VAP-1 and 68Ga-Siglec-9 peptide 

was evaluated in vitro in human plasma samples 

possessing different SSAO levels. The VAP-1 specificity 

was further tested with competition assay in 

mice bearing melanoma xenografts by PET imaging 

and autoradiography. In vivo imaging of inflammation 

was examined in a rat model. All in vivo 

studies were confirmed by ex vivo measurements. 

Standardized uptake value 

a Tumor 

b Tumor 

c 

500 

P


Clinical translation of ex vivo sentinel lymph node mapping for colorectal cancer using 

invisible near-infrared fluorescence light 

Hutteman M. (1) , Choi H. S. (2) , Mieog S. (1) , Van Der Vorst J. (1) , Ashitate Y. (2) , Kuppen P. (1) , Löwik C.W.G.M. (1) , Van De Velde C. (1) , 

Frangioni J. (2) , Vahrmeijer A. (1) . 

(1) Leiden University Medical Center, 

(2) BIDMC / Harvard Medical School. 

m.hutteman@lumc.nl 

Introduction: Sentinel lymph node (SLN) mapping 

in colorectal cancer may have prognostic and therapeutic 

significance, however, currently available 

techniques are not optimal. We hypothesized that 

the combination of invisible near-infrared (NIR) 

fluorescent light and ex vivo injection could solve 

remaining problems in the field. 

Methods: The FLARE imaging system was used 

for real-time identification of SLNs after injection 

of the lymphatic tracer HSA800 in the colon and 

rectum of (n = 4) pigs. A total of 32 SLN mappings 

were performed in pigs, in vivo and ex vivo after oncologic 

resection, using an identical injection technique. 

Guided by these results, SLN mappings were 

performed in ex vivo tissue specimens of 6 colorectal 

cancer patients undergoing resection (Figure 1). 

Results: Using NIR fluorescent imaging, lymph 

flow could be followed in real-time from the injection 

site to the SLN. Pre-clinically, in pigs, the SLN 

was identified in 32/32 (100%) SLN mappings in 

both colon and rectum, under both in vivo and ex 

COLON 

RECTUM 

Color 

Fig1.: NIR Fluorescence-Guided SLN Mapping in Patients with Colorectal Cancer 

vivo conditions. Clinically, SLNs were identified in 

all patients (n = 6) using the ex vivo strategy (Figure 

1). No false negatives were found. SLNs were 

identified within 5 minutes after injection of the 

fluorescent dye. 

Conclusions: The current study shows proof of 

principle that ex vivo NIR fluorescence-guided SLN 

mapping can provide high-sensitivity, rapid, and accurate 

identification of SLNs in colon and rectum. 

This will permit optimized, non-FDA-approved NIR 

fluorescent lymphatic tracers to be translated immediately 

to the clinic. 

NIR Fluorescene Color-NIR Merge 


YIA applicant 

day1 


YIA applicant 

56 


Development of strategies for in vivo MRI and Optical Imaging of neural stem cells distribution 

for the treatment of traumatic spinal cord injury 

Lui R. (1) , Merli D. (1) , Libani I. V. (1) , Madaschi L. (1) , Marra F. (1) , Marfia G. (1) , Carelli S. (1) , Clerici M. S. (2) , Gorio A. (3) , 

Lucignani G. (1) , Ottobrini L. (1) . 

(1) University of Milan, 

(2) University of Milan and Don Gnocchi Foundation IRCCS, 

(3) University of Milan and Humanitas IRCCS, Rozzano, Milan. 

ramona.lui@unimi.it 

Introduction: The use of adult stem cells in cell-mediated 

therapies is an area of considerable interest within 

tissue regeneration research. However, important 

parameters such as the distribution of the injected 

cells, cell survival, target organ localisation, cell proliferation 

and differentiation cannot be evaluated in 

vivo. Here we propose multiple labelling protocols 

for in vivo visualisation by MRI, and Optical fluorescence 

imaging of murine neural stem cells (mNSC) 

originating from the subventricular zone of the adult 

murine brain in spinal cord injury animal models. 

Methods: mNSC were isolated and cultured as 

described[1]. Cells were directly labelled for 24 hours 

with 200 μg Fe/ml of SPIOs (Endorem®) in presence 

of different amount of Protamine Sulphate (PS) (ratio 

Fe/PS 1:0,025; 1:0,05) and immediately analysed 

for viability, iron content (Perl’s Staining and spectrophotometer 

analysis), morphology, staminality 

and differentiation capability. Labelled cells were 

injected into the tail vein of a spinal cord injury 

murine model and followed by MRI for a month to 

visualize their distribution. Initial cell distribution 

was also followed with scintigraphy after cell labelling 

with 111Indium-oxine (60 μCi/106 cells). Cells 

localization, distribution e viability, over time, were 

analysed in vivo with CCD camera after injection of 

mNCS infected with a viral vector expressing Luciferase 

under a PGK promoter (PLW vector). 

Results: mNSC incubated for 24 with 200 μg Fe/ml 

Endorem® did not show significant differences in 

terms of viability and proliferation rate between labelled/non-labelled 

cells in presence or absence of 

different amount of PS[2]. The percentage of viable 

cells was 85% for Fe/PS 1:0.025 ratio, and 77% for 

Fe/PS 1:0.05 ratio compared to the 95% of the control. 

On the contrary, the percentage of iron-positive 

cells increased in proportion to the PS content in the 

medium. In particular we obtained 94,5% of ironpositive 

cells in the samples incubated with Fe/PS 

1:0.025 ratio and 99% iron-positive cells in the samples 

incubated with Fe/PS 1:0.05 ratio. The iron content 

increased from 110 pg Fe/cell to 210 pg Fe/cell 

increasing PS concentration. In both cases, labelled 

cells were able to give rise to floating neurospheres 

imaging life 

as observed by optical microscopy after further 5 

days of culture, demonstrating their maintenance 

of the self-renewal capability. Immune fluorescence 

analysis for Nestin confirmed these data. Differentiation 

capability was also maintained as confirmed 

by β-TubulinIII and GFAP expression. Scintigraphic 

techniques confirmed initial distribution to lung, 

spleen and liver while MRI showed iron signal due 

to stem cell localization into the lesion site at 21 and 

28 days after injection. Neural stem cells, infected 

with the viral vector PLW, were detected up to one 

week after i.v. injection within the lesion of injured 

mice. Infected cells intramedullary injected as control 

were detected at the same timepoint as well. 

Conclusions: These results showed that adult neural 

stem cells can be efficiently labelled with different 

molecules without significantly perturbing physiological 

stem cell features and self-renewal capability. 

This labelling protocols can be applied for 

the in vivo visualisation by MRI, scintigraphy, and 

Luminescence imaging of the distribution of stem 

cells after their transplantation into murine model 

of disease. 

Acknowledgement: this work is supported by CAR- 

IPLO Foundation grant. Dr Lui R is supported by a 

fellowship from the Doctorate School of Molecular 

Medicine, University of Milan. 

References: 

1. Bottai D et al. Mol Med (2008) 14:634-644 

2. Politi LS et al. Neuroradiology (2007) 49:523–534


Validation of bone marrow-derived stromal cell graft position by colocalisation of histology, 

bioluminescence and magnetic resonance imaging. 

De Vocht N., Van Der Linden A. . 

Universiteit Antwerpen – Bio Imaging Lab 

nathalie.devocht@ua.ac.be 

Introduction: The use of stem cell transplantation 

as a therapeutic tool to treat neurodegenerative disorders 

(e.g. traumatic brain injury, stroke, multiple 

sclerosis) has gained increasing interest over the 

last decade. However, a profound knowledge of cell 

implant migration, differentiation and survival will 

be necessary to understand the physiological mechanisms 

involved in regeneration of injured brain tissue. 

For this, the development of multimodal cell 

imaging techniques, both in vivo and post-mortem, 

is currently of highest importance. 

Methods: We optimised a multimodal labelling 

strategy for bone marrow-derived stromal cells 

(BMSC), based on (i) genetic modification with 

both the eGFP and Luciferase reporter genes, and 

(ii) endocytic uptake of blue fluorescent magnetite-containing 

micron-sized particles (MPIO). 

This combined labelling strategy allows unambiguous 

identification of cell localisation, survival 

and differentiation using pre-mortem bioluminescence 

/ magnetic resonance imaging (BLI/MRI) 

and post-mortem histological analysis. 

Results: For this study, we labelled Luciferase/ 

eGFP-expressing BMSC (BMSC-Luc/eGFP, Bergwerf 

et al. 2009) with 1.63 µm fluorescent (Glacial 

Blue) MPIOs from Bangs Laboratories. MPIO labelling 

of BMSC-Luc/eGFP did not influence their 

phenotypic properties and self-renewal capacity. 

In order to evaluate the suitability of these MPIO 

particles for in vivo multimodal imaging, we implanted 

4 x 105 MPIO labelled (n = 15) or unlabelled 

(n = 15) cells by stereotactic injection in the 

right hemisphere (DV: 2mm beneath the dura; ML: 

2mm; AP: -2mm) of syngeneic male FVB mice (10- 

12 weeks old). Survival of unlabelled and MPIOlabelled 

MSC-Luc/eGFP implants was monitored 

during 2 weeks by BLI, which demonstrated equal 

survival of both cell populations. Moreover, T2- 

and T2*-weighted MR images clearly demonstrated 

the presence of iron oxide particles in mouse 

brain. Additionally, combined immunohistochemistry 

and fluorescence microscopy was able to confirm 

cell identity and the presence of MPIO particles 

within BMSC-Luc/eGFP grafts. 

Conclusions: We here demonstrate that combining 

three labelling strategies allows unambiguous identification 

of MSC following implantation in mouse 

brain. The MPIO-labelling allows for pre-mortem 

MRI detection of cell implant localisation. In addition, 

BMSC survival was followed up using BLI up 

to two weeks post-injection. Finally, the combination 

of eGFP expression and MPIO-labelling allows 

for discrimination of cell implants (eGFP+/MPIO+) 

from inflammatory cells (green background fluorescence/MPIO-) 

and free particles (eGFP-/MPIO+), 

enabling an improved qualitative histological analysis 

of cell implantation. 


by: (i) the EC-FP6-NoE funded DiMI (ii) the Flemish 

government-funded IWT-SBO BRAINSTIM 

Project and (iii) the FWO Flanders. 

References: 

1. Bergwerf I, De Vocht N, et al., BMC Biotechnology 

2009; 9:1 


day1 


DAY 

2

• ESMI Plenary Lecture 3: Theodorus W.J. Gadella Jr. (Amsterdam, The Netherlands) 


Chairs: Vasilis Nziachristos (Munich, Germany) , Bengt Långström (Uppsala, Sweden) 

• Parallel Session 5: Neuroscience II 

Chairs: Markus Rudin (Zürich, Switzerland), Veerle Baekelandt (Leuven, Belgium), 

Mathias Hoehn (Cologne, Germany) 

• Parallel Session 6: Cardiovascular I – together with ESR 

Chairs: Tony Lahoutte (Mons, Belgium), Nicolas Grenier (Bordeaux, France) 

• Parallel Session 7: Probes II – together with EANM 

Chairs: Frédéric Dollé (Orsay, France), Denis Guilloteau (Tours, France) 

• Parallel Session 8: Gene and Cell Based Therapies – together with CliniGene 

Chairs: Ludwig Aigner (Salzburg, Austria), Cornel Fraefel (Zürich, Switzerland) 

• ESMI Plenary Lecture 4: Hans-Jürgen Wester (Munich, Germany) 

Molecular imaging of CXCR4 receptors 

Chairs: Adriana Maggi (Milano, Italy), Adriaan Lammertsma (Amsterdam, The Netherlands) 

• Plenary Session on Current Contribution of Imaging Technologies to Drug Development 

Chairs: Adriana Maggi (Milano, Italy), Adriaan Lammertsma (Amsterdam, The Netherlands) 

Day 2 - Friday May 28, 2010

60 


Chairman of section of Molecular Cytology, Director Centre for Advanced Microscopy 

Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands 

www.mc.bio.uva.nl 

Dr. Gadella is founder/director of the Centre for Advanced Microscopy (CAM) at the University of Amsterdam 

and full professor in Molecular Cytology chairing a group of 25-30 scientists including 4 assistant 

professors. The CAM and chairgroup are positioned within the Swammerdam Institute for Life Science at 

the Science Faculty of the University of Amsterdam and fully integrated within the Netherlands Institute for 

Systems Biology founded in 2007. Dr Gadella personally supervises a research team on spatiotemporal cell 

signaling. His team wants to understand how cells can achieve and maintain a local signal in order to drive 

morphogenesis, to define new cytoskeletal anchorage or vesicle-docking sites. The focus is on signal flow 

across and in the plane of the membrane of living mammalian cells. To this end genetic encoded fluorescent 

biosensors are employed and the in situ molecular interactions between signaling molecules including phospholipid-second 

messengers, receptors, G-proteins and downstream targets are analyzed. By multiparameter 

imaging approaches several signaling events are visualized and quantified simultaneously in individual living 

cells with sub-second temporal and submicron spatial resolution. The in situ cellular imaging research 

heavily depends on advanced bioimaging. To this end advanced automated microscopy approaches such 

as fluorescence resonance energy transfer (FRET) microscopy (including bleaching-, spectral-, ratio- and 

fluorescence lifetime-imaging approaches), fast live cell microscopy (including spinning disk, line-scanning 

and controlled light exposure confocal microscopy, and total internal reflection microscopy (TIRF)), and 

photochemical microscopy approaches such as photoactivation, uncaging, fluorescence recovery after photobleaching 

(FRAP), fluorescence loss in photobleaching microscopy (FLIP), fluorescence correlation spectroscopy 

(FCS), cross-correlation (FCCS), lifetime-correlation (FCLS) have been implemented developed 

and applied to quantifying cell signalling phenomena. Currently, the palette of advanced imaging instrumentation 

is expanded with superresolution localization microscopy approaches such as PALM and STORM. 

The research group is funded from university resources (roughly 40%), national grant agencies (NWO-ALW, 

CW, FOM and STW) and several international grants (ESF and EU-FP6/7 projects including integrated projects, 

STREPs and TMR programs). 

The Gadella lab has numerous (inter)national collaborations in the field of signal transduction (Dr. C. Hoffman, 

Univ. Würzburg; M. Wymann, Univ. Basle; K. Jalink, NKI Amsterdam) and collaborations on probe 

development (dr. C. Schultz, EMBL Heidelberg; dr. K. Lukyanov & D. Chudakov, Moscow) and collaborations 

on advanced light microscopy (prof. van Noorden (AMC); dr. A. Houtsmuller, Erasmus MC; dr. H. 

Gerritsen, Utrecht; dr. Dobrucki, Univ. Krakow; prof. M. Carmo Fonseca, Lisbon). In 2007 prof Gadella 

was elected president of the Netherlands Society for Advanced Microscopy. Recently he was appointed as 

national coordinator of the advanced light microscopy activities of the Netherlands for the new large EU 

infrastructure (ESFRI) programme EuroBioimaging that recently entered the startup phase. 

imaging life 

Theodorus W.J. Gadella Jr.



Gadella T.W.J. jr, Goedhart J., Van Weeren L., Crosby, K., Hink M.A. 

Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences & Netherlands Institute for Systems Biology, 

University of Amsterdam, Science Park 904, NL-1098 XH Amsterdam. 

Th.W.J.Gadella@uva.nl 

Since the cloning of the green fluorescent protein 

from Aequoria victoria, numerous fluorescence 

microscopy applications have been described in 

the literature. By exploiting the spectroscopic axis 

in microscopy (excitation/emission wavelength, 

fluorescence lifetime) utilizing spectral variants 

of GFP colocalization and FRET applications 

were enabled. In the other direction, the spectroscopical 

axis in microscopes can also be used to 

obtain novel GFP variants with optimized properties. 

Here we report a screening method that, 

in addition to fluorescence intensity, quantifies 

the excited state lifetime of a fluorescent protein, 

providing a direct measure for the quantum yield 

of the fluorescent protein. The novel approach 

was used to screen a library of cyan fluorescent 

protein (CFP) variants yielding the brightest cyan 

fluorescent protein variant described thus far 

which we dubbed mTurquoise. mTurquoise has a 

marked increased quantum yield of 0.84, and a 

seriously increased lifetime of 3.7 ns. Because of 

its monoexponential decay and high R0 for FRET 

to SYFP2 (5.7 nm), it is the preferred donor in 

FRET studies. In addition, the lifetime screening 

method also yielded several other CFP lifetime 

variants with identical spectra. It is demonstrated 

that three spectrally identical CFP variants can 

be separated in single FLIM experiment in living 

cells and their distribution & molecular stoichiometry 

can be quantified. The triple unmixing 

technique using the new lifetime variants can be 

combined with spectral separation of other GFP 

color variants with a great potential for multiparameter/multiplexed 

imaging. 

We apply the different probes for the study of 

GPCR-triggered signaling in single mammalian 

cells. The design of a Gq FRET sensor reporting 

on activation by endogenous expressed receptors, 

multiparametric imaging of lipid-derived second 

messengers, PtdInsP2-dependent PLC translocation, 

and RhoGEF activation using a variety of 

FRET-, ratio imaging- and TIRF-microscopic applications 

will be presented. 


day2 

ESMI Plenary Lecture 3 by Theodorus Gadella

62 


Potential to use transgenic animals for imaging of neurological diseases 

Aigner L. . 

Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Austria 

ludwig.aigner@pmu.ac.at 

Transgenic animal models represent an elegant and 

innovative tool to image the multitude of events 

including degenerative, inflammatory and regenerative 

responses in the brain. However, there are 

currently no transgenic animal models that would 

image specifically one or another neurological 

disease. Typically, a transgenic reporter animal 

highlights the activity of a gene promoter that is 

specifically active in one or another cell type during 

a certain physiological process, upon response 

to injury, along the course of neurodegeneration 

or during a regenerative response. Thus, transgenic 

animal models allow the imaging of physiological 

and pathophysiological responses that are 

associated with neurological / neurodegenerative 

diseases, but not the disease itself. For example, 

currently available models allow the elucidation of 

several aspects of neurodegeneration: 1) Inflammatory 

events can be visualized using transgenic 

animals with the TLR2 promoter controlling the 

expression of reporter genes; herein, these reporters 

are up-regulated in response to the onset 

imaging life 

of an inflammatory response hand in hand with 

endogenous TLR2 and microglial activation (Lalancette-Hebert, 

Gowing et al. 2007). 2) Reactive 

gliosis may be pictured using GFAP-reporter mice 

as a result of stroke in living animals (Cordeau, 

Lalancette-Hebert et al. 2008). 3) Hypoxia might 

be imaged due to the application of models like 

the HRE-GFP transgenic animals where cells that 

suffer from oxygen deprivation express reporter 

proteins induced by low-oxygen-responsive transcription 

factors (Berchner-Pfannschmidt, Frede 

et al. 2008). 4) Neuroplasticity after brain ischemia 

comprises the recovery of dendritic structure, and 

this event may be imaged using transgenic models 

with fluorescent proteins expressed throughout a 

distinct cell population and including the cellular 

protrusions (Li and Murphy 2008). 5) Neurogenesis 

and the migration of young neurons can be 

visualized using DCX-promo luciferase mice. This 

presentation will provide a summary and an overview 

on the current state of transgenic models in 

the field of neurological diseases.


Specific cell labeling with imaging reporters in vivo by viral vectors: the challenges 

Reumers V. (1) , Ibrahimi A. (2) , Toelen J. (2) , Aelvoet S. A. (1) , Van Den Haute C. (1) , Debyser Z. (2) , Baekelandt V. (1) . 

(1) Neurobiology and Gene Therapy, Katholieke Universiteit Leuven, 

(2) Molecular Virology and Gene Therapy, Katholieke Universiteit Leuven. From MoSAIC, the Molecular Small Animal Imaging Center, K.U.Leuven, Belgium 

Veerle.Baekelandt@med.kuleuven.be 

Viral vectors are extremely efficient tools for gene 

transfer into living cells. We are using lentiviral 

vectors and adeno-associated viral (AAV) vectors 

for stable overexpression and suppression of gene 

expression in rodent brain. Stereotactic injection 

of these viral vectors into different brain regions 

of mouse and rat brain induces overexpression or 

inhibition of disease-related genes, which can be 

used for target validation, functional genomics and 

generation of disease models. We have invested 

over the last years into reporter gene technology 

and non-invasive imaging in rodent brain. We have 

optimized bioluminescence imaging to monitor 

gene expression in mouse brain and to track 

neuronal stem cell migration and differentiation. 

In order to specifically target certain neuronal cell 

populations we have recently developed conditional 

lentiviral and AAV viral vectors based on Cremediated 

recombination. Application of these viral 

vectors in specific cre-transgenic mouse strains 

allows to specifically label endogenous neural stem 

cells in the subventricular zone or dopaminergic 

cells in the substantia nigra. This approach holds 

promise to non-invasively follow up endogenous 

neurogenesis and neurodegeneration over time. 


day2 

Parallel Session 5: NEUROSCIENCE II

YIA applicant 

64 


Optimization of an MRI method to measure microvessel density: application to monitor 

angiogenesis after stroke 

Boehm-Sturm P. (1) , Adamczak J. (1) , Farr T. D. (1) , Jikeli J. (2) , Kallur T. (1) , Hoehn M. (1) . 

(1) MPI for Neurological Research in Cologne, Germany 

(2) Center of Advanced European Studies and Research in Bonn. 

philipp.boehm-sturm@nf.mpg.de 

Introduction: Angiogenesis is the growth of new vessels 

from an existing vascular network and is highly 

upregulated following ischemia. Increases in microvessel 

density (MVD) have been reported to occur 

in the peri-infarct zone of stroke patients, which was 

correlated with increased survival time [1]. Steadystate-contrast-enhanced 

(SSCE) MRI provides 

the ability to monitor MVD in vivo. To do so, the 

changes in relaxivities ΔR2/ΔR2* due to an i.v. injection 

of a paramagnetic blood pool contrast agent are 

evaluated by spin-echo (SE) and gradient-echo (GE) 

imaging, and the MVD index Q=ΔR2/(ΔR2)2/3 is 

mapped [2,3]. However, noise in the MR images can 

significantly distort the results of the method. Therefore, 

the goal of this study is to optimize the SSCE 

MRI parameters in simulations in order to find a 

noise-resistant protocol. We aim to apply the method 

to monitor angiogenesis after stroke in rats and to 

validate this by immunohistochemistry. 

Left: simulated relative error in Q (in %) over TESE and TEGE at a fixed noise level; the arrow indicates the optimal set of TEs. Right: lesion in SE 

image 48 h after MCAO (a) and Q at 7 days prior (b), and 7 (c), and 14 days (d) post MCAO of the same animal. Arrow indicates an area with high Q 

value. Typical RECA (red)/BrdU (green) (e) and Hoechst(blue)/RECA(red)/laminin (green) (f) fluorescence microscopy images close to the infarct. 

Methods: Q-maps were simulated for different levels 

of noise and echo times TESE/TEGE of the SE/ 

GE images. Male Wistar rats (n=7) were scanned 

with SSCE MRI 7 days prior, and 7 and 14 days post 

60 min transient MCAO. An SE image was acquired 

48 h post MCAO to depict the lesion extent. Rats 

received Bromodeoxyuridine (BrdU) injections between 

3-7 days post MCAO in order to label proliferating 

cells. Following the final SSCE MRI scan 

at 14 days, animals were perfused and brain sections 

stained for BrdU, rat endothelial cell antigen 

(RECA), and laminin. 

imaging life 

Results: The simulations revealed that the error in 

Q is minimal for a TESE of 30-40 ms and a TEGE of 

10-15 ms. Areas with increased Q could be detected 

close to the infarct in a few of the rats as early as 7 

days and in most of the rats at 14 days post MCAO. 

BrdU/RECA double staining was established to 

identify newly formed endothelial cells and laminin 

staining confirmed the presence of an intact basement 

membrane in the vessels. 

Conclusions: Our results show that scanner noise 

can severely limit the capabilities of SSCE MRI but 

a set of optimal scan parameters can minimize this 

problem. The method has been shown to monitor 

potential changes of the microvasculature longitudinally 

and non-invasively in the ischemic rat brain. 

It will in the future allow to study neo-angiogenesis 

in the course of (stem)cell-based therapies after cerebral 

lesions. 

Acknowledgements: Generous supply of ENDOREM 

by Drs. C. Corot and P. Robert is gratefully acknowledged. 

This work was supported in part by grants from 

the European Union under FP6 program (StemStroke, 

LSHB-CT-2006-037526), and under FP7 program 

(ENCITE, 201842), and an Alexander von Humboldt 

Research Fellowship to TDF. 

References: 

1. Krupinski et al, Stroke (1994) 25:1794-1798; 

2. Jensen et al., MRM (2000) 44:224–230; 

3. Wu et al., NMR Biomed (2004) 17:507–512


MRI study of intra-arterial bone marrow-derived macrophage administration after acute 

focal ischemic stroke in rats 

Riou A. (1) , Chauveau F. (1) , Cho T.H. (1) , Nataf S. (2) , Berthezene Y. (1) , Nighoghossian N. (1) , Wiart M. (1) . 

(1) CREATIS-LRMN : CNRS UMR5220, INSERM U630, Univ. Lyon1, INSA-Lyon, France 

(2) CNRS UMR5220, INSERM U630. 

adrien.riou@live.fr 

Introduction: In the past few years numerous studies 

have been published to assess if cell-based therapy 

after stroke can enhance long-term recovery. It 

is well established that cerebral ischemia results in 

a complex inflammatory cascade that mainly involves 

cells from the mononuclear phagocyte system1, 

although the beneficial or deleterious effect 

of this activation following stroke is still a matter 

of controversy. Furthermore, therapeutic benefits 

gained from cell-based therapy depend on migration 

and localization of grafted cells within the target 

tissue that is closely related to the cell delivery 

route2 and therapeutic time window3 chosen for 

the therapy. The aim of this study was to assess the 

feasibility of in vivo early intra-arterial (IA) bone 

marrow-derived macrophage (BMDM) administration 

for acute focal ischemic stroke treatment, using 

multiparametric magnetic resonance imaging 

(MRI). 

Methods: BMDM were obtained from a sacrificed 

littermate of recipient rats by flushing out one 

hindlimb. Bone marrow cells were then seeded on 

uncoated flasks at 5 x 105 cells/ml in medium supplemented 

with murine macrophage-colony stimulating 

factor and flt3 ligand (mM-CSF and flt3L at 

10ng/ml) for one week. At day 6, cells were labeled 

with anionic superparamagnetic nanoparticles 

overnight (AMNP, CNRS UMR 7612, Paris, France, 

[Fe]=1mM). Male Sprague Dawley rats (250 to 350g, 

n=17) were subjected to 1-h intraluminal transient 

middle cerebral artery occlusion (tMCAO) (n=12) 

or sham procedure (n=5). IA administration of 

AMNP-labeled cells was performed at the time of 

reperfusion in all tMCAO animals and in 3 shams 

animals (4 million in 1-ml except for one sham: 1 

million in 1-ml). Ischemia / reperfusion and cell 

injection were monitored by transcranial laser doppler 

flowmetry. MRI was performed on a Bruker 

Biospec 7T/12cm magnet at D0 just after cells administration 

and from D1 to D9. The MR exam included 

T2-, T2*-, diffusion- and perfusion-weighted 

imaging and multi-echo 3D imaging. Animals 

were sacrificed after completion of the MRI exams 

and brains were prepared for immunohistological 

analysis with Prussian blue for iron detection and 

Ox-42 for macrophage detection. 

Results: IA administration in tMCAO group lead to 

heterogeneous results: 3 rats died following injection, 

2 had cells detectable only in the temporalis 

muscle and 7 had cells detectable in the ipsilateral 

parenchyma but with heterogeneous patterns: 3 had 

a widespread persistent hypointense signal distribution, 

while 4 had only few local spots of signal 

loss on follow-up scans. Furthermore, IA administration 

in the sham group caused lesion formation 

on follow-up scans in all injected animals (n=3), 

even when cell number was decreased, as opposed 

to the non-injected sham rats that did not have any 

lesions. Immunohistological analysis is in progress 

to ascertain iron-labeled macrophage localization. 

Conclusions: Our results were consistent with that 

of previous studies2, 4 showing that IA delivery 

route efficiently brought a large number of cells to 

the brain soon after transplantation, but severely 

increased mortality. More importantly, lesions observed 

in the sham group (undemonstrated to date 

to our knowledge) suggested that this high mortality 

rate resulted from cell embolisation in cerebral 

vessels leading to formation or worsening of the 

ischemic lesion. This result points out a serious limitation 

for the translation of this cell delivery route 

into the clinics. 

References: 

1. Huang J, et al. Surg Neurol.;66:232-45 (2006). 

2. Li L, et al. J Cereb Blood Flow Metab (2009). 

3. de Vasconcelos Dos Santos A, et al. Brain Res.1306:149- 

58 (2010). 

4. Walczak P, et al. Stroke. 2008;39:1569-74 (2008). 


day2 


YIA applicant 

66 


Inactivated paramagnetic tissue plasminogen activator predicts thrombolysis outcome 

following stroke 

Gauberti M. (1) , Montagne A. (1) , Vivien D. (1) , Orset C. (1) . 

INSERM U919 SP2U Serine protease and pathophysiology of the neurovascular unit, France 

gauberti@cyceron.fr 

Introduction: Ischemic stroke is the third leading 

cause of death in developed countries. Despite numerous 

clinical trials, tissue plasminogen activator 

(tPA) induced thrombolysis remains the only treatment 

of the acute phase. However there is growing 

body of evidences that parenchymal tPA has deleterious 

effects including increased risk of intra cranial 

hemorrhages and neurotoxicity. To limit these side 

effects, only rigorously selected patients (onset of 

symptoms < 4.5 hrs, age < 85 hrs, etc.) benefit from 

thrombolysis, with more than 90% of patients being 

untreated. In previous in vitro studies we showed 

that vascular tPA can cross the intact blood brain 

barrier (BBB) by Low Density Lipoprotein Receptor 

Protein (LRP) mediated transcytosis [1]. Accordingly, 

increasing data of the literature suggest that 

BBB specific permeability to tPA could be critical for 

brain outcome following thrombolysis [2] [3]. 

Figure 1. Ex-vivo NIRF brain imaging of fluorescent tPA and albumin 

administered at 1 or 4 hours after permanent middle cerebral artery 

occlusion in mice. Unlike albumin which has the same molecular 

weight, tPA can cross the BBB at 4 hours post ischemia. 

Results: Here in a model of permanent middle cerebral 

artery occlusion in mice, we show that intravenous 

tPA aggravates ischemic lesion size and 

BBB permeability if administrated at 4 hours but 

not at 1 hour after ischemia. In parallel, although 

BBB remains impermeable to albumin, we provide 

evidences both by microscopic epifluorescence and 

near infrared fluorescence (NIRF) brain imaging of 

a passage of tPA across the BBB only when administrated 

at 4 hours post ischemia. Using MRI and inactivated 

paramagnetic tPA as molecular probe, we 

imaging life 

are tempting to demonstrate that such type of brain 

imaging could allow us to pre-select stroke patients 

who would not be susceptible to haemorrhagic transformations 

following rtPA-induced thrombolysis. 

Conclusions: Non invasive imaging of inactivated 

paramagnetic tPA could allow clinicians to include 

stroke patients for thrombolysis with objective brain 

imaging data independently of the time post-stroke 

onset. As long as the BBB remains impermeable to 

tPA, thrombolysis seems safe. 

References: 

1. Benchenane et al.; Circulation 111:2241-2249 (2005). 

2. Benchenane et al.; Stroke 38:1036 (2005). 

3. Roussel et al.; Thromb Haemost. 102:602-608 (2009).


Matrix metalloproteinase-9 – a possible therapeutic target in intractable epilepsy 

Wilczynski G. . 

Nencki Institute, Poland 

g.wilczynski@nencki.gov.pl 

Introduction: Temporal lobe epilepsy (TLE) is a 

chronic, devastating, pharmacologically intractable 

disease in which aberrant synaptic plasticity 

plays a major role. Recently, MMP-9, a matrix 

metalloproteinase, has been implicated in synaptic 

plasticity, long-term potentiation and learning and 

memory formation. We asked, whether MMP-9 

might play a pathogenic role in epileptogenesis. 

Methods: High resolution light- and electronmicroscopic 

immunocytochemistry, in situ hybridization, 

in situ zymography; use of transgenic 

animals; antibody microarrays 

Results: Our study revealed MMP-9 as a novel 

synaptic enzyme, and a key pathogenic factor 

in two distinct animal models of TLE: kainateevoked-epilepsy 

and pentylenetetrazole (PTZ) 

kindling-induced epilepsy. In particular, sensitivity 

to PTZ-induced epileptogenesis is decreased 

in MMP-9 knockout (KO) mice, whereas it is 

increased in MMP-9-overexpressing rats. Moreover, 

confocal- and immunoelectron-microscopic 

analyses demonstrated that MMP-9 associated 

with hippocampal dendritic spines bearing asymmetric 

(excitatory) synapses. In addition, both 

MMP-9 protein levels as well as its enzymatic activity 

became strongly increased upon seizures. 

Furthermore, MMP-9-deficiency diminished seizure-evoked 

pruning of dendritic spines, and it 

decreased aberrant synapse formation following 

mossy-fibers sprouting. 

We then asked whether MMP-9 could play an important 

role also in human intractable epilepsy. 

Accordingly, we have studied the expression and 

localization of MMP-9 in samples of human epileptic 

brain tissue, obtained upon surgical treatment 

of childhood intractable epilepsy. By antibody 

microarrays, we found MMP-9, but none of 

the other seven MMPs studied, to be consistently 

upregulated in epileptic lesions, as compared to 

autopsy brain tissue. By immunohistochemistry 

of epileptic tissue, increased MMP-9 localized 

specifically to affected neurons. 

Conclusions: Taken together, the aforementioned 

results suggest that the synaptic pool of MMP-9 is 

critically involved in the sequence of events that underlie 

epileptogenesis, both in rodents and humans. 

Therefore, the enzyme should be considered as a 

candidate target for therapeutic pharmacological 

intervention. 

Acknowledgement: The work was supported by 

Polish-Norwegian Research Grant 

References: 

1. Wilczynski et al., Important role of matrix 

metalloproteinase 9 (MMP-9) in epileptogenesis. J Cell 

Biol 180: 1021-1035, 2008. 

2. Konopacki et al., Synaptic localization of seizureinduced 

matrix metalloproteinase-9 mRNA. 

Neuroscience; 150:31-39, 2007 


day2 


68 


Potentials of new contrast agents for vascular molecular imaging in patients 

Douek P., Boussel L., Sigovan M., Cannet E. . 

Creatis, Hopital Louis Pradel, Lyon University, France 

philippe.douek@creatis.univ-lyon1.fr 

Introduction: Atherosclerosis is a diffuse and 

multisystem, chronic inflammatory disorder 

involving vascular, metabolic, and immune systems 

leading to plaque instability and / or vascular 

trombosis. The traditional risk assessment relies on 

clinical, biological and conventional imaging tools. 

However, they fall short in predicting near future 

events particularly in patients with unstable carotid 

artery disease or coronary artery disease. The plaque 

instability is dictated in part by plaque morphology, 

which in turn is influenced by pathophysiologic 

mechanisms at the cellular and molecular level. 

In current clinical practice, anatomic imaging 

modalities such as intravascular ultrasound, highresolution 

magnetic resonance imaging can identify 

several morphologic features supporting the unstable 

plaque, but give little or no information regarding 

molecular and cellular mechanisms. optical imaging, 

PET, Molecular MR imaging, or more recently 

spectral CT may identify some of these molecular 

and cellular processes. 

Methods: We aimed to review 

1) the role of relevant biological, factors and 

advances in atherosclerosis biology, that illuminate 

key biological aspects of atherosclerosis, including 

macrophage activity, protease activity, lipoprotein 

presence, apoptosis, and angiogenesis 

imaging life 

2) imaging agent chemistry (nanotechnology, 

chemical biology screens) 

3) recent advances in the field of molecular 

imaging that have led to the development of novel 

paramagnetic and superparamagnetic targeted 

contrast agents that bind exclusively to cells such as 

macrophages, or molecules such as albumin, fibrin, 

angiogenesis markers... with either PET, MR, US or 

CT .and 

Results: 4) a few clinically promising applications of 

molecular imaging for high-risk atherosclerosis and 

its potential for current and emerging treatments in 

clinical trials. 

Conclusions: A multimodal assessment of plaque 

instability involving the combination of systemic 

markers, high resolution imaging and molecular 

imaging targeting inflammatory and thrombotic 

components and the potential of new drugs 

targeting plaque stabilization may lead to a new 

stratification of the atherothrombotic risk and to 

a better prevention of atherothrombotic stroke or 

myocardial infarction.


Protease specific nanosensors in atherosclerosis 

Schellenberger E. . 

Charité Berlin, Germany 

eyk.schellenberger@charite.de 

Introduction: Atherosclerosis and especially the 

consequences of rupture of atherosclerotic plaques 

are the foremost cause of death in industrialized 

societies. Up to date the identification of vulnerable 

atherosclerotic plaques prone to rupture has not been 

solved for the clinics. The pathological processes 

leading to destabilization of such plaques are highly 

complex and involve inflammatory action and 

pathological tissue remodeling of the extracellular 

matrix performed by proteases. Therefor the imaging 

of specific protease activity by high resolution 

MR imaging could be an important tool to find 

dangerous plaques that need urgent treatment. 

Results: Optical imaging with activatable fluorescent 

smart probes has become the modality of choice 

for experimental in vivo detection of protease 

activity. Recently we introduced a novel highrelaxivity 

nanosensors that are suitable for in vivo 

imaging of protease activity by MRI. Upon specific 

protease cleavage, the nanoparticles rapidly switch 

from a stable low-relaxivity stealth state to become 

adhesive, aggregating high-relaxivity particles. 

To demonstrate the principle, we chose a cleavage motif 

of matrix metalloproteinase 9/2 (MMP-9 and MMP-2), 

proteases that important during the destabilization of 

the extracellular matrix of plaques. Based on clinically 

tested very small iron oxide particles (VSOP), the 

MMP-9-activatable protease-specific iron oxide 

particles (PSOP) have a hydrodynamic diameter of 

only 25 nm. PSOP are rapidly activated, resulting in 

aggregation and increased T2*-relaxivity of the particles. 

Conclusions: PSOP could be useful sensors for the 

in vivo identification of vulnerable plaques in the 

future. 


day2 

Parallel Session 6: CARDIOVASCULAR I - together with ESR

YIA applicant 

70 


Imaging of inflamed carotid artery atherosclerotic plaques with the use of 99mTc-HYNIC- 

IL-2 scintigraphy in the end-stage renal disease patients 

Opalinska M. (1) , Hubalewska-Dydejczyk A. (2) , Stompor T. (3) , Krzanowski M. (4) , Mikołajczak R. (5) , Garnuszek P. (5) , 

Karczmarczyk U. (6) , Maurin M. (5) , Rakowski T. (7) , Sowa-Staszczak A. (2) , Glowa B. (2) . 

(1) Nuclear Medicine Unit, Chair and Department of Endocrinology, Jagiellonian University Medical School, (2) Nuclear Medicine Unit, Chair and Department of 

Endocrinology, Jagiellonian University Medical School, (3) Chair and Department of Nephrology, Hypertesiology and Internal Medicine, University of Warmia 

and Mazury, (4) Chair and Department of Nephrology, Jagiellonian University Medical School, (5) Radioisotope Center POLATOM, (6) Departament of Radiopharmaceuticals, 

National Medicines Institute, (7) 2nd Cardiology Clinic, Institute of Cardiology, Jagiellonian University Medical School. 

mkal@vp.pl 

Introduction: Cardiovascular diseases are the main 

cause of death in developed countries. In some patients’ 

populations, including those with end-stage 

renal disease (ESRD), cardiovascular related mortality 

is 30 times higher compared to the general 

population. 

Such a high cardiovascular mortality is associated 

with significantly accelerated atherosclerosis in 

ESRD mainly due to intense inflammatory state. 

Therefore serum concentrations of inflammatory 

agents that promote atherosclerosis are often used 

to evaluate the cardiovascular risk. 

The histological studies of the atherosclerotic 

plaque revealed that at least 20% of inflammatory 

cells within unstable plaque comprise lymphocytes 

T, which contain receptors for interleukin 2 (IL-2); 

those receptors can be identified by scintigraphy 

with radiolabeled IL-2. 

Methods: 28 patients (13 men, 15 women, aged 55,2 

± 9,6, 17 on peritoneal dialysis, 11 on hemodialysis) 

underwent scintigraphy with the use of 99mTc- 

HYNIC-IL-2. In all cases ultrasound examination 

of the carotid arteries was performed to obtain information 

about localization and morphology of 

atherosclerosis plaques and intima-media thickness 

(IMT) measurement. Furthermore, levels of selected 

proinflammatory factors, atherogenic markers and 

calcium-phosphate balance parameters were measured. 

Finally, target/non-target (T/nT) ratio of IL-2 

uptake in atherosclerotic plaques confirmed by carotid 

USG with IMT, presence of calcifications in 

atherosclerotic plaques and concentration of the 

measured agents were compared. 

Results: Increased 99mTc-IL-2 uptake in atherosclerotic 

plaques previously visualized by neck ultrasound 

in 38/41 (91%) cases was detected. Median 

T/nT ratio of focal 99mTcIL-2 uptake in atherosclerotic 

plaque was 2,35 (range 1,23– 3,63, average 2,35 

± 0,70). Mean IMT value on the side of plaque assessed 

in scintigraphy was 0,79 ± 0,18 mm (median 

0,8, range 0,5 – 1,275). 

imaging life 

No statistically significant association was found 

between 99mTc-IL-2 T/nT ratio and mean value 

of either IMT or classical cardiovascular risk factors. 

Inversely, proportional dependence between 

scintigraphy results and hemoglobin concentration 

(R = - 0,21, p = 0,02) was found. Furthermore, relationships 

between T/nT ratio and homocysteine (R 

= 0,22, p = 0,037), ApoB (R = 0,31, p = 0,008), ApoB/ 

ApoA-I ratio (R= 0,29, p= 0,012) and triglycerides 

concentration (R = 0,26, p = 0,021) were detected. 

Lower T/nT ratio in patients with better parameters 

of nutrition state (hemoglobin, albumin, adiponectin) 

in comparison with patients with worse nutritional 

parameters (3,20 ± 0,5 vs 2,16 ± 0,68, p = 

0,025) was revealed as well as the difference between 

values of T/nT ratio in groups of patients with values 

of ApoB, sCD40L and ADMA above and below median 

(3,18 ± 0,52 vs 2,16 ± 0,68 p = 0,031). An interesting 

relationship between T/nT ratio in atherosclerotic 

plaques with and without calcifications (2,35 ± 

0,68 vs 1,924 ± 0,55 p = 0,088) was observed. 

Conclusions: Scintigraphy with the use of labeled 

IL-2 can be a tool for inflamed atherosclerotic (vulnerable) 

plaque visualization within common carotid 

arteries in ESRD patients. Quantitative results 

of the carotid arteries scintigraphy with labeled IL-2 

correlate with serum concentration of selected cardiovascular 

risk markers 


Polish Committee for Scientific Research (KBN) 

within Research Project 2 P05B 003 28 

References: 

1. Annovazzi A et al; 99mTc-interleukin-2 scintgraphy for 

the in vivo imaging of vulnerable atheroslerotic plaque. 

Eur J Nucl Med Mol Imaging 2006; 33: 117 – 126.


Uptake of 68 Ga-Chloride in Atherosclerotic Plaques of LDLR -/- ApoB 100/100 Mice 

Silvola J. (1) , Laitinen I. (2) , Sipilä H. (1) , Laine J. (3) , Leppänen P. (4) , Ylä-Herttuala S. (4) , Knuuti J. (1) , Roivainen A. (5) . 

(1) Turku PET Centre, University of Turku, 

(2) Nuklearmedizinische Klinik der TU Muenchen, Technische Universitaet Muenchen, 

(3) Department of Pathology, Turku University Hospital, 

(4) A.I. Virtanen Institute, University of Kuopio, 

(5) Turku Centre for Disease Modelling, University of Turku. 

jmuhau@utu.fi 

Introduction: Atherosclerosis is an inflammatory 

disease in which monocytes/macrophages have essential 

role in the development of plaques. Radiogallium 

has been used for decades for in vivo imaging 

of inflammation. 68Ga is a positron emitter with a 

half-life of 68 min and it is suitable for PET imaging. 

Purpose of this study was to explore the uptake of 

68Ga-chloride in atherosclerotic plaques in mice. 

Methods: Uptake of intravenously administered 

68Ga-chloride (17 ± 2 MBq) was investigated in 

9 atherosclerotic LDL-/-ApoB100/100 mice and 

6 control mice at 3 hours after injection. LDL-/- 

ApoB100/100 mice were kept in on a high fat, Western-type 

diet for 3-4 months, starting at 7 months 

of age. Control mice were fed with regular chow. 

The biodistribution of the tracer was evaluated, and 

aortic cryosections were further analysed by digital 

autoradiography. Subsequently, the autoradiographs 

were combined with histological and immunohistological 

analysis of sections [1]. 

Results: According to the autoradiography analysis, 

the radioactivity uptake in atherosclerotic plaques was 

higher compared to healthy vessel wall (ratio 1.8 ± 0.2, 

P = 0.0002) and adventitia (ratio 1.3 ± 0.2, P = 0.0011). 

Some 68Ga-radioactivity was also detected in calcified 

regions of plaques. Autoradiography signal 

co-localized with macrophages as demonstrated by 

Mac-3 immunohistochemistry. In both mice strains, 

the highest level of radioactivity was found in the 

urine and blood. 

Conclusions: We observed a moderate but significantly 

higher 68Ga-chloride uptake in the aortic 

plaques of atherosclerotic mice. While the uptake of 

68Ga-chloride was promising in this animal model, 

the slow blood clearance may limit the usability of 

68Ga-chloride in clinical imaging of atherosclerotic 

plaques. 

Acknowledgement: The study was conducted within 

the Finnish Centre of Excellence in Molecular Imaging 

in Cardiovascular and Metabolic Research, supported 

by the Academy of Finland, University of Turku, Turku 

University Hospital and Åbo Akademi University. 

This work was further funded by the Finnish Foundation 

for Cardiovascular Research, Instrumentarium 

Foundation and the Drug Discovery Graduate School. 

References: 

1. Haukkala J et al; Eur J Nucl Med Mol Imaging. 36:2058– 

2067 (2009) 


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YIA applicant 

72 


Hybrid imaging using dual energy µCT and FMT for characterization of atherosclerotic 

plaques in ApoE -/- mice 

Gremse F. (1) , Rix A. (2) , Bzyl J. (1) , Lederle W. (1) , Schulz R. (2) , Perkuhn M. (3) Schober A. (3) , Weber C. (3) , Kiessling F. (1) . 

(1) Department of Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, 

(2) Chair for Biological Imaging, Technical University Munich, Germany, 

(3) Department of Diagnostic Radiology, Medical Faculty, RWTH Aachen University, 

(4) Institute of Molecular Cardiovascular Research RWTH Aachen University, Germany. 

fgremse@ukaachen.de 

Introduction: To evaluate the ability of combined 

µCT and fluorescence mediated tomography (FMT) 

to characterize early and late stages in atherosclerotic 

plaque formation. 

Methods: Three groups of ApoE -/- mice were 

scanned by dual energy µCT and FMT. Iodine 

based blood pool CT contrast agent and a fluorescent 

probe for active cathepsins were used. CT and 

FMT datasets were fused using automated marker 

detection and registration. Group 1 consisted of 5 

mice with an age of more than 40 weeks fed with 

normal chow. Group 2 included 6 mice (age < 20 

weeks) with 12 weeks of cholesterol diet and group 3 

consisted of 5 mice (age


Long circulating emulsion based ct contrast agents 

De Vries A. (1) , Custers E. (2) , Lub J. (2) , Nicolay K. (1) , Gruell H. (1) . 

(1) Eindhoven University of Technology, The Netherlands 

(2) Philips Research, Eindhoven, The Netherlands 

A.d.Vries@tue.nl 

Introduction: With X-ray and CT moving forward 

into the interventional care, such as stent placement, 

balloon dilatation, vascular surgery, and electrophysiological 

procedures, there is a need for contrast 

agents that have long circulation times [1]. Previously, 

we prepared iodinated emulsion-based CT contrast 

agents that remain in the blood pool for several hours 

[2]. The primary blood clearance occurs via uptake in 

the reticuloendothelial system (RES), which is known 

to be dose dependant. Here, we tested if circulation 

times of these iodinated emulsions can possibly be extended 

by a co-injection with liposomes to slow down 

RES uptake of the contrast agents. We co-injected the 

iodinated emulsion (radiolabeled with 111 Indium) together 

with liposomes (radiolabeled with 177 Lutetium) 

and investigated the biodistribution of iodinated nanoparticles 

and liposomes by in vivo CT and γ-counting. 

Methods: Liposomes were prepared using DPPC, 

cholesterol and DOTA-DSPE (mol ratio 65.7/33.3/1) 

and were radiolabeled with 177 Lutetium. Iodinated 

nanoparticles were prepared using 2% w/w PBD- 

PEO (with 1 mol% PBD-PEO-DOTA) and 20% w/v 

3,7-dimethyloctyl 2,3,5-triiodobenzoate and were 

radiolabeled with 111 Indium. Swiss mice were used 

in the study, in which 2 mL/kg body weight of iodinated 

contrast agent (113 mgI/mL) together with 

2 mL/kg body weight of a) physiological salt (n=5) 

(control) or b) liposomes (n=5) was injected, leading 

to an injected dose of 225 mg I/kg body weight. 

Helical CT scans were acquired pre-injection (reference) 

and up to 3 hours post-injection using a 

dedicated small animal SPECT/CT system (nano- 

SPECT/CT ® , Bioscan). The uptake of 177 Lutetium 

and 111 Indium in dissected organs was measured 

using a γ-counting wizard. 

Results: Fig. 1 shows the Hounsfield Unit (HU) 

increase of blood after the injection of emulsion 

and emulsion together with liposomes as a function 

of time. The blood half life increased by 40 

minutes to t ½ =116 min when liposomes were coinjected 

(t ½ =77 min for pure emulsion; blood half 

time was derived from a single-exponential fit). 

The CT and biodistribution data (γ-counting) 

showed that when co-injecting liposomes, the 

emulsion particles are taken up in a lower degree 

by the spleen as compared to the control group. 

Conclusions: Circulation times of the iodinated 

emulsions as CT contrast agents can be further 

extended when co-injecting liposomes. The liposomes 

are likely taken up by the RES system, 

thereby slowing down the clearance of the iodinated 

emulsion in a dose dependant manner. As 

liposomes are well tolerated with little toxicity at 

high doses, the above strategy can be exploited to 

formulate well tolerated long circulating iodinated 

contrast agents without increasing the administered 

iodine dose. 

Delta Hounsfield Units 

120 

100 

80 

60 

40 

20 

References: 

Emulsion 

Emulsion + Liposomes 

0 

0 25 50 75 100 125 150 175 200 

Time (min) 

1. Vera DR, Mattrey RF; Acad Rad. 9(7):784-792 (2002) 

2. de Vries, A. et al. Biomaterials, submitted (2010) 


YIA applicant 

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74 


Application of microfluidics to the ultra-rapid preparation of fluorine-18 and carbon-11 

labelled compounds 

Miller P. . 

Imperial College London, UK 

philip.miller@imperial.ac.uk 

Introduction: The advantages associated with the 

miniaturisation of chemical reactions have been 

recognised for well over a decade now. Such benefits 

include controlled and predicable mixing regimes, 

efficient heat transfer, reduced reagent consumption, 

small reaction volumes (nL-µL), improved safety 

and enhanced processing capabilities. Microfluidic 

reactors, the devices used to perform and process 

small scale chemical reactions, contain enclosed 

microchannels typically 10-500 µm in diameter and 

can be fabricated from a wide range of materials including 

various polymers, silicon and glass (figure 

1). The use of microfluidic reactors for rapid synthesis 

with the short-lived isotopes 11C (t1/2 = 20.4 

min) and 18F (t1/2 = 109 min), commonly used in 

positron emission tomography, has been recognised 

since 2003. Interest in the application of microfluidics 

to PET radiosynthesis has come about primarily 

because miniaturised reaction systems have the 

potential to address several key challenges in PET 

radiochemistry, including increasing the speed, 

imaging life 

reducing the scale and improving the processing of 

radiolabelling procedures.[1,2] This talk will provide 

a succinct and critical overview, to the nonspecialist, 

of the emerging and developing field of 

using microfluidics for the rapid preparation of 11C 

and 18F labelling compounds. 

Acknowledgement: PWM is grateful to the EPSRC 

for the award of a Life Sciences Interface fellowship 

(EP/E039278/1). 

References: 

Figure 1 

1. P. W. Miller, J. Chem. Technol. Biotechnol. 2009, 84, 

309. [2] P. W. Miller, A. J. deMello and A. D. Gee, Curr. 

Radiopharmaceuticals.accepted for publication.


Radioligand for in vivo measuring neurotransmitters release 

Halldin C. (1) , Finnema S. (1) , Varrone A. (1) , Farde L. (1) . 

Karolinska Institutet, Sweden 

christer.halldin@ki.se 

Introduction: This report deals with an overview 

of radioligands useful for in vivo measuring 

neurotransmitter release. In particular this will 

be exemplified by a PET-study evaluating the 

effect of (±)-fenfluramine- induced serotonin 

release on the binding of the selective serotonin 

5-HT1B receptor radioligand [11C]AZ10419369 in 

cynomolgus monkeys. In a classical displacement 

paradigm after bolus administration of radioligand 

fenfluramine caused a dose-dependent reduction in 

specific binding ratios. The aim of this study was to 

confirm our previous findings by using a bolus plus 

continuous infusion approach in monkey. 

Materials and Methods: A total of 18 PET 

measurements were conducted using a bolus plus 

infusion paradigm of [11C]AZ10419369 in three 

cynomolgus monkeys. On six of the nine experimental 

days a baseline measurement was followed by a 

displacement measurement in which fenfluramine 

(1.0 or 5.0 mg/kg) was infused i.v. between 80 and 

85 minutes after the bolus radioligand injection. 

On three of the nine experimental days a baseline 

measurement was followed by a pretreatment 

measurement in which fenfluramine (5.0 mg/kg) 

was infused i.v. between 30 and 25 minutes before 

the bolus radioligand injection. Emission data were 

acquired for 125 or 155 minutes using the HRRT 

PET-system. For determination of the fenfluramine 

effect the binding potential (BPND) was calculated 

for different time frames with the cerebellum as 

reference region. 

Results: Administration of fenfluramine had no 

evident effect on radioactivity in the cerebellum. 

After administration of fenfluramine (1.0 and 5.0 

mg/kg), the respective binding potentials decreased 

in the occipital cortex by 19% (7-34%) and 47% (40- 

57%) in the displacement paradigm, and by 39% 

(32-45%) in the pretreatment paradigm (5.0 mg/kg). 

Conclusions: This study confirms that the 5-HT1Bligand 

[11C]AZ10419369 is sensitive to endogenous 

serotonin levels in vivo. 


day2 

Parallel Session 7: PROBES II - together with EANM

76 


18f-tracers for amyloid plaques 

Guilloteau D. . 

CHRU Tours, INSERM Imaging and Brain, France 

denis.guilloteau@univ-tours.fr 

As documented by the increasing literature, PET 

imaging using probes binding to amyloid plaques 

represents a major opportunity to investigate 

early symptoms and prodromal phase of AD, and 

in other hand to follow up the efficiency of new 

treatment. 

The most well-known tracer used for this molecular 

imaging is the [11C]-PIB. Numerous studies with 

PIB have demonstrated that the properties of this 

tracer permit the visualization and the quantification 

of amyloid deposits, but its [11C] labelling limits 

considerably its potential use due to its short half-life. 

A strong effort have been performed by different 

groups in order to develop a tracer labelled with [18F] 

for amyloid plaques. Several 18F amyloid binding 

compounds belonging to different families have 

been described. Of these 18F-AV-45 (Florbetapir) 

is most advanced in clinical development (end of 

imaging life 

Phase III) but Phase I / II clinical results have also 

been reported on 18F-FDDNP, 18F-Flumetametamol, 

18F-BAY94-9172 (AV-1). We will present these 

results and discuss the similarities and differences 

regarding the specificity and the sensitivity of the 

brain uptake. 

Some results obtained with [18F]-FDDNP and 

([18F]-AV45 in our University Hospital will be 

presented.


Chelators for radiocopper: biological/pharmacokinetic differences of [Tyr 3 ,Thr 8 ]octreotide 

conjugates 

Abiraj K. (1) , Fani M. (2) , Tamma M. L. (1) , Reubi J. C. (3) , Barnard P. (4) , Schibli R. (5) , Dilworth J. (6) , Maecke H. (2) . 

(1) University Hospital of Basel, Switzerland 

(2) University Hospital of Freiburg, Germany 

(3) University of Bern, Switzerland 

(4) University of Melbourne, 

(5) ETH Zurich, 

(6) University of Oxford. 

keelaraa@uhbs.ch 

Introduction: Copper-64 has attracted great deal 

of attention as radioisotope for targeted positron 

emission tomography (PET) and radionuclide therapy 

due to its dual decay characteristics (β+: 17.4%; 

E β+max= 653 keV; β-: 39%; E β-max= 579 keV), 

favorable half life (t1/2=12.7 h) and increased availability.1 

Owing to the instability of copper complexes 

under ‘in vivo’ condition, continuous efforts 

are being made to develop bifunctional chelators 

(BFC’s) which can form stable copper complexes for 

labeling of biomolecules.1-3 In the present study, 

we conjugated [Tyr3,Thr8]octreotide (TATE), to 

four different chelating systems, radiolabeled with 

64Cu and evaluated the biological/pharmacokinetic 

differences of these radioconjugates. 

Methods: The BFC’s DOTA(tBu)3 and 

NODAGA(tBu)3 are commercially available whereas 

CB-TE2A(tBu) and PHENDAM were synthesized 

using suitable synthetic methodologies. TATE was 

synthesized on solid phase using standard Fmoc 

strategy and coupled to each BFC. The conjugates 

were labeled with 64Cu using 0.1 M ammonium 

acetate buffer (pH 8.0) under different labeling 

conditions. Receptor affinity measurements were 

performed using radioligand assays and autoradiographic 

methods. The radiolabeled conjugates were 

evaluated in vitro and in vivo in tumor-bearing nude 

mice, using the HEK-sst2 cell line. Imaging studies 

were performed using a clinical PET/CT camera. 

Results: Synthesis of orthogonally protected crossbridged 

cyclam-based BFC (CB-TE2A(tBu)) allows 

facile conjugation to targeting biomolecules such 

as peptide on solid phase. Synthesis of PHENDAM 

provides a new N4-macrocyclic ligand for conjugation 

to biomolecule and subsequent labeling with 

64Cu. All the conjugates (DOTA-TATE, NODAGA- 

TATE, PHENDAM-TATE and CB-TE2A-TATE) 

showed high binding affinity to somatostatin receptor 

subtype-2 (sst2) with IC50 values ranging from 

0.6 nM to 2.5 nM. The labeling of NODAGA-TATE 

could be performed at room temperature (radiolabeling 

yield >97% at specific activity of >20 GBq/ 

µmol) whereas other conjugates required elevated 

temperature. All the radioconjugates showed substantially 

high and receptor mediated uptake by 

HEK-sst2 cells. Biodistribution studies with nude 

mice showed high uptake of the radioconjugates in 

HEK-sst2 xenografts at 1h p.i. (64Cu-DOTA-TATE: 

20.29±2.74%, 64Cu-NODAGA-TATE: 29.39±4.13%, 

64Cu-PHENDAM-TATE: 16.97±1.46%, and 64Cu- 

CB-TE2A-TATE: 19.34±2.55% ID/g). The radioactivity 

in tumor persisted at 4h p.i., but 24h p.i. data 

showed fast washout in case of all the conjugates. 

Compared to 64Cu-DOTA-TATE, the other three 

radioconjugates showed improved pharmacokinetics. 

High in vivo stability of radiometal complex in 

case of 64Cu-NODAGA-TATE and 64Cu-CB-TE2A- 

TATE was apparent from their low uptake in liver 

and blood at all time points. Interestingly, 64Cu- 

PHENDAM-TATE showed fast washout from the 

kidneys resulting in high tumor-to-kidney ratio at 

early time points. The PET/CT images acquired at 

8 h p.i. clearly delineated the tumor and showed images 

in accordance with the biodistribution data. 

Conclusions: Among four different chelating systems 

evaluated, 64Cu-NODAGA-TATE and 64Cu- 

CB-TE2A-TATE showed superior in vivo stability 

of the radiocopper complex. The study infers that 

triazacyclononane and cross-bridged cyclam based 

chelating systems might be ideal for 64Cu labeling 

of biomolecules intended to be used in targeted PET 

imaging and radionuclide therapy but PHENDAM 

may be a new 64Cu chelating system of interest for 

protein labeling. 

Acknowledgement: Swiss National Science Foundation, 

European Molecular Imaging Laboratories 

(EMIL), COST BM0607. 

References: 

1. Shokeen W et al; J. Acc. Chem. Res. 42: 832-841 (2009) 

2. Boswell C A et al; J. Med. Chem. 47: 1465-1474 (2004) 

3. Prasanphanich A F et al; PNAS 104: 12462-12467 

(2007) 


YIA applicant 

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78 


[ 11 C]SOMADAM: potential sert ligand for pet studies and comparison with [ 11 C]MADAM 

Gourand F. (1) , Emond P. (2) , Bergström J. P. (3) , Takano A. (3) , Gulyas B. (3) , Guilloteau D. (2) , Barré L. (1) , Halldin C. (3) . 

(1) CEA/DSV CI-NAPS, France 

(2) INSERM U930-Université François Rabelais de Tours CHRU Bretonneau, France 

(3) Karolinska Institutet, Sweden 

gourand@cyceron.fr 

Introduction: Although [ 11 C]MADAM is an 

appropriate PET radioligand for the visualization 

and quantification of SERT in vivo, metabolite 

analysis in human and non-human plasma samples 

using HPLC separation have shown that [ 11 C] 

MADAM was rapidly metabolized. 1,2 

A possible metabolic pathway is the S-oxidation 

which could lead to SOMADAM and SO 2 MADAM. 

In vitro evaluation of these two potential metabolites 

have shown that SOMADAM exhibited a good 

affinity for SERT and a good selectivity for SERT 

over NET and DAT. 3 In view of its in vitro biological 

properties, SOMADAM has been labelled with 

carbon-11 and evaluated as a potential radioligand 

for in vivo quantification of SERT in the monkey 

brain using PET. 

Methods: The labeling of [ 11 C]SOMADAM is based 

on the N-alkylation reaction of the N-desmethyl 

precursor using [ 11 C]methyl triflate in acetone. 

Comparative PET imaging studies in cynomolgus 

monkey with [ 11 C]MADAM and [ 11 C]SOMADAM 

were carried out and plasma samples were analyzed 

using reverse phase HPLC. 

Results: The incorporation of [ 11 C]methyl triflate to 

[ 11 C]SOMADAM was in the range of 50% and after 

purification by reverse phase HPLC, [ 11 C]SOMADAM 

was obtained with a radiochemical purity higher 

than 99%. PET imaging studies in monkey using 

[ 11 C]SOMADAM showed 1) a rapid and high brain 

uptake and 2) an homogenous distribution of the 

imaging life 

radioactivity. A rapid washout in all regions was 

observed, indicating a high non-specific binding. As 

it has already been shown in previous PET studies, 

the radioactivity accumulation of [ 11 C]MADAM is 

consistent with the known densities of SERT sites. 

HPLC analysis of plasma samples obtained after 

either [ 11 C]MADAM or [ 11 C]SOMADAM injection 

gave radiochromatograms with a similar profile 

where all the radioactive metabolites detected were 

more hydrophilic than the parent compound. After 

[ 11 C]MADAM injection, one radioactive species has 

been identified as [ 11 C]SOMADAM and the fraction 

was approximatively 5% at 4 min and 1% at 15 min. 

Conclusions: From PET imaging studies in 

monkey, it appears that [ 11 C]SOMADAM does not 

present any advantages over [ 11 C]MADAM, either 

in terms of brain kinetics and specific binding. 

Nevertheless, [ 11 C]SOMADAM has been identified 

in plasma sample as a minor labeled metabolite of 

[ 11 C]MADAM. 

References: 

1. Halldin C et al; Synapse. 58:173-183 (2005) 

2. Lundberg J et al; J Nucl Med. 46:1505-1515 (2005) 

3. Vercouillie J et al; Bioorg Med Chem Lett. 16:1297- 

1300 (2006)


MR imaging of extracellular redox by a thiolsensitive Gd(III)-DO3A derivative 

Menchise V. (1) , Gianolio E. (2) , Digilio G. (3) , Cittadino E. (2) , Napolitano R. (2) , Fedeli F. (2) , Catanzaro V. (2) , Aime S. (2) . 

(1) Institute for Biostructures and Bioimages (CNR) c/o Molecular Biotechnology Center University of Turin, Italy 

(2) Department of Chemisty IFM & Center for Molecular Imaging, University of Turin, Italy 

(3) Department of Environmental and Life Sciences, University of Eastern Piedmont “A. Avogadro”, Alessandria, Italy 

valeria.menchise@unito.it 

Introduction: The characterization of the microenvironment 

around and within tumors is of great 

importance to evaluate the invasiveness of cancers 

and to predict resistance to radio- and chemotherapy. 

Severe hypoxia and acidosis has been related 

to aggressive cell phenotype, poor clinical outcome 

and low likelihood of patient survival. 1 More reducing 

microenvironment conditions are related 

to increased cancer cell proliferation and increased 

resistance to chemotherapies. Any method for the 

imaging of the parameters characterizing the tumor 

microenvironment would be therefore of immense 

value for the characterization/grading of the tumor 

and for the choice and calibration of therapies. We 

have recently described a series of Gd-DO3A based 

compounds that exploit exofacial protein thiols 

(EPTs) to be taken up by cells. The most effective 

of these compounds, named Gd-L1A, can reach an 

intracellular concentration as high as 1.2x10 10 Gd 

atom per single cell. 2 The fact that the uptake of Gd- 

L1A by cells is proportional to the amount of EPTs 

makes it an interesting candidate as a probe for the 

MR molecular imaging of the extracellular redox 

microenvironment. 

Methods: In labeling experiments, cells grown to a 

confluence of 80% were subjected to a 4 hours incubation 

with Gd-L1A (0.5 to 3mM) at 37 °C, washed 

three times, mechanically harvested and finally subjected 

to mineralization and quantitative analysis of 

Gd(III) by a relaxometric assay. MR images were acquired 

on mice grafted with B16 tumor before and after 

intratumor/intramuscle injection of Gd-L1A using 

a T1-weighted multislice multiecho protocol (TR/TE 

250/7.7) on an ASPECT spectrometer operating at 1T. 

Results: Uptake experiments of GdL1A carried 

out on B16 melanoma cells treated with chemicals 

known to block EPTs have shown that the extent of 

Gd uptake increases with the concentration of free 

EPTs on the cell membrane. Imaging of EPTs has 

been performed in an animal tumor model obtained 

by inoculating about 1 million of B16 melanoma 

cells subcutaneously in B57Bl/6 mice. Gd-L1A has 

been delivered to tumor areas in these mice models 

and signal enhancement monitored over time and 

compared to that found in non-tumor bearing tissue. 

Gd-DO3A has been used as a control. 

A significant signal enhancement has been found in 

the tumor area of mice treated with Gd-L1A, with 

wash-out kinetics of the contrast agent consistent 

with its internalisation by tumor cells. 

Conclusions: These results show that Gd-L1A is 

suitable to image EPTs, that are in turn sensitive and 

responsive to the extracellular redox. 

Acknowledgement: This work is supported by FP6 

Project NMP4-CT-2006-026668 (MediTrans) and by 

Regione Piemonte (PIIMDMT) 

References: 

1. Tatum et al. Int J Radiat Biol., 82: 699-757 (2006) 

2. Digilio G. et al., Chem. Commun. 893–895 (2009) 


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80 


Everything but not cell replacement with somatic neural stem cell transplantation 

Pluchino S. . 

San Raffaele Scientific Institute Milano, Italy 

pluchino.stefano@hsr.it 

Introduction: Since the first transplant of stem cells 

into the spinal cord of rodents in which an acute demyelinating 

lesion was induced, we have witnessed 

a spur of experimental cell-based transplantation 

approaches aimed at fostering biological and molecular 

mechanisms underlying CNS repair. Theories 

assuming that very little renewing potential is 

identified within the adult CNS have been contravened, 

new promising sources of myelinogenic cells 

for transplantation purposes have been characterized, 

and new cell-replacement strategies have been 

proposed and established. A better understanding of 

the dynamics of endogenous remyelination has been 

achieved, and insights concerning the process of remyelination 

driven by site-specific myelin-forming 

cell transplantation have been discovered. 

Methods: Some major limitations have – however – 

not been overcome yet: 

(i) the limited amount of highly myelinating cells 

that can be grown in vitro and 

(ii) the limited migratory capacity of myelinating 

cells once transplanted. Somatic stem cells might 

represent therefore an alternative and promising area 

of investigation with some potential in its essence. 

Results: New hopes have been recently raised by the 

encouraging preliminary results obtained by transplanting 

CNS-derived NPCs and bone marrow mesenchymal/stromal 

stem cells in rodents with experimental 

MS. However, most of the results with stem 

cells as therapeutic weapons for MS have consistently 

challenged the sole and limited view that stem cells 

therapeutically work exclusively throughout cell replacement. 

Indeed, the transplantation of somatic 

(non-hematopoietic) stem cells promotes substantial 

CNS repair via a number of bystander mechanisms, 

mainly exerted by undifferentiated stem cells releasing 

in vivo a milieu of tissue-trophic and immune 

modulatory molecules, whose release is likely to be 

temporally and spatially orchestrated by specific 

(micro)environmental cues. These molecules are indeed 

are pleiotropic and redundant in nature as well 

as ‘constitutively’ secreted by stem cells. In this view, 

the therapeutic plasticity of stem cell can be viewed 

imaging life 

as the capacity of stem cells to adapt their fate and 

function(s) to specific environmental needs occurring 

as a result of different pathological conditions. 

Conclusions: While further studies are certainly required 

to assess the overall safety, efficacy and in vivo 

therapeutic plasticity of NPCs, the great challenge for 

any future human application of NPC-based protocols 

in MS will be to develop more reliable and reproducible 

approaches optimizing both (tissue) trophic 

as well as immune regulatory capacities of stem cells 

for functional and anatomical rescuing of myelin architecture 

in MS patients. 

Detailed review of the most recent data on the highly 

peculiar immune regulatory stem cell signature will 

be here provided.


Mesenchymal stem cell transplantation: an option to promote remyelination? 

Aigner L. . 

Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg, Austria 

ludwig.aigner@pmu.ac.at 

Mesenchymal stem cells (MSCs) have been 

used previously in a number of animal models 

for neurological / neurodegenerative diseases. 

Moreover, clinical studies that are using MSCs for 

therapy of CNS diseases are currently ongoing. In 

animal models, systemic or local transplantation 

of MSCs provided functional improvement. The 

therapeutic effects of MSCs are derived from their 

immuno-modulatory activity and from the fact 

that MSCs secrete a number of neuroprotective 

cytokines. Moreover, there is evidence that MSCs 

might be able to influence the pool of endogenous 

stem or progenitor cells. Along this line, we have 

recently demonstrated that MSCs secrete activities 

that promote oligodendroglial fate and differentiation 

of neural progenitors (Rivera et al., 2006). 

Here, we will present data on the effects of MSC 

transplantation on endogenous spinal cord 

progenitors after spinal cord injury. Like in the 

in vitro situation, the presence of MSCs in the 

lesioned spinal cord promoted oligodendroglial and 

inhibited astroglial differentiation of endogenous 

progenitors. This supports the possibility of use 

MSC transplantation for strategies to remyelinate 

axons in the diseased CNS. 


day2 

Parallel Session 8: GENE and CELL based therapies - together with CliniGene

82 


Viral vector-mediated transcriptional targeting of dendritic cells for antigen-specific 

tolerance induction in EAE/MS. 

Dresch C. . 

Univesity of Zürich, Switzerland 

cdresch@vetvir.uzh.ch 

Introduction: The cause of multiple sclerosis (MS) 

is unknown and the pathogenic processes leading 

to disease development is incompletely understood. 

Current knowledge supports a T cell 

mediated autoimmune pathogenesis targeting myelin 

components or myelin-producing cells. Immunization 

of susceptible animals with myelin 

antigens or transfer of myelin antigen-reactive T 

cells induces experimental autoimmune encephalomyelitis 

(EAE), an inflammatory disorder of 

the CNS which closely resembles MS. Because the 

ethiology of MS is not yet completely understood, 

there is no curative treatment available at present. 

Methods: The aim of this project was to induce permanent, 

antigen-specific tolerance in EAE/MS. The 

strategy includes the ex vivo modification of autologous 

hematopoietic stem cells (HSC) with lentiviral 

vectors that express antigens involved in EAE/MS 

from a dendritic cell-specific promoter. After reinfusion, 

the modified HSC will give rise to all cells 

of the immune system including antigen expressing 

dendritic cells. As lentivirus vectors mediate the genomic 

integration of transgenes in HSC, there is a 

constant supply of antigen expressing “steady-state” 

dendritic cells. We hypothesized that the stable 

antigen presentation by these cells in thymus and 

periphery in a non-inflammatory condition would 

tolerize self-reactive T cells and, therefore, prevent/ 

revert disease development. 

Results: We demonstrated the effectiveness of this 

strategy for inducing myelin oligodendrocyte glycoprotein 

(MOG)-specific tolerance in an EAE 

model in mice. We show the efficient deletion of 

MOG specific T cells in chimeras that received HSC 

transduced with a MOG-expressing lentivirus vector. 

Also, 0% of mice which received HSC transduced 

with the MOG-expressing lentivirus vector 

developed EAE upon induction (clinical score 0), 

while 100% of mice that received BM cells transduced 

with a GFP-expressing control lentivirus vector 

developed EAE. 

Conclusions: We confirmed the potential of lentivirus 

vectors that transcriptionally target transgene 

imaging life 

expression to dendritic cells for antigen-specific 

tolerance induction in autoimmune diseases. This 

strategy completely prevented MOG induced EAE 

in mice. We will further investigate the mechanisms 

of vector mediated tolerance induction, in particular 

the involvement of regulatory T cells and cytokines. 

The strategy presented here is particularly promising 

for clinical applications. Moreover, these tools 

will also prove useful for studying disease mechanisms 

and for addressing fundamental questions in 

immunity and tolerance. 

Acknowledgement: Swiss-MS Society.


Delivery of a bioluminescent transgene to a tumor via bone marrow engraftment and local 

control of gene expression by non invasive local hyperthermia 

Fortin P. Y. (1) , Lepetit-Coiffe M. (1) , Genevois C. (1) , Debeissat C. (1) , Quesson B. (1) , Moonen C. (1) , Couillaud F. (1) . 

(1) University of Bordeaux/CNRS, France 

py.fortin@imf.u-bordeaux2.fr 

Introduction: The success of a gene therapy strategy 

depends on several parameters including the 

right choice of therapeutic gene, efficient delivery 

method and reliable control of gene expression. In 

the present study, we investigate an in vivo strategy 

to deliver a transgene around a tumor and to control 

the expression of the transgene. This strategy 

includes bone marrow engraftment of genetically 

engineered cells into a wild type mouse to create 

a chimera with nucleated circulating blood cells 

(CBC) expressing the firefly luciferase (lucF) reporter 

gene under the transcriptional control of a 

thermosensitive promoter. Later on, a subcutaneous 

tumor is induced and as part of the physiological 

inflammatory process, CBC accumulate into 

and around the tumor. Finally, lucF expression 

detected by bioluminescence imaging (BLI) is 

induced “on demand” by local hyperthermia using 

MR guided high-intensity focused ultrasound 

(MRgHIFU). 

Methods: Bone marrow cells (BMCs) were obtained 

from homozygote C57/BL6 (CD45.2) transgenic 

mice (NLF-1) containing the lucF transgene under 

transcriptional control of the heat shock protein 70 

(Hspa1b) promoter. BMCs were transplanted into 

a congenic mouse (CD45.1) pre-treated with Busilvex®, 

an injectable form of busulfan (2 busulfan IP 

injections, 25 mg/Kg) to induce medullar aplasia. 

The level of engraftment was measured 2 months 

later by measuring CD45.1/CD45.2 ratio using Fluorescence 

Activated Cell Sorting (FACS). Carcinoma 

Mouse Tumor 93 (CMT-93, ATCC) were implanted 

subcutaneously (2 millions cells) to generate a tumor 

on the left leg. Tumors were heated (44°C, 8 

min) for local gene activation using either a water 

bath or MRgHIFU. LucF expression was evaluated 

in vivo 6 hours post heating by BLI. 

Results: After 2 months, about 80% of engrafted 

mice exhibited more than 65% of CBC from the 

donor mouse as demonstrated by FACS. CMT-93 

cells implanted into engrafted mice formed tumors 

ranging from 5 and 10 mm in one month. Tumors 

heated (44°C, 8 min) by dipping the tumor-bearing 

leg into a water bath, in mice exhibiting more 

than 65% of engraftment, induced lucF activation 

and transient light emission 6 hours later. Light 

emission was found in and around the tumor, for 

about 50% of the mice. The remaining mice did 

not exhibit any light emission. A week later lightemitting 

mice did not produce light anymore. 

They were heated again (44°C, 8 min) but using 

the MRgHIFU device. Six hours later, light emission 

was found around the heated focal region corresponding 

to both tumors and the surrounding 

region (n = 15). Light emission was also detected 

in an additional location (n = 6) corresponding to 

head and apical region of bone both on ipsi and 

contralateral legs. 

Conclusions: The bioluminescent chimera mice 

express the lucF reporter under transcriptional 

control of a thermosensitive promoter in hematopoietic 

cells. This model allows for studying 

gene delivery to tumor using circulating blood cells. 

Local gene expression was induced “on demand” 

by local hyperthermia. MRgHIFU heating reveals 

both expected (inside and around the tumor) and 

unexpected (contralateral leg) activation patterns 

further to be explored. 

Acknowledgement: This work was supported in 

part by Diagnostic Molecular Imaging and the 

Conseil Régional d’Aquitaine. 


day2 


YIA applicant 

84 


Dendritic cell labelling with paramagnetic nanoparticles and 111 In-oxine for in vivo magnetic 

resonance imaging and scintigraphic imaging 

Martelli C. (1) , Borelli M. (1) , Rainone V. (1) , Ottobrini L. (1) , Degrassi A. (2) , Russo M. (2) , Texido G. (3) , Pesenti E. (2) , Fiorini C. (4) , 

Clerici M. (5) , Trabattoni D. (1) , Lucignani G. (1) . 

(1) University of Milan , 

(2) Nerviano Medical Sciences, 

(3) Pharmacology Department, BU Oncology, Nerviano Medical Sciences, Nerviano, MI, 

(4) Politecnico of Milan, 

(5) University of Milan and on Gnocchi Foundation. 

cristina.martelli@unimi.it 

Introduction: Better understanding of the biology 

and the role of dendritic cells (DCs) in regulating 

immune responses is driving the development of innovative 

anti-neoplastic DC based immunotherapies 

both at clinical and pre-clinical levels [1] . The aim of 

this study is the development and the evaluation of a 

tumour-specific DC vaccine, tested on a transgenic 

murine model of breast cancer (MMTV-v-Ha-Ras). 

MRI and SPET methodologies were tested for their 

feasibility in showing the migration to local draining 

lymph nodes (DLNs) of DC, properly labelled 

with paramagnetic nanoparticles (MNPs, Endorem ® ) 

or 111 In-oxine [2] . 

Methods: Total bone marrow cells were extracted 

from wt mice. DC differentiation was studied by 

flow cytometry; at the 6 th day of culture DCs were 

labelled with commercial MNPs (200 ugFe/ml, for 

16h). Labelling efficiency was checked by optical 

microscopy after Perl’s staining and relaxometric 

analysis. Tumour lysates from breast cancer lesions 

of transgenic mice were used to load immature DCs 

(iDCs), and maturation was monitored by flow cytometry. 

Stimulatory activity of Ag-loaded DCs and 

migratory ability were evaluated in vitro. MNP labelled 

and Ag loaded DCs were then injected into 

the footpad of a transgenic tumour bearing mice. 

The same cells were labelled after antigen loading 

with 111 In-Oxine (30 uCi/10 6 cells). MRI and SPET 

imaging were performed at 4, 24 and 48h after cells 

injection. MRI was performed on a 7T Bruker Pharmascan 

instrument, and MSME and FLASH sequences 

were used. SPET imaging was carried out 

with a new prototype of gamma camera developed 

within an European Project by Politecnico of Milano. 

Ex vivo Perl’s staining and gamma counting of 

explanted DLNs was performed to validate imaging 

data and DC migration. 

Results: Perl’s staining showed constant iron content 

of DCs in all the experiments. Mean Iron 

content was 240 pg/cell. DCs labelling with 111 Inoxine 

showed a very high efficiency (83%). Vitality 

was not significantly affected by both labelling 

strategies. Labelling with MNPs did not affect DC 

imaging life 

immune-phenotype or functionality, as demonstrated 

by CD86 and CD83 expression levels, T-cell proliferation 

and INF-γ production. Migration assays 

showed that Ag-loaded DCs were able to migrate 

in the presence of stimulatory chemokines (6Ckine 

and MIP3β). MRI evidenced the presence of an hypointense 

signal in both axillary and popliteal LN, 

in relation to the injection site, 4h after cell injection. 

Signal was visible up to 24 h after DC injection. 

Perl’s staining of LN sections after in vivo injection 

of labelled DCs loaded with the antigens, showed 

the presence of iron within the node, indicating that 

mature and labelled DCs migrate in vivo from the 

site of injection to the DLN. SPET imaging demonstrated 

as well DCs migration from the injection 

site to the DLN. Ex vivo analysis of different organs 

showed a statistically significant higher signal within 

the LN omolateral to the injection site in comparison 

with the controlateral one. 

Conclusions: Labelling protocols do not perturb 

DC physiology and functionality. Dynamic in vivo 

MRI and SPET imaging monitoring of DCs distribution 

will shed light on the fundamental parameters 

responsible for anti-neoplasic efficacy [3] , while 

the use of clinically approved MNPs and tracers will 

speed up the transfer from pre-clinical studies to 

clinical trials. 

Acknowledgement: this work is supported by the FP6 

funded Hi-CAM project (LSHC-CT-2006-037737), 

PRIN (20082NHWH9) and AIRC (IG2009-9311) 

and by fellowships from the Doctorate School of 

Molecular Medicine, University of Milan. 

References: 

1. de Vries IJ et al. Nature Biotech, 2005,23(11):1407- 

1413 

2. Baumjohann D et al. Eur. J. Immunobiology 2006; 36: 

2544-2555 

3. Lucignani G et al. TRENDS in Biotechnology 2006; 

24(9): 410-418


The type 2 cannabinoid receptor as a new PET reporter gene for the brain 

Vandeputte C. (1) , Evens N. (1) , Toelen J. (1) , Deroose C. (1) , Ibrahimi A. (1) , Verbruggen A. (1) , Debyser Z. (1) , Bormans G. (1) , 

Baekelandt V. (1) , Van Laere K. (1) . 

(1) K.U. Leuven, Belgium 

caroline.vandeputte@med.kuleuven.be 

Introduction: Reporter genes play an important role 

in the understanding of gene expression and function 

in living subjects. However, for the brain no 

successful PET reporter systems are available with 

low endogenous background gene expression and 

good blood-brain-barrier (BBB) penetration of the 

PET probe. The aim of this study was to develop 

a new PET reporter gene system which can be applied 

to the brain. The type 2 cannabinoid receptor 

(CB 2 ) has a very low brain expression in physiological 

conditions and CB 2 PET radioligands crossing 

the BBB were recently developed. 

Methods: We constructed lentiviral (LV) and 

adeno-associated viral vector (AAV) transfer plasmids 

encoding human CB 2 , harboring a point mutation 

at position 80 (D80N) referred to as CB 2 (D80N), 

as such or in combination with enhanced green fluorescent 

protein (eGFP) or firefly luciferase (FLuc). 

Rats were stereotactically injected with either 5 µl of 

AAV-eGFP-T2A-CB 2 (D80N) in the right striatum 

and 5 µl of AAV control vector in the left striatum 

or 5 µl of AAV-fLuc-T2A-CB 2 (D80N) in the right 

striatum. At different time points (6, 13, 18, 73, 96 

and 252 days) after stereotactic injection of the 

AAV, a CB 2 selective carbon-11 labeled radioligand 

[ 11 C]GW405833 was injected intravenously and 

dynamic µPET images (Focus 220, Siemens) were 

acquired. BLI scans were performed at 16, 58 and 

281 days after surgery. Time-activity curves (TAC) 

and parametric binding potential maps were determined. 

The animals were sacrificed and perfused 

and double immunohistochemical staining against 

CB 2 and eGFP of the brain slices was performed. 

Results: The observed CB 2 binding potential increased 

over time and reached a maximum in right 

striatum between 18 and 58 days after vector injection 

in the brain. The time-activity curves persistently 

expressed an increased uptake in right 

striatum compared to control left striatum and 

cerebellum. Immunohistochemical analysis showed 

colocalization of both CB 2 and eGFP in right striatum. 

In contrast, only eGFP expression was seen in 

the contralateral hemisphere. 

Conclusions: We have successfully developed a new 

PET reporter gene system consisting of a lentiviral 

or an adeno-associated viral vector expressing 

the CB 2 receptor as the reporter gene which can be 

quantified for several months. 

Acknowledgement: We gratefully acknowledge the 

financial support by the European Commission for 

EC-FP6-STREP-STROKEMAP, BRAINSTIM SBO- 

IWT-060838, DIMI LSHB-CT-2005-512146 and 

the K.U. Leuven Center of Excellence ‘MoSAIC’ 

(Molecular Small Animal Imaging Center). 


YIA applicant 

day2 


86 


Department of Nuclear Medicine , Klinikum rechts der Isar 

Technische Universität München, Ismaninger Straße 22 

D-81675 München, Germany 

Phone: +49-(0)89-4140-4586, Fax: +49-(0)89-4140-4841 

Graduation: 

1981-1985 Study of Chemistry, University of Cologne 

1992-1996 Diploma and PhD, Radiochemistry, 

Res. Center Juelich, Germany 

1995-1997 Scientific Assistant, Department of Nuclear Medicine, 

Technische Universität München (TUM) 

1997-2004 Research Associate, Department of Nuclear Medicine (TUM) 

Academic Appointments 

since 2003 five national and international offers (W3 Radiopharm. Chem. , C4 Bioinorg. and Radiopharm. 

Chem., C3 Radiopharm. Chem., Assoc. Prof. for Radiology, Assoc. Prof. for Radiat. Oncol.). Since 2004 

Professor for Radiopharmaceutical Chemistry at the TU München, Faculty of Medicine) 

Scientific Focus 

Development of specific molecular probes for the non-invasive imaging of diseases, new strategies for 

production of radiopharmaceuticals, radionuclide therapy, tissue selective targeting of imaging probes and 

therapeutic compounds, transfer of genomic and proteomic information into new imaging methods. 

imaging life 

Hans-Jürgen Wester

Molecular imaging of CXCR4 receptors 


Wester H.J. (1) , Demmer O. (2) , Dijkgraaf I. (1) , D´Alessandria C. (1) , Kessler H. (2) . 

(1) Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany 

(2) Institute for Advanced Study, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany 

H.J.Wester@lrz.tu-muenchen.de 

Chemokines play a key role in tumor metastasis and are 

also involved in tumor growth. The receptor subtype 

CXCR4 has been found to be highly expressed in >30 

different types of cancer. Similar to the homing of hematopoietic 

progenitor and stem cells, the chemokine 

receptor subtype CXCR4 and its endogenous ligand, 

the stromal cell derived factor 1-alpha (SDF1-alpha or 

CXCL12), have been found to be responsible for “hijacking” 

circulating tumor cells in organs and tissues 

expressing CXCL12. In primary tumors, expression 

and binding of CXCL12 to CXCR4 promotes tumor 

proliferation and activates cell migration. In addition, 

CXCL12 induces recruitment of progenitor cells, which 

allows for tumor angiogenesis. It has been demonstrated 

that CXCR4 gene expression in mammary cancer 

cells is coregulated by Her2/neu, and also activated by 

HIF-1-alpha, implicating hypoxia-induced metastasis. 

A high CXCR4 density has also been reported for cancer 

stem cells. Consequently, CXCR4 directed therapies 

are currently under development aiming to inhibit 

tumor growth and metastasis by blocking CXCL12 

binding to CXCR4 using antibodies, large and small 

peptides as well as small molecules. CXCR4-directed 

PET probes should be useful tracers for monitoring 

those CXCR4-directed therapies in the future, i.e. for 

patient selection, therapy planning and monitoring 

response and may also independently allow for imaging 

the ´metastatic potential` of primary tumors. 

Antibodies, radiolabeled SDF, medium sized peptides 

and newly developed cyclic CXCR4 peptides antagonists 

are currently assessed as probes for monitoring 

CXCR4 expression or CXCR4 targeted therapies 

using metastasizing tumor models in rodents. 

With the aim to develop suitable radiolabeled probes to 

image CXCR4 receptor expression, we initially started 

with the evaluation of 4-[ 18 F]Fluorobenzoyl-TE14011- 

Me. Initial experiments showed unfavourable in vitro 

characteristics and low labelling yields. Using a similar 

approach by Hanoka et al (2006), Ac-TZ14011 

(Acetyl-Arg 1 -Cit 6 -Arg 7 -Lys 8 (In-DTPA)-T140-amide), 

labelled with In-111, showed high uptake in liver and 

kidney of tumor bearing mice reached, whereas tumor 

uptake was only negligible. 

Exploiting our experience in the development of small 

peptides, i.e. on cyclic RGD- pentapetides as high affinity 

ligand for the alpha(v)beta(3) integrin, we focussed 

our research on small CXCR4-binding pentapeptides. 

We have synthesized approximately 150 

monomeric and dimeric peptides and evaluated their 

binding characteristics on Jurkat cells and transfected 

CMS5 cells stably expressing the hCXCR4 receptor. 

Selected candidates were labelled with 123,125 I-iodine, 

18 F-fluorine and 68 Ga-gallium and investigated in nude 

mice bearing CMS5/CXCR4+ fibrosacoma and metastasizing 

OH-1 SCLC tumors. Based on c(Gly-D-Tyr- 

Orn-Arg-NaI), 4-fluorobenzoylated and 2-fluoropropionylated 

versions showed IC 50 of 11±2 nM and 35±1 

nM ( 125 I-CXCL12 as competitor), and radioiodinated 

c(Gly-D-Tyr-Arg-Arg-NaI) exhibited an IC 50 of 4nM. 

We could demonstrate that N-methylated peptides 

showed an improved IC 50 when compared to the nonmethylated 

analogues. A variety of linkers were investigated 

allowing for conjugation of chelators without 

affecting the affinity of the entire molecule. The 

DOTA-Ahx-Asp (Ahx= aminohexanoic acid) conjugate 

of a cxclic D-Orn-peptide version showed an IC 50 

of 38nM (with free chelator) and 13.7nM after complexation 

with indium. A variety of dimers were synthesized, 

the linkers and linker length optimized and 

the generated constructs assessed. C4-C16 dicarbonic 

acid spacers showed a continuously increasing affinity 

in the series C4 (6.6nM) – C10 (2.3nM), whereas 

longer spacers were unsuitable. The first of a series of 

promising 68 Ga-labeled peptides is renally excreted 

leading to high tumor-to-background contrast and 

high contrasting small animal PET-imaging approx. 

1h post injection. 

Compared to the other CXCR4-probes described, 

the small cyclic peptides developed offer significant 

advantages and will allow for broadening state-ofthe-art 

high contrast peptide receptor imaging to 

another important class of GPCRs. 


day2 

ESMI Plenary Lecture 4 by Hans-Jürgen Wester

88 


Evaluation of the temporal window for drug delivery following ultrasound mediated 

membrane permeability enhancement 

Yudina A. (1) , Lepetit-Coiffé M. (1) , Moonen C. (1) . 

Laboratoire IMF CNRS UMR 5231 / Université Bordeaux 2, France 

anna@imf.u-bordeaux2.fr 

Introduction: Ultrasound (US)-mediated delivery 

is a new therapeutic option to locally facilitate the 

passage of drugs through cell plasma membrane 

by reversibly altering its permeability[1] possibly 

via the formation of pores that are known to spontaneously 

reseal within a short time – seconds to 

minutes[2]. However under certain conditions the 

effects of US for drug delivery last for hours after 

the exposure[3]. The present work is the first in 

vitro attempt to confirm and quantitatively assess 

the temporal window for the US-mediated intracellular 

drug delivery by live-cell imaging. 

Methods: Cell-impermeable optical chromophores 

with fluorescence intensity increasing 100-1000 

fold upon intercalation with nucleic acids served 

as smart agents for reporting cellular uptake. Opticell 

chambers with a monolayer of C6 cells were 

subjected to ultrasound in the presence of microbubbles 

followed by varying delays between 

0 and 24 hours before addition of Sytox Green 

optical contrast agent. Micro- and macroscopic 

fluorescence imaging was used for qualitative and 

quantitative analysis. 

Results: Strong enhancement of the fluorescence 

signal upon binding to nucleic acids allowed efficient 

visualization of the local effect of US on internalization 

of cell-impermeable intercalating dyes. 

Up to 25% of viable cells showed uptake of contrast 

agent with a half time of 8 hours, with cellular uptake 

persisting even at 24 hours (Figure 1). Only 

cells exposed to ultrasound showed the effect. 

imaging life 

Conclusions: Optical imaging showed that temporal 

window of increased membrane permeability is 

much longer than previously suggested. This may 

have important repercussions for in vivo studies in 

which membrane permeability may be temporally 

separated from drug administration to better adapt 

to pharmacokinetic / pharmacodynamic properties 

of the drug or drug carrier. 


EC-project FP7-ICT-2007-1-213706 SonoDrugs and 

Foundation InNaBioSanté-project ULTRAFITT. Microscopy 

was performed in the Bordeaux Imaging 

Center of the Neurosciences Institute of the University 

of Bordeaux II. 

References: 

Figure 1. In the absence of US 

only occasional cells with the 

compromised membrane show 

the uptake of cell-impermeable 

Sytox green (A). However the 

effects of the US on membrane 

permeability can last up to 

24h (B), with the fluorescence 

intensity increasing as the time 

between US application and 

fluorophore administration 

shortens (C-F). 

1. Hernot S, Klibanov AL; Adv Drug Deliv Rev. 60(10):1153- 

66 (2008) 

2. van Wamel A et al.; J Control Release. 112(2):149-55 

(2006) 

3. Hancock HA et al. Ultrasound Med Biol. 35(10):1722-36 

(2009)


Integrisense: a novel near-infrared fluorescent probe for α v β 3 integrin and its applications in 

drug discovery 

Sur C. (1) , Lin S.A. (1) , Gleason A. (1) , Kossodo S. (1) , Pickarski M. (1) , Coleman P. (1) , Rajopadhye M. (2) , Duong L. T. (1) , Yared W. (2) , 

Peterson J. (2) , Bednar B. (1) . 

(1) Merck Research Laboratories, Westpoint US 

(2) ViSen Medical. 

cyrille_sur@merck.com 

Introduction: The α v β 3 integrin belongs to the superfamilly 

of dimeric transmembrane cell adhesion 

receptors involved in critical biological processes 

such as cell-to-cell, and cell-to-extracellular 

matrix binding. As these activities are central to 

pathological conditions such as inflammation and 

tumor progression, the RGD-binding α v β 3 integrin 

has been considred as clinically-relevant biomarkers 

of these disease states. Although, RDG peptides 

have been developed for PET and SPECT imaging, 

the operating cost of these modalities limits their 

widespread deployment in drug discovery teams. In 

contrast optical molecular imaging allows the noninvasive 

monitoring and quantification of fluorescent 

probes in vivo with less expensive equipment 

that can be installed at multiple research sites. In 

order to take full advantage of the rich biology of integrins, 

a partnership has been established between 

Merck Research Laboratories and VisEn Medical to 

develop a novel near-infrared (NIR) fluorescent integrin 

probe, IntegriSense TM . The pharmacological 

and biological characterization of IntegriSense TM , 

a peptidomimetic antagonist-based molecule with 

improved specificity and binding affinity will be 

presented together with research applications in the 

oncology and atherosclerosis research fields. 

Methods: The in vitro cell biology and pharmacological 

profiling of IntegriSense TM were conducted 

with recombinant HEK-293 cells expressing α v β 3 

and included confocal microscopy for cellular localization 

and flow cytometry for binding and kinetic 

constant determination. In vivo pharmacodynamics 

and tumor localization studies were performed 

in female NU/NU mice bearing different human 

tumor xenografts. IntegriSense TM NIR fluorescent 

signal was acquired with a Fluorescent Molecular 

Tomography imaging system (FMT2500). The potential 

application of IntegriSense TM in atherosclerosis 

was evaluated in apolipoprotein E-deficient 

(ApoE-/-) and human cholesteryl ester transfer 

protein (CETP) knockin/LDL receptor-deficient 

(C57BL/6-Tg(CETP)-Ldlrtm1) mice. Transgenic 

as well as control C57Bl/6 mice were fed a cholesterol 

enriched diet (9% fat, 0.15% cholesterol) to 

induce atherosclerosis. IntegriSense TM NIR signal 

was measured in vivo 24 and 48 hours after probe 

injection and its localization in inflamed aortas was 

further evaluated by ex-vivo imaging and histology. 

Results: Integrisense-680 has absorption/emission 

spectra centered at 674nm/692nm and an extinction 

coefficient of 2.02x10 5 M -1 cm -1 . Binding of IntegriSense 

TM to HEK-293 stably expressing recombinant 

human α v β 3 yielded a Kd of 4.2±0.6 nM and 

a dissociation constant koff of 1.08x10 -4 s -1 . Plasma 

pharmacokinetic analysis revealed a two compartmental 

profile with t1/2 of 6 min and 210 min corresponding 

to clearance of free and bound IntegriSense 

TM , respectively. Biodistribution studies in 

mice with xenograft tumors showed a specific uptake 

of IntegriSense TM by tumor cells and supported 

using IntegriSense TM to monitor tumor growth in 

animal models. 

In vivo measurements with FMT2500 in mice atherosclerosis 

models detected a longitudinal increase in 

IntegriSense TM fluorescence in the thorax area that 

was three to five fold higher when compared with 

control animals. This NIR signal originated from 

atherosclerotic lesions in the aortic arch, carotid, 

and subclavian arteries as confirmed by ex vivo imaging 

of the dissected vessels and histopathology. 

Conclusions: This report demonstrated that the α v β 3 

integrin optical reporter, IntegriSense TM developed 

by Merck and VisEn partnership can be used as a 

molecular imaging biomarker for in vivo imaging 

studies. For instance, IntegriSense TM can be used to 

monitor tumor growth as well as to detect inflammation 

in atherosclerosis plaques. The ability of 

this biomarker to report on several important cellular 

and physiological functions makes it a tool of 

choice to evaluate preclinically the therapeutic potential 

of novel anticancer and atherosclerosis drugs. 


day2 

Plenary Session on current contribution of IMAGING TECHNOLOGIES to DRUG DEVELOPMENT

90 


Harnessing the power of bioluminescence to cross the in vitro–in vivo divide 

Watson J. (1) , Klaubert D. (1) , Biserni A. (2) , Ciana P. (2) , Maggi A. (2) , Allard S. (1) . 

(1) Promega 

(2) TOP (Transgenic Operative Products)srl 

John.watson@promega.com 

Introduction: Bioluminescence imaging is accomplished 

by sensitive detection of light emitted following 

chemical reaction of the luciferase enzyme 

with substrate. The imaging process in animal 

models requires a reporter construct that leads to 

production of luciferase enzyme. The most commonly 

used reporter for this purpose is a construct 

that can express firefly luciferase (1). Data will be 

presented that shows how a number of biomarkers 

can be measured using bioluminescence in vitro, 

including caspase-3/7, ATP and transcriptional 

activation. We will also describe the in vivo characterization 

of VivoGlo Caspase-3/7 Substrate, a 

modified firefly luciferase substrate that in apoptotic 

cells is cleaved by caspase-3 to liberate aminoluciferin, 

which can be consumed by luciferase 

to generate a luminescent signal. Evidence will be 

shown illustrating the possibility to merge a line of 

reporter mice (repTOP) engineered for the ubiquitous 

expression of the luciferase reporter gene 

(2-4) and the modified luciferase substrate, Vivo- 

Glo Caspase-3/7 Substrate, to measure molecular 

apoptotic events in whole living animals. We will 

also demonstrate that this substrate can be used 

to non-invasively observe the apoptotic affects of 

chemotherapy several days before they are able to 

be detected by traditional methods (5). 

Methods: In this study, liver apoptosis was induced 

in luciferase reporter mice by a single i.p. injection 

of D-galactosamine (D-GalN; 800mg/kg) and 

Lipopolysaccharide (LPS; 100mg/kg) (6). Six hours 

after LPS/D-GalN or vehicle administration, mice 

were treated i.p. with increasing doses (17-150mg/ 

kg) of the VivoGlo Caspase-3/7 substrate (Z- 

DEVD-Aminoluciferin, Sodium Salt) and subjected 

to the bioluminescence in vivo imaging procedure. 

Results: The in vivo imaging data obtained clearly 

showed a dose-dependent increase of photon 

emission in the hepatic area of mice treated with 

D-GalN/LPS. No photon emission was observed in 

organs not affected by the apoptotic treatment. A 

complete analysis of pro-apoptotic effects induced 

by the treatment was carried out by ex vivo imaging 

acquisition of photon emission in several dissected 

imaging life 

tissues. This investigation confirmed that light 

emission observed in vivo was produced selectively 

by liver and adipose tissues. Western blot analysis 

and the measure of Caspase-3/7 enzymatic activity 

fully supported in vivo imaging data. 

Conclusions: The work here shows the power of 

a modified luciferin substrate such as VivoGlo 

Caspase-3/7 Substrate to detect apoptotic cells in 

living animals, providing an important advancement 

over current imaging methodologies based 

on fluorescence, nuclear or magnetic resonance 

including: virtually no background, high sensitivity 

and simple instrumentation needed for the in vivo 

imaging measurement of the Caspase-3/7 activity. 

The present application carried out in reporter 

mice genetically engineered to develop specific 

cancers will open the way to novel, more predictive 

approaches for the study of anti-cancer treatments 

that will enable researchers to measure drug efficacy 

in space and time, providing relevant information 

to be rapidly translated to human therapy. 

References: 

1. Zinn, KR et al; ILAR Journal. 49:103-115 (2008) 

2. Ciana, P et al; Nat. Med. 9:82-86 (2003) 

3. Maggi, A and Ciana, P; Nat. Rev. Drug Discov. 4:249-255 

(2005) 

4. Maggi, A et al; Trends Pharmacol. Sci. 25:337-342 

(2004) 

5. Hickson, J et al; Cell Death Differ. doi:10.1038/ 

cdd.2009.205 (2010) 

6. Nakama, T et al; Hepatology. 33:1441-1450 (2001)


In vivo imaging in drug discovery: the example of application in the development of novel 

estrogenic compounds. 

Biserni A. (1) , Rando G. (2) , Ciana P. (2) , Komm B. (3) , Maggi A. (2) . 

(1) TOP (Transgenic Operative Products)srl, 

(2) University of Milan, 

(3) Wyeth Research . 

andrea.biserni@top-mice.com 

Introduction: Intracellular Receptor (IR) signalling 

is temporally organized at different levels: at the 

cellular level, post-translational modification and 

proteolysis were shown to be responsible for IRdependent 

recruitment of co-regulators and proteins 

of the transcriptional apparatus necessary to 

modulate IR activity in time. At the whole organism 

level, circadian rhythms and pulsatility of hormone 

release may be responsible for maintaining 

a state of activity of IR that is critical for hormone 

action. With hormone therapy, the reinstatement 

of the receptor oscillatory activity may be a key element 

for achieving the desired beneficial effect. To 

date, the lack of appropriate models to study the 

response to pharmacological treatments in time 

has been a limiting factor in the study of spatiotemporal 

activity of drugs. The availability of the 

ERE-Luc reporter mouse model where the application 

of BLI-based imaging methodologies allows 

one to study estrogen receptor activity in living animals, 

opens the way to a more in-depth analysis of 

the effect of estrogen therapy (ET). The aim of this 

study was to set up a protocol for the study of ET 

and SERM (Selective Estrogen Receptor Modulator) 

therapies to verify their ability to efficaciously 

restore the effects of the natural endocrine state in 

reproductive and non reproductive organs. 

Methods: The study was carried out in the ERE-Luc 

reporter mouse model where the luciferase reporter 

gene is expressed under the transcriptional control 

of an ER responsive promoter. In this model, MARs 

(Matrix Attachment Regions) insulator sequences 

flanking the transgene guarantee a ubiquitous and 

tightly regulated expression of the biosensor making 

it suitable for pharmacological studies. In the present 

study, we treated ovariectomized, female mice with 

CE (3 mg/kg/day), raloxifene (RAL; 10 mg/kg/day) 

and bazedoxifene (BZA, 10 mg/kg/day) for 21 days. 

To measure ER state of activity, mice were subjected to 

in vivo imaging. Photon emission was measured by the 

use of a segmentation algorithm enabling the automatic 

quantification of photon emission in different body regions 

of mouse images. At the end of the study a further 

analysis was carried out on tissue extracts by luciferase 

enzymatic assay. 

Results: In the initial analysis, we evaluated the 

effect of each treatment in the genital, hepatic, 

abdominal area and limbs at 0, 3, 7, 14 and 21d. 

CE had a major effect in the genital and hepatic 

areas shown by a significant increase of ER activity. 

As expected the effect of SERMs was significant 

in the skeletal (limbs) where ER activity 

started to be significantly increased only after 

day 14. In the body areas affected by treatments, 

we observed time-dependent changes: i.e. in the 

liver CE activity decreased with time, while in 

the limbs ER activity was shown to increase with 

the time of exposure to SERMs. This observation 

prompted us to perform a more in-depth analysis 

of ER activity by measuring photon emission 

daily. This second analysis demonstrated that ER 

activity oscillates in time with a period and amplitude 

that is tissue specific and is differentially 

affected by the hormonal treatments. Furthermore, 

the analysis of ER oscillation revealed that 

ovariectomy does not fully disrupt the fluctuation 

observed in intact, cycling mice indicating 

the existence of mechanisms other than circulating 

hormone responsible for ER transcriptional 

activity. The treatments evaluated partially restored 

the physiological oscillatory behavior of 

the receptor. 

Conclusions: Our study: Provides evidence for a 

novel, long paced, oscillatory activity of ERs that is 

independent of ovarian function. This oscillatory 

ER activity may be relevant for the whole body homeostatic 

control and may be of relevance for the 

identification of safer and more efficacious therapies. 

Points to the importance of whole animal imaging 

for drug development and for the study spatio-temporally 

drug activity on targets. 

Novel protocols for a wider application of BLI to 

the study of molecular events in vivo should be 

developed. 

Acknowledgement: Grants from: NoE DIMI 

LSHB-CT-2005-512146; IP CRESCENDO LSHM- 

CT-2005-018652), National Institutes of Health 

(RO1AG027713), and Wyeth Pharmaceutical Co. 


day2 

Plenary Session on current contribution of IMAGING TECHNOLOGIES to DRUG DEVELOPMENT

DAY 

3

• ESMI Plenary Lecture 5: Jagat Narula (Irvine, USA) 

Molecular Imaging of unstable coronary plaques 

Chairs: Uwe Haberkorn (Heidelberg, Germany), Helmut Maecke (Freiburg, Germany) 

• Parallel Session 9: Cardiovascular II 

Chairs: Klaas Nicolay (Eindhoven, The Netherlands), Michael Schäfers (Münster, Germany) 

• Parallel Session 10: Cancer II – together with COST action BM0607 

Chairs: Marion de Jong (Rotterdam, The Netherlands), Fabian Kiessling (Aachen, Germany) 

• Plenary Session and Closing Ceremony 

Chairs: John Clark (Edinburgh, UK), Bertrand Tavitian (Orsay, France) 

Day 3 - Friday May 29, 2010

94 


imaging life 

Jagat Narula 

Jagat Narula MD, PhD, FACC, FRCP [Edin, Hon] 

Professor of Medicine and Chief, Division of Cardiology 

Director, Memorial Heart & Vascular Institute 

Medical Director, Edwards Lifesciences Center for Advanced Cardiovascular Technology 

University of California, Irvine School of Medicine 

Jagat Narula completed his cardiology training in India at the All India Institute of Medical Sciences, Delhi, 

and relocated to Massachusetts General Hospital and Harvard Medical School in 1989. After fellowships in 

heart failure, transplantation, and cardiovascular imaging, he joined the Massachusetts General cardiology 

faculty. In 1997, he moved to Hahnemann University School of Medicine in Philadelphia. At Hahnemann, 

he was the Thomas J. Vischer Professor of Medicine, Chief of Division of Cardiology, and Vice-Chairman 

of Medicine. He joined University of California, Irvine in 2003 as Professor of Medicine Chief, Division 

of Cardiology, and Associate Dean for Research. Dr. Narula is involved in clinical and basic research in 

the fields of heart failure and atherosclerosis, with major emphasis on development of novel noninvasive 

imaging techniques. He has made vital contributions to the imaging of apoptotic cell death in heart 

muscle, and to the imaging of atherosclerotic plaques that are vulnerable to rupture. His research is not 

limited by any one imaging modality; and uses integrated imaging approaches for better identification 

of cardiovascular pathology. His research is funded, in part, by grants from the National Institutes of 

Health. Dr. Narula has authored more than 700 research publications or presentations and edited 25 books 

and journal supplements. He has been awarded “Best Young Investigator” on several occasions by the 

Cardiovascular Council, the Society of Nuclear Medicine, and the American Society of Nuclear Cardiology. 

He serves on various committees of the American Heart Association. He is the editor-in-chief of the Journal 

of the American College of Cardiology- Imaging. He has also been an Associate Editor of the Journal of 

the American College of Cardiology and the Founder Editor of Heart Failure Clinics of North America.


Molecular imaging of unstable coronary plaques 

Narula J. . 

University of California, USA 

narula@uci.edu 

Sudden cardiac death and acute myocardial infarction 

often occur as the first manifestation of coronary 

artery disease. Otherwise asymptomatic individuals 

with subclinical atherosclerosis almost always 

have a classic risk-factor profile and it is essential 

that they are identified before the occurrence of an 

acute coronary event. The ability to recognize such 

individuals requires the development of noninvasive 

strategies that can localize unstable atherosclerotic 

lesions. Plaques that are vulnerable to rupture demonstrate 

distinct histological characteristics, including 

large plaque and necrotic core volumes covered 

by attenuated fibrous caps and extensive remodeling 

of the vessel at the lesion site. The morphologic features 

can be characterized by CT angiography and 

magnetic resonance imaging. 

In addition to characteristic morphological profile, 

monocyte-macrophage infiltration of the fibrous 

cap is one of the strongest determinants of plaque 

instability, and amenable to molecular inaging. After 

crossing the intimal border, infiltrating monocytes 

express receptors for chemoattractants (such as 

MCP-1 or adhesion molecules), and subsequently develop 

scavenger receptors in the subintimal layers for 

ingestion of oxidized LDL. Although multiple cellular 

processes can be targeted for the identification of 

plaque inflammation only a few molecular imaging 

strategies have been successfully employed for clinical 

imaging. The inflammatory cells in plaques have 

high metabolic activity. In contrast to granulocytes, 

which carry a supply of glycogen to provide energy 

for their phagocytic activities, the macrophages in 

an atheroma require exogenous glucose for their 

metabolism. Incidental uptake of a glucose analog, 

[18F]-FDG, commonly used to characterize malignant 

tumors, has been demonstrated in the aorta, 

carotid arteries and, rarely, the coronary vessels of 

patients with tumors. Carotid endarterectomy specimens 

have demonstrated that uptake correlates with 

macrophage content, and is reduced in patients 

treated with statins. A prospective study has demonstrated 

that FDG uptake can be seen in the coronary 

arteries if background FDG uptake in the myocardium 

is adequately suppressed. Following coronary 

interventions, intense FDG uptake has been seen at 

the sites of stent implantations in patients with acute 

coronary events; no uptake was observeat the stent 

sites in patients with stable disease. Concurrent CT 

angiography was performed in these patients for precise 

anatomical localization of FDG uptake on PET 

imaging. Another clinical approach with molecular 

imaging has utilized radiolabeled annexin A5 to 

target apoptotic macrophages in the atherosclerotic 

lesions. A large proportion of macrophages in the 

attenuated fibrous caps of vulnerable and ruptured 

plaques undergo apoptosis. The annexin uptake in 

the carotid vessels has been predominantly seen in 

symptomatic carotid disease. Annexin imaging of 

coronary vessels has not been attempted. Various other 

molecules targeting evolution of receptors such as 

MCP-1 and scavenger receptors, as well as those targeting 

the products of inflammation such as metalloproteinases 

are being successfully employed for the 

imaging of atherosclerosis in experimental models. 


day3 

ESMI Plenary Lecture by Jagat Narula

96 


Advances in contrast-enhanced MRI of the mouse heart 

Strijkers G. . 

Biomedical NMR Eindhoven, The Netherlands 

g.j.strijkers@tue.nl 

Introduction: In preclinical cardiovascular research 

using mouse models, MRI has proven to 

be the imaging modality of choice for studying 

cardiac pathology. This is mainly thanks to its 

high temporal and spatial resolution, enabling 

accurate determination of cardiac functional 

parameters. 

MRI contrast agents are used to differentiate between 

viable and infarct myocardium. Recently, it 

has become possible to use MR contrast agents for 

the evaluation of the mouse myocardial perfusion 

status. Molecular MR imaging techniques are being 

developed, allowing contrast agents to specifically 

target disease markers, providing additional 

information about the infarction. The use of contrast 

agents in the mouse myocardium requires innovative 

mouse cardiac MRI techniques to quantify 

contrast agent kinetics and concentration in the 

myocardium. 

Results: In this presentation I will present recent 

advances in the contrast-enhanced MR imaging of 

the mouse heart. 

First, I will discuss new MR technology, which enables 

the assessment of mouse myocardial perfusion. 

This methodology involves intravenous injection of 

an MRI contrast agent, after which the first pass of 

the contrast agent through the heart is monitored 

in a time-series of images. The combination of the 

high heart rate (400-600 bpm), small heart size (5-6 

mm left ventricle (LV) diameter) and fast systemic 

blood circulation time (4-5 s) require a very fast 

imaging protocol while preserving adequate spatial 

resolution to visualize the regional myocardial inflow 

of the contrast agent. 

Secondly, I will discuss recent advances in the T1 

quantification of the mouse myocardium, which 

enables quantification of contrast agent concentration. 

The T1 quantification method uses a combination 

of 3D IntraGate FLASH steady-state imaging 

together with DESPOT1 analysis. The 3D IntraGate 

FLASH makes use of a navigator echo that retrospectively 

triggers the acquisition to the heartbeat 

and respiration cycle. 

imaging life 

Acknowledgement: Grant sponsor: Dutch Technology 

Foundation STW, applied science division of 

NWO and the Technology Program of the Ministry 

of Economic Affairs; Grant numbers: 07952 and 

10191. Grant sponsor: European Union Network of 

Excellence Diagnostic Molecular Imaging; Grant 

number: 512146 (LSHB-CT-2005-512146).


Molecular imaging of α v β 3 integrin expression with 18 F-galacto-RGD after experimental 

myocardial infarction: comparison with left ventricular remodeling and function. 

Saraste A. (1) , Sherif H. (1) , Nekolla S. (1) , Weidl E. (1) , Higuchi T. (1) , Reder S. (1) , Tapfer A. (1) , Botnar R. (1) , Wester H. J. (1) , 

Schwaiger M. (1) . 

Technische Universität München, Germany 

antti.saraste@utu.fi 

Introduction: Myocardial infarction (MI) and subsequent 

left ventricular (LV) remodeling are the 

most frequent cause for development of chronic 

heart failure. Expression of α v β 3 integrin receptors 

is increased early after MI as part of the healing 

process. Poor infarct healing can contribute to progressive 

LV remodeling. 18 F-galacto-RGD (RGD) is 

a PET tracer that binds to α v β 3 integrin receptors. 

We studied whether molecular imaging of myocardial 

RGD uptake early after MI can predict longterm 

LV remodeling in rat. 

Methods: Wistar rats underwent permanent left 

coronary artery (LCA) ligation to induce myocardial 

infarction (MI) or sham operation. One week 

post-MI, rats were imaged with a small animal PET 

scanner after injection of 13N ammonia to define 

perfusion defect area, and 90 minutes post injection 

of RGD in order to measure α v β 3 integrin expression. 

Myocardial RGD uptake (%ID/cc) in the 

infarcted (defect area) and remote myocardium 

were measured in co-registered polar maps. Cardiac 

magnetic resonance imaging (MRI) with 1.5T 

scanner, small animal coil and ECG triggering was 

used to measure LV remodeling and function repeatedly 

1 week and 12 weeks post-MI. 

Results: One week after LCA ligation, RGD uptake 

was significantly higher in the defect area than 

in the remote myocardium of MI rats and in controls 

(0.2±0.05 vs. 0.06±0.03 and 0.07±0.04 %ID/ 

cc, respectively, p20% increase in EDV, early RGD uptake 

in the defect area was 29% lower than in rats with 

less than

98 


Existing and emerging animal models mimicking cardiovascular disease and their relevance 

for molecular imaging 

Schäfers M. . 

European Institute for Molecular Imaging – EIMI, University of Münster 

schafmi@uni-muenster.de 

Appropriate animal models, mimicking the pathology 

of cardiovascular human diseases is essential 

for progress in the field of cardiovascular research 

and for the transfer of preclinical results into the 

clinical setting. In principle, a variety of animal 

species can be used in cardiovascular research. Although 

larger animal such as pigs (a classical model 

for myocardial infarction) are used in cardiovascular 

research, the focus has been shifted to small 

imaging life 

animal models such rats and mice recent years. 

Animal models of cardiovascular diseases can rely 

both on genetic manipulations or surgical interventions 

and do nowadays cover the wide spectrum 

of cardiovascular human diseases. In this talk, examples 

for relevant human cardiovascular diseases, 

myocardial infarction and atherosclerosis, already 

being studied by small animal imaging is given.


Imaging of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques with 

Multispectral Optoacoustic Tomography 

Razansky D. (1) , Harlaar N. J. (1) , Hillebrands J. L. (2) , Taruttis A. (1) , Herzog E. (1) , Zeebregts C.J. (2) , Van Dam G. M. (2) , 

Ntziachristos V. (1) . 

(1) Technical University of Munich and Helmholtz Center Munich, Germany 

(2) University Medical Center Groningen, The Netherlands. 

tdr@tum.de 

Introduction: The indication for a carotid endarterectomy 

is nowadays mainly based on symptomatology 

or, alternatively, on the degree of stenosis in 

carotid arteries (>80%). Those indications however 

do not provide an accurate assessment of plaque 

vulnerability and therefore only a small percentage 

of patients do actually benefit from the surgical intervention 

by preventing a major cerebro-vascular 

event especially in the asymptomatic group [1]. 

High activity levels of tissue biomarkers, such as 

cathepsins, integrins, and matrix metalloproteinases 

(MMPs), have been associated with atherosclerotic 

plaque instability, thus can potentially be 

used for highly specific diagnosis [2]. While fluorescent 

tagging of such molecules has been amply 

demonstrated, no imaging method has so far been 

shown capable of resolving inflammation-associated 

tags with high fidelity. 

Methods: We showcase herein the ability to resolve 

activation of common probes sensitive to matrix 

metallo-proteinases with unprecedented image 

quality and resolution utilizing multi-spectral optoacoustic 

tomography (MSOT) [3], thus revealing 

atherosclerotic activity. Human carotid plaque specimens 

from patients were incubated ex vivo with 

an MMP-sensitive activatable fluorescent probe 

(MMPsense 680TM) directly after endarterectomy. 

The specimen were subsequently imaged using an 

MSOT scanner capable of simultaneous high resolution 

visualization of morphology and molecular 

activity with better than 200 micron resolution. 

Results: MSOT analyses identified heterogeneous 

MMP activity throughout the plaques, revealing 

hot and cold spot regions indicative of relatively 

high and low MMP activity respectively. The results 

from intact specimen corresponded well with 

epi-fluorescence images made on thin cryosections. 

Elevated MMP activity in the hot spot regions resolved 

was further confirmed by in situ zymography, 

accompanied by increased macrophage influx. 

Results from both MSOT investigations and histological 

sections confirmed that most of the plaque 

formation activity occurs, as expected, close to the 

bifurcation area of the carotid artery. 

Conclusions: We show, for the first time to our 

knowledge, the ability of multispectral optoacoustic 

tomography to deliver volumetric images of activatable 

molecular probe distribution deep from optically 

opaque tissues. High resolution mapping of MMP 

activity in the vulnerable plaque of human carotid 

specimen was demonstrated. This ability directly relates 

to clinical potential as it can allow highly specific 

visualization and staging of plaque vulnerability 

in atherosclerosis during surgical intervention or by 

intravascular or potentially non-invasive imaging; 

thus impacting therapeutic clinical decision. 

Acknowledgement: This research was partially supported 

by the German Research Foundation (DFG) 

Research Grant (RA 1848/1), ERC Senior Investigator 

Award, and the Medizin Technik BMBF award for 

excellence in medical innovation. 

References: 

1. Chambers BR et al; Cochrane Database Syst Rev. 

2:CD001923 (2000). 

2. Libby P.; Inflammation in atherosclerosis. Nature. 

420:868-874 (2002). 

3. Razansky D et al.; Nature Phot. 3:3525-3529 (2009). 


day3 

Parallel Session 9: CARDIOVASCULAR II

100 


c-Jun N-terminal kinase promotes inflammation at atherosclerosis-prone sites by enhancing 

expression and activity of NF-κB transcription factors 

Evans P. (1) , Cuhlmann S. (1) , Van Der Heiden K. (1) , Haskard D. (1) , Krams R. (1) , Gsell W. (1) , Carlsen H. (2) , Jones H. (1) . 

(1) Imperial College London, 

(2) University of Oslo, Norway. 

paul.evans@imperial.ac.uk 

Introduction: Atherosclerosis is a chronic inflammatory 

disease of arteries that causes heart attack or 

stroke. Early lesions contain monocytes and T lymphocytes 

which are recruited from the circulation 

to activated vascular endothelial cells (ECs). This 

process relies on NF-κB transcription factors which 

induce pro-inflammatory molecules (e.g. VCAM-1) 

in ECs. Atherosclerosis develops predominantly at 

branches and bends of arteries that are exposed to 

low, oscillatory shear stress (frictional force exerted 

by flowing blood on ECs). We previously demonstrated 

that c-Jun N-terminal kinase (JNK), a MAP 

kinase, is activated in ECs at atherosusceptible but 

not atheroprotected sites (Chaudhury et al 2010). 

Here we combined in vitro studies with molecular 

imaging techniques in murine models to study the 

function of JNK in arterial EC. 

Methods: We studied the function of JNK by 

performing microarray analysis of cultured 

ECs treated with a pharmacological JNK inhibitor. 

En face immunostaining was used to determine 

the expression of particular proteins in 

ECs at atheroprotected and atherosusceptible 

sites of murine arteries. In addition, we assessed 

NF-κB transcriptional activity in murine 

arteries of transgenic NF-κB-luciferase reporter 

mice by measuring luminescence using an ultrasensitive 

camera (Xenogen). Inflammation 

was also assessed by F18-FDG PET/CT imaging 

of murine arteries and identification of 

CD68-positive macrophages by immunostaining. 

Results: Transcriptome profiling of cultured ECs 

treated with a pharmacological inhibitor revealed 

that JNK functions as a positive regulator of NFκB 

transcription factors. This observation was 

confirmed by silencing of JNK1 and ATF2 (a 

downstream transcription factor) which led to 

reduced NF-κB expression in cultured ECs. We 

validated our findings by studying ECs in the aorta 

of wild-type and JNK1-/- mice. En face immunostaining 

revealed that EC expression of NF-κB 

and VCAM-1 and accumulation of CD68-positive 

macrophages was enhanced at atherosusceptible 

imaging life 

compared to atheroprotected sites in wild-type 

mice. Genetic deletion of JNK1 suppressed NF-κB 

and VCAM-1 expression at the atherosusceptible 

site, indicating that JNK1 positively regulates 

NF-κB expression and inflammation. Similarly, 

studies of transgenic NF-κB-luciferase mice revealed 

that NF-κB activity in ECs was enhanced 

at atherosusceptible compared to atheroprotected 

sites. To determine whether a causal relationship 

exists between shear stress and vascular inflammation 

we altered blood flow in the murine carotid 

artery by placing a flow altering cuff. We 

observed that low, oscillatory shear stress enhanced 

JNK activity, increased the expression of 

NF-κB and VCAM-1, promoted the accumulation 

of macrophages and enhanced arterial uptake of 

F18-FDG. 

Conclusions: We conclude that JNK1-ATF2 signalling 

promotes EC activation and inflammation 

at atherosusceptible sites exposed to low, oscillatory 

shear stress by enhancing NF-κB expression. 

Our findings illuminate a novel level of cross-talk 

between the NF-κB and JNK signalling pathways 

that may influence the spatial distribution of atherosclerotic 

lesions. 

Acknowledgement: Funded by the European Union 

FP6 NoE DiMI (LSHB-CT-2005-512146) and British 

Heart Foundation. 

References: 

1. Chaudhury et al. 2010 Arterioscler. Thromb. Vasc. Biol. 

30(3):546-53.


Absolute Quantification in Small Animal Pinhole Gated Myocardial Perfusion SPECT 

Goethals L. (1) , Devos H. (1) , Vanhove C. (1) , De Geeter F. (1) , Lahoutte T. (1) . 

(1) VUB Brussels, Belgium 

lode.goethals@vub.ac.be 

Introduction: Previously, our group has demonstrated 

the feasibility of absolute quantification using SPECT 

[1] imaging by incorporating a CT derived non-uniform 

attenuation correction (AC) map and a triple energy 

scatter window correction in the reconstruction 

algorithm of SPECT images. In this study we apply 

these techniques to the rat myocardium, which has a 

size below the resolution of our imaging system, thus 

suffering from a partial volume effect, which results in 

an underestimation of the activity concentration in the 

target tissues. To compensate for this underestimation 

we performed ultrasound imaging prior to SPECT imaging 

to determine myocardial wall thickness. We then 

derived a recovery coefficient (RC) from phantom studies 

to correct for the partial volume effect. 

Methods: SPECT/CT scans were performed in 9 

healthy Wistar rats 30 min after injection of 99mTc- 

Tetrofosmin. Prior to SPECT/CT imaging, 2D cardiac 

ultrasound images were acquired in the midventricular 

segments to determine diastolic and systolic 

myocardial wall thickness. After SPECT/CT imaging, 

animals were sacrificed and 6 midventricular segments(anterior-anterolateral-inferolateral-inferiorinferoseptal-anteroseptal) 

of the left ventricle were 

excised and counted in a gamma well counter. Using 

6 midventricular ROI’s in AMIDE, the effect of scatter 

correction (SC) and attenuation correction (AC) was 

determined in every midventricular segment. 

The activity in every ROI was expressed in Curie per 

gram (taking into account the density of myocardial tissue 

of 1,055 Kg/L). These image-derived activities were 

compared to the ex vivo activity concentrations 

counted in the gamma counter. To correct for 

the partial volume effect, a RC was determined from 

a phantom study. All values obtained after AC and SC 

were corrected by this RC and compared to the ex vivo 

counting data. 

Results: AC leads to a significant increase in counts in 

every midventricular segment, whilst SC results in a 

decrease in counts in every midventricular segment. 

The combination of both S and AC leads to a significant 

increase in counts in the inferior midventricular segment 

(paired Student’s T test, p < 0,05). 

After combined AC and SC, there remains a vast underestimation 

of activity in the myocardium, compared 

to the ex vivo data. Incorparation of a recovery 

coefficient derived from the phantom study, alleviates 

this problem: in the sub 5mm region, a RC of Y 

= 17,252 . X (with X = myocardial size in mm, Y = RC 

in %) was derived from the phantom studies. Dividing 

the combined AC and SC values by a RC derived 

from the average wall thickness (diastolic + systolic 

/ 2) leads to an adequate estimation of activity, correlating 

well with the ex vivo data. Use of the diastolic 

thickness as source of the RC leads to an overestimation 

of activity, use of the systolic thickness 

as a source of the RC leads to an underestimation 

of activity on the non-gated images. Use of the appropriate 

RC on the corresponding gated images also 

leads to an adequate estimation of activity on images. 

Conclusions: Combining attenuation, scatter and 

partial volume effect corrections in small animal 

myocardial perfusion SPECT allows for non invasive 

absolute quantification of myocardial perfusion. 

Acknowledgement: Researh at ICMI is funded by the 

Interuniversity Attraction Poles Program - Belgian 

State - Belgian Science Policy. Tony Lahoutte is a Senior 

Clinical Investigator of the Research Foundation - Flanders 

(Belgium) FWO. Lode Goethals is a PhD fellow of 

the research foundation – Flanders (Belgium) -FWO. 

References: 

1. Improved quantification in single-pinhole and 

multiple-pinhole SPECT using micro-CT information. 

Vanhove C, Defrise M, Bossuyt A, Lahoutte T. Eur J Nucl 

Med Mol Imaging. 2009 Jul;36(7):1049-63. 


YIA applicant 

day3 

Parallel Session 9: CARDIOVASCULAR II

102 


Cancer imaging 

Haberkorn U. . 

University Heidelberg, Germany 

Uwe.Haberkorn@med.uni-heidelberg.de 

Increased metabolism has been found to be one 

of the most prominent features of malignant 

tumors. This property led to the development of 

tracers for the assessment of glucose metabolism 

and amino acid transport and their application for 

tumor diagnosis and staging. Prominent examples 

are fluorodeoxyglucose, methionine and tyrosine 

analogs which have found broad clinical application. 

Since quantitative procedures are available these 

imaging life 

techniques can also be used for therapy monitoring. 

Another approach may be based on the noninvasive 

detection of apoptosis with tracers for 

phosphytidyl-serine presentation and/or caspase 

activation as surrogate marker for therapeutic 

efficacy. Finally, the use and development of 

specific ligands to target structures overexpressed 

in tumor tissue may be a valuable tool for diagnosis 

and novel therapeutic interventions.


Controlled drug delivery under image guidance 

Gruell H. (1) , Langereis S. (1) , De Smet M. (2) , Sanches P. (2) , Hijnen N. (2) , Rossin R. (1) , Bohmer M. (1) , Keupp J. (1) , Tiemann K. (3) , 

Heijman E. (1) . 

(1) Philips Research, 

(2) Eindhoven University of Technology, 

(3) University Hospital Münster. 

holger.gruell@philips.com 

Introduction: Focused ultrasound (FU) is an excellent 

method for local triggered drug delivery 

using either pressure or temperature sensitive delivery 

systems. Temperature sensitive liposomes 

(TSLs) as drug carriers are promising to maximize 

delivery of drugs to tumors that are heated 

slightly above body temperature using FU. Besides 

heating, the pressure pulses of ultrasound 

waves can be exploited to disrupt microbubbles, 

which can lead to formation of transient pores in 

the endothelial wall. The latter is termed sonoporation 

and was shown to mediate cellular uptake 

of therapeutic agents. Either way, imaging based 

method to visualize, follow, and control drug delivery 

in-vivo are of importance to establish more 

quantitative treatment protocols. 

Methods: TSLs of different lipid compositions 

and a release temperature slightly above body 

temperature were loaded with T1, CEST or fluorinated 

MRI contrast agents and the drug doxorubicin. 

MRI was used to assess the signal change 

upon heating the TSLs from T=310 K to T=315 K 

using FU. For pressure induced drug delivery, microbubbles 

were injected into tumor bearing mice. 

Tumor and muscle tissues were treated with FU. 

Subsequently, radiolabeled albumin was injected 

and the extravasation in tumor and muscle tissue 

was followed and quantified using single photon 

computed tomography (SPECT). 

Results: Focused ultrasound is used to locally 

heat tissue, while MRI provides spatial and temperature 

control of the process. Co-encapsulation 

of drugs and MRI contrast agents allows to image 

the drug carrier system as well as to quantify 

release of drugs under hyperthermia conditions 

with MRI, making this modality the perfect 

choice for temperature induced drug delivery 

under image guidance. The temperature of drug 

release but also contrast properties can be tuned 

by nature and composition of the phospholipid 

bilayer. The rapid release of drugs and contrast 

agents can be explained by the formation of transient 

pores in the lipid membrane upon heating. 

For pressure induced drug delivery, the cavitation 

of microbubbles by ultrasound creates transient 

pores in the endothelial layer allowing macromolecules 

to efficiently extravasate. This effect 

is found to be more pronounced in muscle tissue 

than in tumors. In healthy muscle tissue the 

intact endothelial layer prevents extravasation of 

macromolecules while the leaky and premature 

vasculature in tumors is more permeable for macroscopic 

compounds. 

Conclusions: Focused Ultrasound allows to noninvasively 

trigger drug delivery in vivo, either using 

a local heating or focused pressure pulses in 

combination with suited drug delivery vehicles. 

The drug delivery process can be planned, controlled 

and followed using diagnostic imaging 

modalities such as MRI or ultrasound in combination 

with smart drug delivery vehicles. Above 

approach allows local non-invasive image guided 

treatment of a tissue that potentially leads to an 

improvement of the therapeutic window or even 

enables delivery of completely different classes of 

drugs such as siRNA or plasmid DNA. 

Acknowledgement: This project was funded in 

part by the EU Project Sonodrugs (NMP4-LA- 

2008-213706) 

References: 

1. Langereis et al., J. Am. Chem. Soc. 131, 1380 (2009). 

2. De Smet et al. J. Contr. Release 143, 120 (2010). 


day3 

Parallel Session 10: CANCER II - together with COST

104 


Comparative biodistribution of twelve gastrin/CCK2 receptor targeting peptides 

De Jong M. . 

Erasmus MC Rotterdam, The Netherlands 

m.hendriks-dejong@erasmusmc.nl 

Introduction: Molecular targeted radionuclide cancer 

therapy is becoming of increasing importance, 

especially for disseminated diseases. Systemic chemotherapies 

often lack selectivity; targeted radionuclide 

therapy has important advantages as the 

radioactive cytotoxic unit of the targeting vector is 

specifically directed to the cancer, sparing normal 

tissues. The basis of COST Action BM0607 is the 

great potential of targeted radionuclide therapy 

using a variety of vectors and radionuclides. This 

Action brings together the different disciplines involved 

and provides a reliable and rapid means for 

developing new (fundamental) knowledge, method 

standardization and products. 

Minigastrin and CCK analogs have been developed 

for radionuclide imaging and therapy of CCK2R 

expressing tumors. One project in COST Action 

BM0607 aims to select the optimal gastrin/CCK 

analogs for targeting MTC and SCLC expressing 

the CCK2R. In the present study, twelve DOTA- 

conjugated analogues were labeled with In-111 

and their in vitro and in vivo CCK2R targeting 

properties were studied in nude mice with CCK2R 

expressing A431 tumors. 

Methods: All peptides were labeled with In-111 

at high specific activity. Receptor affinity and internalization 

were tested in vitro using CCK2Rtransfected 

A431 tumor cells. Mice with CCK2Rtransfected 

A431 tumors in the left flank and with 

mock-transfected A431 tumors in the right flank 

received radiolabeled peptide intravenously for 

imaging and biodistribution studies. 

Results: All peptides demonstrated high receptor affinity 

and receptor-specific internalization in vitro 

and tumor targeting in the CCK2R expressing tumor 

in vivo. Tumor uptake at 1 h p.i. ranged from 

2.53 ± 0.47 %ID/g for CP07 to 13.3 ± 4.9 %ID/g for 

CP10, whereas uptake at 4 h p.i. ranged from 1.88 ± 

1.12 %ID/g to 9.90 ± 2.0 %ID/g, respectively. Uptake 

in non-target tissues was low for all peptides, except 

for high kidney uptake for CP10 and CP11. Highest 

tumor-to-kidney ratios (3-5) were obtained with 

the gastrin analogues CP01, CP02, CP03 and CP09. 

imaging life 

Conclusions: All peptides showed good CCK2Rmediated 

tumor-targeting in vitro and in vivo, 

with low uptake in non-target organs except for 

the kidneys in some cases. CP01, CP04 and CP05 

displayed favorable in vivo characteristics, combing 

high tumor uptake with low kidney retention. 

Acknowledgement: This study is part of COST Action 

BM0607


Targeting cancer stem cells using radiolabeled Sonic Hedgehog 

Tworowska I. (1) , Delpassand E. S. (1) , Sims-Mourtada J. (1) . 

(1) RadioMedix Housten, USA 

itworowska@radiomedix.com 

Introduction: Sonic Hedgehog (SHH) is an extracellular 

signalling protein involved in embryo 

development and morphogenesis [1]. Constitutive 

activation of the hedgehog pathway is observed in 

several types of cancers [2], especially in the most 

aggressive cancer stem cells and it is associated 

with their chemo/radiation resistance [3]. This 

work reports results on characterization of the SHH 

conjugates and their application to the cancer imaging 

using positron emission tomography (PET). 

Methods: 6xHis-tagged recombinant SHH 

(19.5kDa) with terminal NH2 group was conjugated 

to the DOTA (1,4,7,10-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic 

acid) in phosphate 

buffer and purified by dialysis. Alternatively 

SHH ligand was coupled to the maleimido-monoamide-DOTA 

chelator through thiol group of 

cysteine. Protein conjugates were further characterized 

using MALDI-MS and HPLC. Radiolabeling 

of the DOTA-SHH was performed in 0.1M 

NH4OAc (pH=4.4) using 68GaCl3 eluted from 

68Ge/68Ga generator (iThemba). In vitro bioactivity 

was evaluated by cellular uptake studies of 

the radiolabeled ligand in the several cancer cell 

lines and enriched stem cancer-cells population 

(mammospheres). 

Results: DOTA-SHH was obtained with total 

yield of 74%-89%. Reaction proceeded with radiochemical 

purity of >98% and specific activity 

of 3.38E+05 MBq/g, (theoretical specific activity 

= 3.64E+11 MBq/gram) as assessed by radio-TLC 

and radio-HPLC. 68Ga-DOTA-SHH have shown 

high affinity to Patched receptors (PTCH) in cancer 

cells (BT-474, MDA-MB-231, MCF-7) and its 

cellular uptake correlates with PTCH receptor expression 

levels. Cellular uptake of the SHH conjugates 

in the presence of the hedgehog pathway inhibitor, 

cyclopamine, was reduced by more than 

50%. Competitive uptake studies in the presence 

of the cold SHH, have also shown reduced cellular 

uptake by approximately 90%. In vitro uptake 

of 68Ga-DOTA-SHH was approximately 12-fold 

higher in cancer stem-cells (MCF-7 cultures) 

compared to the total cell population. 

Conclusions: Our studies have shown that 68Ga 

DOTA-SHH conjugates have potential application 

to non-invasive imaging of cancer, especially 

the most aggressive cancers. This is 

the first report on application of radiolabeled 

SHH conjugates for identification of the cancer 

stem-cell from the total tumor population. 

SHH conjugates may be also useful to evaluate 

new therapies targeting cancer stem-cells. 

References: 

1. Hammerschmidt M., Brook A., McMahon A.P., Trends 

Genet., (1997), 13, 14-2. 

2. Beachy P.A., Karhadkar S.S., Berman D.M., Nature (2004), 

432, 324-331; Mullor JL, Sánchez P, Altaba A.R., Trends 

Cell Biol.,(2003), 12 (12): 562–9. 

3. Al-Hajj M., Becker M.W., Wicha M., Weissmann I., Clarke 

M.F., Curr. Opin. Genet. Dev., (2004), 14(1), 43-47 


day3 

Parallel Session 10: CANCER II - together with COST

YIA applicant 

106 


Evaluation of the photosensitizer Bremachlorin for photodynamic treatment of breast 

cancer bone metastasis. 

Van Driel P. (1) , Que I. (1) , Snoeks T. (1) , Mol I. (1) , Xie B. (1) , Keereweer S. (1) , Kaijzel E. (1) , Löwik C.W.G.M. (1) . 

Leiden University Medical Center 

tpieter_v_driel@hotmail.com 

Introduction: Photodynamic therapy (PDT) is a 

promising treatment for tumors in which harmless 

light is used for the irradiation of nontoxic photosensitizers 

to create highly toxic reactive oxygen 

species for the destruction of certain tumors. In 

this study, the photosensitizer Bremachlorin TM 

(Brema Pharma Int.), derived from the seaweed 

spirulina, consisting of the compounds Chlorin e6, 

Purpurin 5 and Chlorin p6, was evaluated for PDT 

of breast cancer derived bone metastasis. 

Methods: The uptake of Bremachlorin in the human 

breast cancer cell line MDA-MB231 luc 

D3H2LN was quantitatively measured with flow 

cytometry and visualized with confocal laser scan 

microscopy. Cell viability after photodynamic 

treatment was measured with a luciferase assay 

and colorimetric MTS cell proliferation assay to 

identify the cytotoxic potential of Bremachlorin 

on these cells.The tumor cells were injected in the 

bone marrow cavity in femurs of immunodeficient 

mice to form osteolytic tumors. In vivo effects of 

PDT with Bremachlorin were followed by whole 

body bioluminescent imaging (BLI). 

Results: Flow cytometry analysis showed 

Bremachlorin uptake by the tumor cells was (close 

to) 100%. Also, fluorescent signal increased with 

an increasing dose of Bremachlorin. Complete 

cell death in vitro was already achieved after an 

incubation time of 5 hours with a concentration 

of 2 mg/l Bremachlorin. In vivo BLI experiments 

showed the partial or total eradication of the osteolytic 

tumors after a single pulse treatment of 

imaging life 

Bremachlorin-treated animals. In case of partial 

cure, the tumors were successfully eradicated by a 

second PDT treatment 

Conclusions: Our in vitro results show the tumor 

selective uptake of the photosensitizer Bremachlorin 

and its cytotoxic effects on the human 

breast cancer cells. Furthermore, with whole body 

BLI we show that PDT treatment of Bremachlorin-treated 

animals is an interesting therapy to 

eradicate aggressive tumors deep down in bone 

marrow. These results are very promising and 

interesting for future clinical use of photodynamic 

treatment of tumors with the photosensitizer 

Bremachlorin. 

Acknowledgement: Dhr. H. Vink and Andrei Reshetnickov 

from Brema Pharma


In vivo targeting of HEK-hsst 2/3/5 Xenografts by 111 in-labeled [(DOTA)Ser 1 ,Leu 8 ,trp 22 ,Tyr 25 ]-SS- 

28 in SCID mice 

Maina T. (1) , Tatsi A. (1) , Marsouvanidis I. P. (1) , Krenning E. P. (2) , De Jong M. (2) , Nock B.A. (1) . 

(1) IRRP, NCSR “Demokritos”, 

(2) Dept. of Nuclear Medicine, EMC Rotterdam. 

maina_thea@hotmail.com 

Introduction: Radiolabeled pansomatostatin-like 

analogs are expected to enhance the diagnostic 

sensitivity and to expand the clinical indications 

of currently applied sst 2 -specific radioligands 

(like OctreoScan ® ) [1,2]. We have previously reported 

on [(DOTA)Ser 1 ,Leu 8 ,trp 22 ,Tyr 25 ]SS-28 

displaying a high affinity for all five hsst 1-5 . In 

mice, the 111 In-radioligand showed specific uptake 

in AR4-2J tumors which express the rat sst 2 

[3]. We were further interested to investigate the 

ability of [( 111 In-DOTA)Ser 1 ,Leu 8 ,trp 22 ,Tyr 25 ]SS- 

28 to target human sst x expressing tumors in vivo. 

Methods: HEK-293 cells stably transfected with 

one of the hsst 2A , the hsst 3 or the hsst 5 were cultured 

in a humidified-5% CO 2 atmosphere at 37 0 C 

in DMEM GLUTAMAX-I supplemented with 

10%FBS, 100 U/mL penicillin / 100 µg/ml µg/mL 

streptomycin and 400 µg/mL G418. Cells were 

cropped, suspended in PBS and cell-suspension 

was equally divided in Eppendorf tubes (1 – 2 x 

10 7 cells, 150 mL). Inoculation was performed 

by subcutaneous injection of a cell suspension 

bolus in the left flank of young SCID mice. After 

10-18 days well-palpable tumor masses developed 

at the inoculation site and biodistribution 

experiments were performed. On the day of 

the experiment the radioligand was injected as 

a bolus (100 µL, 2 µCi) in the tail vein, alone or 

together with a high excess sst x -binding peptide 

(blocker). Animals were sacrificed in groups of 

four at 4 h postinjection (pi) and tissues were 

excised, weighed and counted in a gamma-counter. 

Values were calculated as percent injected 

dose per gram (%ID/g) tissue and are expressed 

as mean ± SD. 

Results: [( 111In-DOTA)Ser 1 ,Leu8 ,trp22 ,Tyr 25 ]SS-28 

showed significant and specific uptake in the hsst - 2A 

positive HEK implants of 4.43 ± 1.5%ID/g vs. 0.49 ± 

0.0%ID/g block (50 µg Tate) at 4 h pi (the previously 

+ reported values in the rsst -AR4-2J tumor, were 

2 

9.35 ± 1.31%ID/g and 0.35 ± 0.0%ID/g, respectively 

[3]). Equally high and specific uptake was displayed 

+ in the hsst -HEK xenografts with a 4.88 ± 1.1%ID/g 

3 

vs. 0.52 ± 0.05%ID/g block at 4 h pi (100 µg Demo- 

+ Pan 2 [2]). Preliminary values in the hsst -HEK 

5 

xenografts were slightly inferior (

poster

• MI in Cancer Biology – Visualisation of Extra- and Intracellular Processes 114 

Poster number P001 – P012 

• Imaging in Drug Development 126 


• Cancer from Bench to Bedside – Translational Research in Oncology 136 


• Imaging in Cardiovascular Disease: from Bench to Bedside 144 


• Molecular Neuroimaging: from Bench to Bedside 147 


• Imaging-guided Gene and Cell based Therapies 161 


• Probe Design – Innovative Approaches to Smart Contrast Agents 171 


• Technology – Technical Advances in MI Instrumentation 183 


• Imaging in Endocrine Diseases 192 


• MI of Infection and Inflammation 197 


• Imaging for Targeted Therapy 205 


• MI Data Analysis Methods 210 


• Late Breaking Abstracts 218 


Poster Session

110 


Guided Poster Sessions – Poster Walks 1 to 7 

Guided Poster Session 1, Day 1: Thursday May 27, 2010 from 14:30 to 16:00 

Poster Walk 1: Imaging Cancer Biology – P001 to P012 & P105 to P106 

Co-Chairs: Fabian Kiessling (Aachen, Germany), Markus Rudin (Zürich, Switzerland) 

Poster Walk 2: Technology & Data Analysis Methods – P070 to P078 & P097 to P104 

Co-Chairs: Serge Maitrejean (Paris, France), Adriaan Lammertsma (Amsterdam, The Netherlands) 

Poster Walk 3: Molecular Neuroimaging – P034 to P047 & P107 

Co-Chairs: Sabina Pappata (Naples, Italy), Andreas Jacobs (Muenster, Germany) 

Poster Walk 4: Probe Design – P058 to P069 

Co-Chairs: Frédéric Dollé (Orsay, France), Helmut Maecke (Freiburg, Germany) 

Guided Poster Session 2, Day 2: Friday May 28, 2010 from 14:30 to 16:00 

Poster Walk 5: Imaging in Cancer & Drug Development – P013 to P022 & P023 to P030 

Co-Chairs: Peter Brader (Graz, Austria), NN 

Poster Walk 6: Imaging in Other Diseases – P031 to P033 & P079 to P083 & P084 to P091 

Co-Chairs: Bertrand Tavitian (Orsay, France), Nicolas Grenier (Bordeaux, France) 

Poster Walk 7: Imaging-guided Gene and Cell based and Targeted Therapies – 

P048 to P057 & P092 to P096 

Co-Chairs: Ludwig Aigner (Salzburg, Austria), NN 

imaging life


overview poster presentations – per title 

MI in CANCER BIOLGY 

In vivo MRI multicontrast kinetic analysis of intracellular trafficking of liposomes 114 

Diffusion-weighted MRI for differentiation of breast lesions at 3.0 Tesla: a new biomarker for breast imaging 115 

Imaging tumour apoptosis with 68Ga-labelled AnnexinA5 derivatives early after cancer therapy 116 

68 Ga-RGD-based PET tracers for imaging αν β 3 expression: a comparative study 117 

Influence of anaesthetics on tumor tracer uptake in radiopeptide receptor imaging (PRI) and radionuclide therapy (PRRT) 118 

Optical imaging of oral squamous cell carcinoma and cervical lymph node metastasis using near-infrared fluorescent 

probes in a mouse model – a pilot study 119 

Spectral unmixing of red and green luciferases for in vivo bioluminescence imaging 120 

High resolution redox imaging of intact cells by rxYFP, a ratiometric oxidation- sensitive fluorescent protein 

Assessment of antiangiogenic therapy effects in preclinical tumour models: Implementation of a novel contrast - 

121 

enhanced 3 D scanning technique and comparison to established ultrasound imaging protocols 

Influence of the BMP pathway on cell cycle regulation and its differentiation inducing potential on brain 

122 

tumor initiating cells 123 

A near infrared fluorescent-based method for imaging breast cancer induced osteolysis 124 

Near infrared fluorescent probes for whole body optical imaging of 4T1-luc2 mouse breast cancer development 

and metastasis 125 

IMAGING in DRUG DEVELOPMENT 

Improved animal models to study tumor growth and spontaneous metastases: bioluminescence imaging 

characterization of the HT-29 human colorectal cancer cell line 

Development of dorsal skin-fold window chamber for the analysis of blood vessel modifications induced by 

126 

electropermeabilization 

Characterization and evaluation of a tumor specific RGD optical probe for time-domain near-infrared 

127 

fluorescence imaging 128 

18F labeling of insulin via click chemistry 

Whole-body distribution, pharmacokinetics and dosimetry of radioiodinated fully humanized anti-VAP-1 

129 

antibody – a PET imaging study of rabbits 130 

Pharmacological characterization of iodine labeled adenosine kinase inhibitors 

Determining the nasal residence time of protein-polymer conjugates for nasal vaccination using a novel 

131 

imaging technique 132 

Nanotubes as mutli-modality vehicles for imaging and therapy of cancer 133 

Early assessment of temozolomide treatment efficacy in glioblastoma using [ 18F]FLT PET imaging 134 

Sensitive time-gated FRET microscopy of G-Protein coupled receptors using suicide enzymes, lanthanide cryptates and 

fluorescent ligands 135 

CANCER from BENCH to BEDSIDE 

Selection of a nanobody scaffold with low renal retention 136 

In vitro assessment of androgen mediated uptake of 18F-FDG, 11C-choline and 11C-acetate in prostate cancer 137 

Comparison of two 68Gallium-labeled octreotide analogues for molecular PET(CT) imaging of neuroendocrine tumours 138 

Real time per operative optical imaging for the improvement of tumour surgery in an in vivo micro-metastases model 139 

Fluorescence imaging modalities for imaging gastrointestinal tumor models 

Assessment of effectiveness and toxicity of the therapy with somatostatin analogue labelled 90Y-DOTATATE in 

140 

patients with non-functional pancreatic neuroendocrine tumours (PNT) 

Intra operative near-infrared fluorescent imaging of colorectal liver metastases using clinically available Indocyanine 

141 

Green in a syngene rat model 142 

Molecular imaging of resistance to EGFR tyrosine kinase inhibitors by 18F-FLT PET/CT and its reversal in non small cell 

lung cancer 143 

MI in CARDIOVASCULAR DISEASE 

Synchronised Cardiac and Lung CT in Rodents Using the Mobile CT Scanner LaTheta 144 

Molecular imaging of neurovascular inflammation in a mouse model of focal cerebral ischemia using Ultra small 

Superparamagnetic Particles of Iron Oxide (USPIOs) targeted to vascular cell adhesion molecule-1 (VCAM-1) 145 

Scintigraphy with the use of 123I-IL-2: a new promising tool for cardiovascular risk assessment in patients with high 

cardiovascular risk 146 


poStEr

112 


imaging life 

NEUROIMAGING from BENCH to BEDSIDE 

MEMRI-DTI study of focal transient ischemia in immature rat brain 147 

A clinically relevant model of in situ embolic stroke in the anesthetized monkey (macaca mulatta): long-term 

electrophysiological and mri analyses 148 

Automated radiosynthesis of [ 18F]MPPF derivatives for imaging 5-HT receptors 1A 

The vitamine E analogue CR6 protects against the long-term microstructure damage induced by MCA occlusion: a 

149 

longitudinal Diffusion Tensor Imaging study 150 

Design and synthesis of fluorescent probes for serotonin 5-HT1A receptors 151 

Comparative evaluation of cerebral blood flow in a rat model of cerebral ischemia using 15O-H 0 positron emission 

2 

tomography and 99mTc-HMPAO single-photon emission tomography 

Comparison of dopamine transporter density in Parkinson’s disease patients with and without autonomic dysfunction 

152 

using F-18 FP-CIT PET/CT 153 

Synthesis of tosylate and mesylate precursors for one-step radiosynthesis of [ 18F]FECNT 154 

Sex differences inDopamine D Receptor Occupancy in the Amygdala using AMPT with PET and [ 2 18F]fallypride 155 

Manual versus automatic delineation of VOIs for analysis of nuclear medicine images 156 

PET Amyloid and Tau Ligand [18F]FDDNP uptake in early Alzheimer disease 

Cryogenic brain injury as a model of brain trauma: Use of GFAP-luc mice to assess GFAP expression as an indication 

157 

of neural injury 158 

Plaque burden in the APPPS1 mouse picked up with diffusion kurtosis magnetic resonance imaging 159 

In vivo Imaging of Rat Glioma using the TSPO-ligand [ 18F]DPA-714 160 

GENE and CELL based THERAPIES 

A novel 19F MRI-based migration assay: application to primary human dendritic cells 

In vivo magnetic resonance imaging reveals altered migration of endogenous neural progenitor cells following 

161 

cuprizone-induced central nervous system demyelination 162 

Evolution pathways of compartmentalization and distribution of labeling iron-oxide particles in tumor tissue 163 

Acupuncture Works on Endorphins via Activating Stretch-Activated Cation Channels 164 

Labeling protocols for MRI and optical imaging of human muscle cells precursors 165 

Studying molecular processes in-vivo: A framework for quantifying variability in molecular MRI 166 

Clinically applicable cell tracking by MRI in cartilage repair using Superparamagnetic Iron Oxide (SPIO) 

Labeling of HUVEC with different iron oxide particles: An in vitro study about incorporation, distribution, 

167 

retention and toxicity 

Visualization of aberrant migration in the YAC 128 mouse model for Huntington’s disease by in situ labelling of 

168 

neural progenitor cells with iron oxide particles 169 

Optimization of in vitro radiolabeling of mesenchymal stem cells with 18F-fluorodeoxyglucose 170 

PROBE DESIGN 

Responsive MRI contrast agent for specific cell imaging of inhibitory, GABAergic neurons 171 

Novel ultrasound contrast agents for drug delivery 172 

Methods to study the interaction between aptamer probes and cell surface biomarkers 173 

MRI intelligent contrast agents as enzyme responsive nanosystems 174 

Standardization of molecular PBCA-microbubbles for routine use 175 

Novel Gd(III)-based probes for MR molecular imaging of matrix metalloproteinases 176 

Functionalization of nanoparticles for molecular imaging; a covalent approach 

Comparison of different chelating systems for the synthesis of Ga-68 labelled peptides for molecular imaging using 

177 

RGD-peptides as model compound 178 

GdDOTA-PIB: a potential MRI marker for Alzheimer’s disease 

A comparative study of the self-elimination of para-aminobenzyl alcohol and hemithioaminal-based linkers. 

179 

Application to the design of Caspase-3 sensitive pro-fluorescent probes 180 

Olefin Metathesis for the functionalization of superparamagnetic nanoparticles 

Pyridine-based lanthanide complexes : towards bimodal agents operating as near infrared luminescent and 

181 

MRI reporters 182 

TECHNOLOGY 

Delivery of multiple F-18 tracers from a single automated platform (FASTlab TM synthesizer) 183 

Monte-carlo modelling of a silicon detector insert combined with a PET scanner 184 

Autofluorescence corrected multispectral red-shifted fluorescent protein tomography 185


Acquiring the sample surface from co-registered FMT/MR measurements of a murine tumor model 186 

fDOT/ PET/CT imaging of biological processes in tumors 187 

Deep tissue molecular imaging with fluorescent biomarkers using multispectral optoacoustic tomography. 

A simulation study 188 

Microfluidic [ 11C]-carbonylation reactions for the rapid synthesis of radiolabelled compounds for PET 189 

Boosting image quality in low-dose RC-gated 5D cone-beam micro-CT 190 

Methodological approach using microscopy for quantitative evaluation of neo-angiogenesis and tumour 

progression in pre-clinical cancer models 191 

ENDOCRINE DISEASES 

Changes of heart stroke volume index established by 99mTc MIBI GSPECT scintigraphy after radioiodine treatment 

of patients with subclinical hyperthyroidism 192 

PET/CT investigations with 68Ga-DOATATE in neuroendocrine tumors - first clinical experience 193 

Influence of number of subsets and iterations of Ordered Subsets Expectation Maximization (OSEM 3D Flash) 

reconstruction on quantitative assessment of small and medium detected lesions in SPECT study with used in 

99mTc[EDDA/HYNIC]Octreotate for patients with GEP-NET 194 

The precise localization of metastatic lesion with used SPECT/CT with CoDe system after 131I – MIBG therapy in patients 

with disseminated pheochromocytoma 195 

Multimodal in vivo imaging of pancreatic beta-cells via antibody mediated targeting of beta-cell tumors 196 

INFECTION and INFLAMMATION 

6-[ 18F]Fluoro-PBR28, a novel TSPO 18 kDa radioligand for imaging neuroinflammation with PET 197 

Neuroinflammation is increased in the brain of ageing corpulent (JCR:LA-cp) rats: a positron emission tomography study 198 

Detection of inflammatory diseases by NIRF imaging with specific probes targeting leukotriene receptor CysLT R 1 199 

PET imaging of Hypoxia by 18F-Fluoromisonidazole ([ 18F]FMISO) to detect early stages of experimental arthritis 

Intracellular [ 

200 

64Cu]PTSM and extracellular [ 64Cu]DOTA-antibody labelling of ovalbumin-specific Th1 cells for in vivo PET 

investigations of Th1 cell trafficking in OVA-specific lung inflammation 201 

[ 18F]DPA-714, [ 18F]PBR111 and [ 18F]FEDAA1106: Radiosyntheses on a TRACERLAb FX-FN synthesizer 

In vivo near-infrared fluorescence imaging of lung matrix metalloproteinases in an acute cigarette smoke-induced 

202 

airway inflammation model in different mice strains 

Development and pre-clinical evaluation of a novel class of 

203 

18F labelled PET ligands for evaluation of PBR/TSPO 

in the brain 204 

Local administration of adeno-associated virus into the mammary gland ductules 205 

TARGETED THERAPY 

Copper-64- and Gallium-68- NODAGA-conjugated bombesin antagonists as new PET tracers 206 

A novel indocyanine green nanoparticle probe for non invasive fluorescence imaging in vivo 

Evaluation and optimization of the concept of an antibody directed enzyme-prodrug therapy using noninvasive 

207 

imaging technologies 208 

A novel [ 18F] PET imaging agent for the epidermal growth factor receptor 209 

MI DATA ANALYSIS METHODS 

Structural methods in subcellular image analysis 210 

In vivo-post mortem multimodal image registration in a rat glioma model 211 

Feasibility and success of Independent Component Analysis of resting state fmri data from the rat 212 

A fast and robust acquisition scheme for CEST experiments 

Binding potential estimation in 11C-PE2I PET brain striatal images: impact of partial volume correction under 

213 

segmentation errors 

Automated quantification scheme based on an adapted probabilistic atlas based segmentation of the brain basal 

214 

nuclei using hierarchical structure-wise registration 215 

VHISTdiff - comparing workflow histories (VHIST/VINCI) 216 

Fast matrix-free method for fluorescence imaging 217 

Late Breaking 

PET and MRI studies applied on characterization of Fisher/F98 rat glioma model 218 

Non-invasive “E2F sensing” system for monitoring DNA damage alteration induced by BCNU 

Multi-tracer PET imaging of a mouse model of Alzheimer´s disease to assess microglial activation related to ageing 

219 

and anti-inflammatory treatment 220 


poStEr 

Poster Session

P-001 

114 


In vivo MRI multicontrast kinetic analysis of intracellular trafficking of liposomes 

Aime S. , Delli Castelli D. , Dastrù W. , Terreno E. , Cittadino E. , Torres E. , Mainini F. , Spadaro M. . 

University of Torino, Italy 

silvio.aime@unito.it 

Introduction: Liposomes are mainly used in the 

pharmaceutical field as drug delivery systems. 

Despite their large use, there is still a lack of 

information about the interaction between the 

nanovesicles and the cells in tumor environments 

and their intracellular fate after the cellular uptake. 

Most of the information on this topic comes out from 

in vitro cellular studies that are not always reliable 

models for mimicking the in vivo system. The aim 

of this work is the visualisation of the metabolic 

pathway of these vesicles directly in vivo by means 

of MRI. Since MRI does not provide enough spatial 

resolution to directly observe events at subcellular 

level, we have developed a multicontrast analysis 

that provides indirect evidence about the uptake 

and the intracellular trafficking of the nanovesicles. 

The method relies on the peculiarity of nanovesicles 

encapsulating paramagnetic Ln(III)-based complexes 

that may act as T1, T2, and CEST agents. In order to 

account for the observed MRI data, a kinetic model 

able to describe the underlying biological processes 

has been developed. The fit of the data provides a 

rough estimate of the kinetic constants for each 

process considered in the model. 

Methods: Non targeted, stealth or pH-sensitive, 

liposomes encapsulating paramagnetic 

lanthanide(III) complexes were prepared and in vitro 

characterized. The liposomes were locally injected in 

B16 melanoma tumor xenografted on C57 mice. The 

temporal evolution of T1, T2 and CEST MR contrast 

was followed at 7 T until 48 h post-injection. 

Results. The evolution over time is different among 

the three contrast modes (T1,T2, CEST). The process 

taking place (cellular uptake, intracellular release, 

esocytosis, and wash out) have been modeled and 

rough estimates for the kinetic constants have been 

determined upon the simultaneous interpolation of 

all the data. Moreover, the intracellular trafficking of 

stealth vs pH sensitive liposomes has been compared. 

The comparison among the evolution of the three 

contrast modalities for the two different liposomes 

is quite different. In particular, the maximum T1 

contrast was observed at 5hrs post injection while for 

the stealth one it occurs at 24hrs post injection. 

imaging life 

Conclusions: The MRI multicontrast analysis 

developed in this work represents an innovative way 

to get deeper insight into the in vivo detection of 

sub-cellular process. In particular, the intracellular 

trafficking of two liposomal formulations (pHsensitive 

and conventional stealth) of great relevance 

in the field of drug delivery have been compared. The 

kinetic analysis revealed that both liposomes are taken 

up quite fast from Tumor Associated Macrophages 

but the intracellular release of the imaging 

reporters is much faster for the pH sensitive one.


Diffusion-weighted MRI for differentiation of breast lesions at 3.0 Tesla: a new biomarker for 

breast imaging 

Bogner W. , Gruber S. , Pinker K. , Grabner G. , Moser E. , Trattnig S. , Stadelbauer A. , Helbich T. . 

Department of Radiology, Vienna, Austria 

thomas.helbich@akhwien.at 

Introduction: To compare the diagnostic quality of 

different diffusion weighting schemes with regard 

to apparent diffusion coefficient (ADC) accuracy, 

ADC precision, and diffusion-weighted image 

(DWI) contrast-to-noise ratio (CNR) for different 

types of lesions and breast tissue. Based on these 

assessments a new biomarker for breast imaging 

will be defined. 

Materials and Methods: Institutional Review Board 

approval and written, informed consent were 

obtained. Fifty-one patients with histopathologic 

correlation or follow-up were included in this study 

on a 3.0 Tesla MR scanner. There were 112 regions 

of interest (ROIs) drawn in 24 malignant, 17 benign, 

20 cystic, and 51 normal tissue regions. ADC maps 

were calculated for combinations of ten different 

diffusion-weightings (b-values), ranging from 0 

to 1250 s/mm². Differences in ADC among tissue 

types were evaluated. The CNR of lesions on DWI 

was compared for all b-values. A repeated measure 

ANOVA was used to assess lesion differentiation. 

Results: ADC (mean±SD x10-3mm²/s) values 

calculated from b=50 and 850 s/mm² were 

0.99±0.18, 1.47±0.21, 1.85±0.22, and 2.64±0.30 

for malignant, benign, normal, and cystic tissue, 

respectively. An ADC threshold of 1.25 x10-3mm²/s 

allowed discrimination of malignant from benign 

lesions with a diagnostic accuracy of 95% (p

P-003 

116 


Imaging tumour apoptosis with 68Ga-labelled AnnexinA5 derivatives early after cancer 

therapy 

De Saint-Hubert M. (1) , Bauwens M. (1) , Devos E. (1) , Deckers N. (2) , Reutelingsperger C. (2) , Verbruggen A. (1) , Mortelmans L. (1) , 

Mottaghy F. (3) . 

(1) KULeuven, Heverlee, Belgium 

(2) University Maastricht, The Netherlands 

(3) RWTH Aachen, Germany 

marijke.desainthubert@med.kuleuven.be 

Introduction: Molecular imaging of apoptosis offers 

a direct and early measurement of response to 

cancer therapy which allows a fast decision making 

in cancer treatment. One of the early characteristics 

of apoptosis is externalization of phosphatidylserine 

(PS) on cell membranes. Annexin V 

(AnxA5) binds with a high affinity to membranebound 

PS. Due to suboptimal imaging quality of 

99m Tc labelled AnxA5 we aimed to specifically label 

AnxA5 with an emerging radioisotope, 68 Ga, allowing 

Positron Emission Tomography (PET). 

Methods: We radiolabelled AnxA5 with 68 Ga using 

two mutated forms of AnxA5 with a single cysteine 

residue at position 2 or 165, respectively Cys2- 

AnxA5 and Cys165-AnxA5, allowing site-specific 

coupling of 68 Ga-Dota-maleimide. In vitro cell 

binding of both radiotracers was studied in healthy 

and anti-Fas treated Jurkat cells. Biodistribution 

and pharmacokinetics were studied with µPET in 

healthy mice and in a hepatic apoptosis model (anti-Fas 

Ab treated) up to 60 min p.i.. Daudi (Burkitt 

lymphoma) tumour bearing mice were scanned 

before and after treatment with combined chemotherapy 

(125 mg/g Endoxan) and radiotherapy (10 

Gy/tumour) using µPET and µMRI. Tracer uptake 

was measured and imaged ex vivo using autoradiography 

and correlated to histological evidence of 

apoptosis (TUNEL). 

Results: 68 Ga-Dota-maleimide labelling yield was at 

least 98% and coupling yield of 68 Ga-Dota-maleimide 

to Cys2-AnxA5 and Cys165-AnxA5 was ~70%. 

Labelling and purification took about 60 min, with 

a final radiochemical purity of at least 98%. In vitro 

binding of 68 Ga-Cys2-AnxA5 and 68 Ga-Cys165- 

AnxA5 to anti-Fas treated tumour cells was 5 times 

higher compared to normal cells. 

Dynamic PET images in normal mice revealed that 

both tracers showed a fast clearance from the blood 

towards the kidneys, with no significant change in 

biodistribution from 30 min p.i. on. Dissection data 

confirmed clearance was mainly via the urinary tract. 

imaging life 

Dynamic PET images of anti-Fas treated animals 

revealed a major shift of radioactivity from the 

kidneys to the (apoptotic) liver for both 68 Ga- 

Cys2-AnxA5 and 68 Ga-Cys165-AnxA5. Compared 

to normal mice anti-Fas treated animals showed a 

7 to 9 times higher liver uptake (for respectively 

68 Ga-Cys2-AnxA5 and 68 Ga-Cys165-AnxA5) as 

compared to healthy animals. Autoradiography 

images confirm the higher uptake in anti-Fas 

treated livers, corresponding to TUNEL positive 

cells. 

MRI-PET fusion images allowed clear delineation 

of each tumor. Using this technique, we noted that 

the post-therapy SUV values significantly exceeded 

those prior to therapy. The absolute tumour uptake 

of 68 Ga-Cys2-AnxA5 and 68 Ga-Cys165-AnxA5 

was respectively only 0.5 ± 0.1 % ID/g and 1.0 ± 

0.3 % ID/g and significantly increased to 1.5 ± 0.2 

% ID/g and 1.6 ± 0.1 % ID/g after therapy. Autoradiography 

confirms an increased activity in 

treated tumors with several regions showing 10-20 

times higher uptake compared to the tumor uptake 

prior to treatment while other regions were 

found relatively unaffected. The same heterogeneous 

distribution of apoptosis was observed with 

TUNEL stainings. 

Conclusions: 68 Ga-Cys2-AnxA5 and 68 Ga- 

Cys165-AnxA5 can be prepared with a high yield 

and within a reasonable time period. Apoptosis 

targeting capacity was clearly demonstrated in 

a model of hepatic apoptosis. PET-MRI fusion 

images demonstrated a higher tumor uptake after 

cancer therapy, indicating that 68 Ga-AnxA5 

may be useful for the early evaluation of tumor 

therapy. 

Acknowledgement: This work was financially 

supported by the European Union through the 

grant Euregional PACT II by the Interreg IV program 

of Grensregio Vlaanderen-Nederland (IVA- 

VLANED-1.20).


68 Ga-RGD-based PET tracers for imaging αν β 3 expression: a comparative study 

Dumont R. (1) , Maecke H. (1) , Haubner R. (2) , Behe M. (1) , Weber W. (1) , Fani M. (1) . 

(1) University Clinic Freiburg, Germany 

(2) Medical University Innsbruck, Austria 

rebecca.dumont@uniklinik-freiburg.de 

Introduction: The integrin α ν β 3 is a cellular adhesion 

molecule that is frequently expressed on 

malignant tumors and acts as an important receptor 

affecting tumor angiogenesis, local invasiveness, 

and metastatic potential. For imaging of the 

α ν β 3 integrin, cyclic pentapeptides containing the 

tripeptide sequence arginine-glycine-aspartate 

(RGD) have been developed. These peptides specifically 

bind to α ν β 3 in its activated state and recent 

clinical trials have shown that PET imaging 

with radiolabelled RGD peptides allows quantitative 

determination of activated α ν β 3 integrin in 

patients with various malignant tumors [1,2]. Development 

of a 68 Ga-RGD-based tracer would be 

of great clinical utility given the increasing clinical 

importance of oncologic PET imaging and the excellent 

imaging properties and availabilty of 68 Ga. 

Thus, we developed [ 68 Ga]-DOTA-c(RGDfK) [3] 

and most recently [ 68 Ga]-NODAGA-c(RGDfK) 

[4]. The aim of this study was to compare the 

tracer properties in a U87MG human glioblastoma 

xenograft model. 

Methods: The conjugate DOTA-c(RGDfK) was 

synthesised according to the literature [3]. An 

alternative head-to-tail cyclization protocol of 

the linear RGDfK was followed for the conjugate 

NODAGA-c(RGDfK) using a 50% solution 

of 1-propanephosphonic acid cyclic anhydride 

(T3P), triethylamine, and 4-di(methylamino)pyridine 

(DMAP). The two conjugates NODAGAc(RGDfK) 

and DOTA-c(RGDfK) were labelled 

with 68 Ga in sodium acetate buffer 0.2 N, pH 4.0 

using the Modular-Lab PharmaTracer module by 

Eckert & Ziegler. Biodistribution studies were performed 

in balb-c nude mice bearing subcutaneous 

U87MG human glioblastoma xenografts 1 h postinjection 

of the radiotracers (600 pmol / 4 MBq / 

mouse). To evaluate specificity of the compounds, 

blocking studies were done with an excess of the 

commercially available c(RGDfV). Static images 

were concurrently accquired with a microPET Focus 

small animal scanner and all results were evaluated 

comparatively. 

Results: Both conjugates were labelled with 68 Ga 

with radiochemical purity > 97% and specific activity 

of 8-10 MBq/nmol at room temperature 

(NODAGA-c(RGDfK)) or elevated temperatures 

(DOTA-c(RGDfK)). The biodistribution profile 

of the two radio-conjugates was similar. However, 

[ 68 Ga]-NODAGA-c(RGDfK) showed faster blood 

clearance and higher tumor uptake compared to 

[ 68 Ga]-DOTA-c(RGDfK) (5.2 ± 1.4 %IA/g vs 3.5 ± 

0.8 %IA/g) and improved tumor-to-blood and tumor-to-kidney 

ratios (27.7 ± 7.0 vs 9.2 ± 1.1 and 2.6 

± 0.3 vs 1.6 ± 0.1, respectively). Specificity of both 

tracers was demonstrated by successful blocking of 

tumor uptake in the presence of excess c(RGDfV), 

present on both biodistribtion and imaging studies. 

PET imaging quantitatively confirmed tumor targeting 

and biodistribution results of both conjugates. 

Conclusions: Compared to [ 68 Ga]-DOTA-c(RGDfK), 

the superior labelling conditions and in vivo profile 

of [ 68 Ga]-NODAGA-c(RGDfK) make this 

compound a compelling radiotracer candidate 

for use in PET imaging of α ν β 3 -expressing tumors. 

References: 

1. Haubner R et al., PLoS Med. 2:e70 (2005) 

2. Beer AJ et al., Clin Cancer Res. 12:3942-9 (2006) 

3. Decristoforo C et al., Eur J Nucl Med Mol Imaging. 

35:1507-1515 (2008) 

4. Knetsch P et al., J Label Compd Radiopharm. 52: S413 

(2009) 


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118 


Influence of anaesthetics on tumor tracer uptake in radiopeptide receptor imaging (PRI) and 

radionuclide therapy (PRRT) 

Haeck J. , De Poel M. , Bijster-Marchand M. , Verwijnen S. , De Swart J. , Bernsen M. , De Jong M. . 

Erasmus Medical Centre, Oegstgeest, The Netherlands 

j.haeck@erasmusmc.nl 

Introduction: Neuro-endocrine tumors originating 

from various organs are known to over express somatostatin 

receptors (SSR) on the cell membrane (1). 

PRI and PRRT enable selective imaging and treatment 

of tumor cells over expressing SSR using radiolabelled 

somatostatin analogues, e.g. the SPECT 

tracer 111In-DTPA-octreotide. In experimental studies, 

in vivo imaging and measurement of tracer uptake 

in tumors and in normal organs is generally 

performed in anaesthetised laboratory animals. Different 

methods of anaesthesia are used according to 

the possibilities, experience and common practice 

in a laboratory. However, in many in vivo experiments 

the influence of changes in physiology due to 

anaesthetics is not taken into account. The aim of 

the current study was to determine the influence of 

different anaesthetics on bio-distribution of radioactive 

111In-DTPA-octreotide, a somatostatin analogue, 

5 

in rats bearing SSR positive tumors. 

Methods: Lewis rats (n=8) were inoculated with 0.5 

million CA20948 (SSR-positive) pancreatic tumor 

cells in the flank. The tumors were grown to approximately 

1.5 cm2 3 

2 

before imaging. Prior to i.v. injection 

of the radiolabelled peptide (50 MBq in 0.4 1 µg) the 

animals were anaesthetized with either inhalant-an- 

0 

aesthetic isoflurane (n=4) or a mixture of injectable 

anaesthetics consisting of sufentanil (300µg/kg) and 

medetomidine (300µg/kg) injected intra-peritoneally 

(n=4). The rats were kept under anaesthesia during 1h 

tracer circulation and subsequent SPECT/CT imaging. 

After imaging the rats were euthanized and tumor and 

organs were removed to study the peptide biodistribution 

ex-vivo. Biodistribution was performed at 3h p.i. 

Results: A significantly higher tumor uptake of 

111 In-DTPA-octreoscan was found when the rats 

were anaesthetized with isoflurane in comparison 

to medetomidine/sufentanil (figure 1), p


Optical imaging of oral squamous cell carcinoma and cervical lymph node metastasis using 

near-infrared fluorescent probes in a mouse model – a pilot study 

Keereweer S. (1) , Mol I. (2) , Snoeks T. (2) , Kerrebijn J. (1) , Van Driel P. (2) , Xie B. (2) , Kaijzel E. (2) , Baatenburg De Jong R. J. (1) , 

Löwik C.W.G.M. (2) . 

(1) Erasmus Medical Center, Rotterdam, The Netherlands 

(2) Leiden University Medical Center, The Netherlands 

s.keereweer@erasmusmc.nl 

Introduction: In head and neck cancer surgery, intra-operative 

assessment of the tumor-free margin 

is critical to completely remove the tumor, thereby 

improving the prognosis of the patient. Currently, 

this mostly relies on visual appearance and palpation 

of the tumor. However, optical imaging techniques 

provide real-time visualization of the tumor, 

warranting intra-operative, image-guided 

surgery. The use of the near-infrared (NIR) light 

spectrum offers two additional advantages: increased 

tissue penetration of light and an increased 

signal-to-background-ratio of contrast agents. We 

performed a pilot study to assess the possibilities 

of optical imaging of oral squamous cell carcinoma 

and cervical lymph node metastasis using 

near-infrared fluorescent probes in a mouse model. 

A 

B 

Fig1. A. Bioluminescence image of oral carcinoma and cervical 

lymph node metastasis. B. Fluorescence image showing increased 

signal of Prosense680TM in the tongue and cervical lymph nodes. 

Methods: An oral tumor model was developed using 

luciferase-bearing OSC19 (human oral squamous 

cell carcinoma) cells that were injected directly into 

the tongue of 8 Balb/C nu/nu female mice. Tumor 

progression was followed by bioluminescence imaging 

(BLI) using the IVIS-100 (Caliper LS) and by 

inspection of the tongue. At day 21, different NIR 

fluorescent probes were systemically injected, targeting 

cathepsins (Prosense680TM, VisEn Medical), 

matrix metalloproteinases (MMPsense 680TM, 

VisEn Medical), increased glucose uptake (800CW 

2-DG, LI-COR) and increased epidermal growth 

factor expression (800CW EGF, LI-COR) of the tumor. 

Fluorescence imaging of the mouse and organs 

(after surgical removal) was performed using the 

Maestro (CRi) and Odyssey (LI-COR). A control 

group of 8 Balb/C nu/nu mice without tumor was 

used to compare light intensity between healthy and 

cancer tissue. 

Results: After 7 days, oral squamous cell carcinoma 

was established in all 8 mice. After 11 days, all 

mice had developed cervical lymph node metastasis, 

which was confirmed by a BLI signal (figure 1,A). 

The primary tumor and cervical lymph node metastases 

were successfully detected by the use of all NIR 

fluorescent probes, with increased tumor-to-control 

ratios that varied per probe (figure 1,B). 

Conclusions: This preliminary study shows the establishment 

of an oral squamous cell carcinoma 

mouse model, which progress could be followed by 

bioluminescence. Moreover, real-time visualization 

of the primary tumor and cervical lymph node metastases 

was achieved by fluorescence imaging using 

various NIR fluorescent probes. This technique 

can be used for intra-operative, image-guided surgery, 

which could improve complete removal of oral 

squamous cell carcinoma. 


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P-007 Spectral unmixing of red and green luciferases for in vivo bioluminescence imaging 

120 

Mezzanotte L. (1) , Kaijzel E. (2) , Michelini E. (1) , Que I. (2) , Calotti F. (2) , Hoeben R. (2) , Roda A. (1) , Löwik C. W.G.M. (2) . 

(1) University of Bologna, Bologna, Italy 

(2) Leiden University Medical Center, Leiden, The Netherlands 

laura.mezzanotte@libero.it 

Introduction: Amongst the numerous luciferase reporter 

genes cloned from different animal species 

and mutated to achieve different emission properties 

only a few have been employed for in vivo bioluminescence 

imaging (BLI). Good thermostability, high 

and stable photon emission and codon optimization 

of the luciferase gene are required (1,2). Moreover, 

spectral overlap prevents them from being measured 

simultaneously. Recently, spectral unmixing 

methodologies have been employed to unmix and 

quantify signals from images obtained from the 

collection of light emitted from proteins with different 

emission spectra (3,4). Here we investigated 

the combined use of green click beetle luciferase 

(CBG99, max. emission 537nm) and a red codonoptimized 

mutant of P.pyralis (Ppy-RE8, max emission 

618nm) for in vivo BLI purposes. 

Methods: Both in vitro and in vivo studies were carried 

out to analyse the different luciferases. Lentiviruses 

expressing Red Ppy-RE8 and Green CBG99 

luciferase reportergenes were generated and human 

embryonic kidney (Hek293) cells were subsequently 

Fig1.: CBG99 and PpyRE8 expressing cells were mixed in different 

ratios and measured in vitro (a) and in vivo at 540nm (c) and 

620nm (d).The graph (b) shows the max. emission peaks of the 

different luciferases. 

imaging life 

transduced to express the different variants of luciferase. 

Cell population of red and green emitting 

were mixed in different proportions and BLI measurements 

were carried out in a plate luminometer 

with appropriate filters. 

In addition, Hek293 cells co-expressing the mentioned 

luciferases were imaged using the IVIS Spectrum system 

(Caliper LS) by collecting BLI signals at different 

wavelengths. Finally, the cells expressing Ppy-RE8 or 

Green CBG99 were injected subcutaneously into immunodeficient 

mice and BLI imaging was performed 

in vivo using the same system. Signals from the two 

luciferases were unmixed using Living Image 3.2 

software. 

Results: Both in vitro as in vivo results demonstrated 

the feasibility to use Red Ppy-RE8 and Green CBG99 

luciferase reporter genes, simultaneously. These luciferase 

variants demonstrated to be a good couple for 

dual luciferase applications employing the same substrate 

luciferin. Using a pair of band pass filter (535nm 

and 628nm) contributions of red- and green-emitting 

luciferases can be calculated using a luminometer. 

Confirmative results were obtained using the Living 

image software to spectrally unmix the images composed 

by the signals of the two different luciferases and 

obtained from both in vitro and in vivo experiments. 

Conclusions: The possibilities of a combined use of 

the selected red and green luciferases have been demonstrated. 

Preliminary in vivo data envisage the future 

application of these couple of luciferases for monitoring 

of multiple events simultaneously by means of bioluminescence 

imaging. 

References: 

Branchini BR et al. Anal Biochem. 2010 Jan 15;396(2):290-7. 

Mezzanotte L, et al. Mol Imaging Biol. 2009,Nov 25. 

Gammon ST, et al. Anal Chem. 2006 Mar 1;78(5):1520-7. 

Michelini E, et al. Anal Chem. 2008 Jan 1;80(1):260-7.


High resolution redox imaging of intact cells by rxYFP, a ratiometric oxidation- sensitive 

fluorescent protein 

Pani G. , Maulucci G. , Mele M. , Labate V. , Panieri E. , De Spirito M. . 

Catholic University Medical School, Rome, Italy 

gpani@rm.unicatt.it 

Introduction: Plasmid encoded redox sensitive 

fluorescent probes promise to pave new avenues 

for realt time imaging of oxidative signaling in live 

cells. We have recently shown that rxYFP, a redoxsensitive 

variant of the Yellow Fluorescent Protein, 

can be used ratiometrically to construct high resolution 

redox maps of live cells. Confocal analysis of 

cell fluorescence at two excitation wavelenghts allows 

in fact to monitor, voxel by voxel, the distribution 

of the probe between its reduced and oxidised 

states, while normalizing fror probe concentration 

and photobleaching. 

Methods: rxYFP was transiently transfected in 293T 

human kidney carcinoma and B16F10 murine melanoma 

cells plated on glass bottom dishes (IbiDi). 

Cell fluorescence was analysed by confocal microscopy 

at two different excitation wavelengths (450 

and 488nm) and fluorescence ratios calculated by a 

dedicated software and converted into pseudocolors 

to construct redox-based cell maps. 

Results: By imaging cells based on rxYFP distribution 

between reduced and oxidised states, we were 

able to confirm that, in human and murine malignant 

cells, the nucleus is significantly more reduced than 

the cytosol; additionally, simple deconvolution of redox 

images constructed with untargeted rxYFPallows 

to identify hypereduced perinuclear spots that correspond 

to mitochondria, as further confirmed by the 

use of a mitochondrially targeted form of the probe. 

In 2D scans, the cell border of adherent cells consistently 

appears to be significantly more oxidised than 

the inner cytosol, while 3D cell reconstruction oxidation 

is polarized towards the bottom of the cells; finally, 

in a spontaneously migrating cell the leading edge 

appears to me more oxidised than the trailing one. 

Conclusions: This set of obserbations suggests, in 

keeping with our previous biochemical studies, that 

integrin signaling and cuytoskeleton rearrangement 

are associated with increased prooxidant activity, with 

relevant implications for cell invasion and metastasis. 

Acknowledgement: Work partially supported by DiMI 

FP6 European NoE DiMI (LSHB-CT-2005-512146) 

References: 

1. Maulucci G, Labate V, Mele M, Panieri E, Arcovito G, 

Galeotti T, Østergaard H, Winther JR, De Spirito M and 

Pani G High resolution imaging of redox signaling 

in live cells through an oxidation-sensitive yellow 

fluorescent protein Sci. Signal. 1, pl3 (2008). 

2. Maulucci G, Pani G, Labate V, Mele M, Panieri E, Papi M, 

Arcovito G, Galeotti T, De Spirito M. 2009 Investigation 

of the spatial distribution of glutathione redox-balance 

in live cells by using Fluorescence Ratio Imaging 

Microscopy. Biosens Bioelectron. 25:682-687. 

3. Maulucci G, Pani G, Fusco S, Papi M, Arcovito G, Galeotti 

T, Fraziano M, De Spirito M. 2009 Compartmentalization 

of the redox environment in PC-12 neuronal cells. Eur 

Biophys J. in press 

4. P. Chiarugi, G. Pani , E. Giannoni, L. Taddei, R. Colavitti, 

G. Raugei, M. Symons, S. Borrello, T. Galeotti, and G. 

Ramponi 2003 Reactive oxygen species as essential 

mediators of cell adhesion:The oxidative inhibition of a 

FAK tyrosine phosphatase is required for cell adhesion 

J. Cell. Biol. 161, 933-944 


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P-009 Assessment of antiangiogenic therapy effects in preclinical tumour models: Implementation 

of a novel contrast - enhanced 3 D scanning technique and comparison to established 

ultrasound imaging protocols 

Rix A. , Lederle W. , Bzyl J. , Gaetjens J. , Fokong S. , Grouls C. , Kiessling F. , Palmowski M. . 

Helmholtz Institute for biomedical engineering , Aachen, Germany 

arix@ukaachen.de 

Introduction: Assessment of antiangiogenic therapy 

effects in rodent tumours plays an important role 

in drug development. Different ultrasound imaging 

protocols are described, predominantly using 2 D 

contrast-enhanced techniques. However, a limitation 

of 2 D scans is the reduced reproducibility of 

the data. Even if the acquired slice might be representative 

for the entire tumour, it will be impossible 

to find the identical slice position in longitudinal 

studies. In our study, we implemented a novel contrast-enhanced 

3 D scanning technique, evaluated 

its sensitivity and compared it to established 2 D 

and 3 D ultrasound imaging protocols. 

Methods: S.c epidermoid carcinoma xenografts in 

nude mice were scanned using a small animal ultrasound 

system (40 MHz). B-mode images with a 

low mechanical index and a slice thickness of 300 

µm were acquired and merged into a 3 D dataset. 

Prior to administration of the contrast agent, only 

one image was acquired per slice. During stable 

blood levels of polybutylcyanoacrylate-microbubbles, 

5 frames per slice were acquired (frame rate: 8 

Hz). The maximum intensity of each voxel per slice 

was recorded and compared to the pre-contrast 

dataset. To compare the potential role of this technique, 

a non contrast-enhanced 3 D Power Doppler 

scan (HF-VPDU: High frequency - volumetric 

power Doppler ultrasound [1]) and a 2 D dynamic 

contrast-enhanced scan (analyzed using the MIOT 

post-processing technique [2]) were performed. 

The sensitivity of all methods for assessing the antiangiogenic 

effects of SU11248 were examined at 

day 0, 1, 2 and 4 after treatment start. Tumour vascularisation 

was validated by determining the vessel 

density on corresponding histological sections. 

Mann-Whitney test was used for statistical analysis. 

Results: The novel 3 D contrast-enhanced imaging 

protocol could be applied to all animals successfully. 

The vascularisation of untreated control tumours 

slowly increased during the study, whereas 

a reduction was observed in tumours of treated 

animals. Differences in vascularisation between 

imaging life 

both groups were significant at day 1 (p


Influence of the BMP pathway on cell cycle regulation and its differentiation inducing 

potential on brain tumor initiating cells 

Rudan D. (1) , Monfared P. (1) , Viel T. (1) , Hadamitzky M. (1) , Euskirchen P. (1) , Knödgen E. (1) ,tSchneider G. (1) , Jacobs A.H. (1,2) . 

(1) Max Planck Institute for Neurological Research, Cologne, Germany 

(2) European Institute for Molecular Imaging, , Germany 

daniel.rudan@nf.mpg.de 

Introduction: Glioblastoma multiforme is the 

most common type of brain tumor, and several alterations 

of the cell cycle and DNA repair mechanisms 

have led to increasing resistance against 

chemotherapy. Latest findings suggest that a subset 

of tumor cells, the brain tumor initiating cells 

(BTIC) with stem cell like properties, are responsible 

for initiation and maintenance of the disease. 

Approximately 1-1000 out of 1.000.000 cells 

within a glioma is a BTIC. These cells are thought 

to bring about relapse and metastasis through 

their tumorigenic potential and chemotherapeutic 

resistance1,2. Design of a specific therapeutic 

strategy against BTICs may improve overall survival 

and quality of life for patients with gliomas. 

The aim is to examine a novel, experimental therapy 

against malignant gliomas by differentiating 

their BTIC population. Building on our previous 

work3, where we demonstrated that BMP-7 treatment 

decreases the proliferation of Gli36ΔEGFR- 

LITG glioma cells up to 50% via a cell cycle arrest 

in G1 phase, we will further characterize and 

compare the effect of the BMP-pathway on cell 

cycle regulation of different glioma cell lines and 

“glioma stem cells”. 

Methods: We want to determine (i) whether BMP 

treatment, alone or in combination with state-ofthe-art 

chemotherapeutic agents, could be applied 

in a disease-tailored therapy against gliomas by 

depleting their BTIC pool and (ii) whether our 

reporter construct (LITG) can quantify the responses 

to such a treatment in culture and in vivo. 

Glioma cell lines, primary glioma cell cultures 

and BTICs with different genetic profiles are being 

utilized to analyze the influence of BMP-7 

and BMP pathway inhibitors on cell cycle regulating 

protein expression, cell viability and caspase 

activation as well as its differentiation-induction 

potential in culture. BTIC will be transduced 

with lentiviral reporter vectors to image changes 

in cell cycle regulation, differentiation status 

and BMP pathway activity non-invasively in vivo 

upon treatment. 

Results: Western blot data indicates that BMP-7 

treatment causes an arrest in cell cycle progression 

in the established glioma cell line A172 by influencing 

the expression of the key cell cycle regulators 

(p53�, p21�E2F-1�). Furthermore we have shown 

the differentiation-induction potential of BMP-7 on 

BTICs in culture optically and on the protein level. 

Upon BMP-7 treatment and respective growth-factor 

withdrawal the expression of the neural stem cell 

marker Mshi1 is clearly downregulated. Currently 

we focus on the design of a lentiviral reporter vector 

for imaging the observed BMP-Pathway reponses. 

Conclusions: Antagonizing the proliferative potential 

of BTICs by targeting the BMP pathway, thereby 

triggering cellular differentiation of malignant stem 

cells, could provide a promising means of improving 

the prognosis for patients with gliomas. 

Acknowledgement: This work is supported in part by 

the FP6 European NoE DiMI (LSHB-CT-2005-512146). 

References: 

1. Singh SK, Hawkins C,Clarke ID, Squire JA, Bayani J, Hide 

T, Henkelman RM, Cusimano MD, Dirks PB, Nature 432, 

396 (2004) 

2. Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda 

E, Broggi G, Brem H, Olivi A, Dimeco F, Vescovi AL, 

Bone morphogenetic proteins inhibit the tumorigenic 

potential of human brain tumour-initiating cells, 

Nature 444, 761 (2006) 

3. Klose A, Waerzeggers Y, Klein M, Monfared P, Vukicevic 

S, Kaijzel EL, Winkeler A, Löwik CW, Jacobs AH, Imaging 

bone morphogenetic protein induced cell cycle arrest 

in experimental gliomas, In Submission 


P-010 

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124 


P-011 A near infrared fluorescent-based method for imaging breast cancer induced osteolysis 

Snoeks T. , Que I. , Mol I. , Kaijzel E. , Löwik C.W.G.M. . 

Leiden University Medical Center, The Netherlands 

t.j.a.snoeks@lumc.nl 

Introduction. In vivo bioluminescence imaging 

(BLI) of luciferase (Luc) expressing tumor cell lines 

has become a well accepted method to quantitatively 

follow tumor growth over time. Fluorescence 

imaging (FLI) can be used to visualize and quantify 

specific structures, molecules and even enzymatic 

activity using targeted dyes and enzyme activated 

smart probes. Two of such targeted fluorescent dyes 

are OsteoSense-680TM (VisEn Medical) and Bone- 

Tag-680TM (LI-COR Biosciences) both of which are 

directed specifically to bone. 

Traditionally, osteolytic lesions and subsequent bone 

loss are quantified using x-ray radiographs and μ-CT 

scans. These procedures are often time consuming 

and both methods possibly irradiate the animal limiting 

the number of repeated measurements. 

The aim of this study was to assess the possibility 

to quantify osteolysis by a loss of fluorescent signal 

after pre-labelling the skeleton with a bone-specific 

fluorescent probe. In this setup, we compared the 

performance of the two aforementioned commercially 

available bone specific fluorescent probes; 

OsteoSense-680TM and BoneTag-680TM. 

Fig1.: Decrease of fluorescence signal on osteolysis. 

Representative mouse labelled with BoneTag-680, imaged with 

the Pearl imager. A) Background FLI image before labelling 

with BoneTag-680. B-E) FLI images at day 2, 8, 19 and 42 after 

tumor cell inoculation in the right leg (*). F) Quantification of 

the fluorescent signal of the tumor bearing leg compared to 

the healthy leg. 

imaging life 

Methods. Immunodeficient mice received an intra 

venous injection with either OsteoSense-680 (2 

nmol, n=5) or BoneTag-680 (8 nmol, n=5). Three 

days after injection of the fluorescent probes the 

human breast cancer cell line MDA-BO2-Luc was 

inoculated in the femur bone marrow cavity of the 

right leg to form an osteolytic tumor. Tumor growth 

was followed by weekly BLI measurements using the 

IVIS Spectrum (Caliper LifeSciences) over a period 

of 6 weeks. Fluorescent data was obtained weekly using 

the IVIS Spectrum and at day 2, 8, 19 and 42 using 

the Pearl Imager (LI-COR Biosciences). In addition, 

μCT scans of each animal were made at day 21 

and 42 using the SkyScan-1076 μCT (SKYSCAN). 

Results. All inoculated mice developed a tumor in 

the right leg confirmed with BLI. Both bone specific 

probes remained detectable throughout the experiment, 

45 days after injection, in the healthy leg. The 

fluorescence signal decreased faster in the tumor 

bearing legs than in the healthy legs, this loss of signal 

could be quantified (Fig 1). The analysis of the 

x-ray and μCT data and the subsequent correlation 

between oslteolytic lesion size and the loss of fluorescence 

signal is still ongoing. 

Conclusions. Preliminary results show the possibility 

to follow osteolysis over time using an animal 

pre-labelled with a bone specific probe. 

Acknowledgement: Supported by the Dutch Cancer 

Society (Grant UL2007-3801)


Near infrared fluorescent probes for whole body optical imaging of 4T1-luc2 mouse breast 

cancer development and metastasis 

Xie B. , Snoeks T. , Mol I. , Van Driel P. , Keereweer S. , Kaijzel E. , Löwik C.W.G.M. . 

Leiden University Medical Center , The Netherlands 

b.xie@lumc.nl 

Introduction: The rapid development of molecular 

imaging has improved early diagnosis and treatments 

for various diseases including cancer. The 

imaging modalities vary broadly in their sensitivitie, 

and the high sensitivity of optical imaging, especially 

near infrared fluorescent (NIRF) imaging, 

makes it an excellent experimental tool for imaging 

small animals like mice. NIRF light has advantages 

such as considerably low tissue absorption coefficient 

in the NIR region (700-900 nm) and low tissue 

autofluorescence so that deeper light penetration 

can be achieved. These advantages make NIR light 

ideal for whole body optical imaging. Furthermore, 

NIRF probes are becoming available for clinical applications, 

e.g. imaging-guided surgery. In the current 

work, we have implanted mouse mammary 

gland cancer cell line 4T1-luc2 in nude mice, and 

then assessed 2 commercially available NIRF probes 

in their ability and specificity of detecting tumor 

progression and metastasis by whole body FLI in 

comparison with BLI. 

Methods: The following NIRF probes were tested 

and validated on detecting the mouse mammary 

gland cancer cell line 4T1-luc2 both in vitro and in 

vivo: Prosense680 TM (VisEn Medical) and 800CW 

Figure: Upper panel shows BLI and FLI Prosense680TM imaging. 

Lower panel shows BLI and FLI Prosense680TM imaging of LNs. 

2-DG(LI-COR Biosciences). In vitro, studies included 

cell-based fluorescent assay by Odyssey measurements, 

flow cytometry analysis, and visualization 

of probe uptake by confocal laser scan microscopy 

analysis. In vivo, 20.000 4T1-luc2 cells were implanted 

into the upper mammary fat pad (MFP) of 

immunodeficient mice. BLI was used as an internal 

control to evaluate the luciferase expression level in 

tumor areas, and also for co-locolization of the NIRF 

probes. After time-dependent BLI and FLI measurements, 

thoracic cavities of mice bearing tumors 

were surgically opened and reimaged to reveal the 

metastasis of surrounding tissues and axial lymph 

nodes (LNs). Organs were quickly removed, and ex 

vivo BLI and FLI were performed. Tissues with positive 

signals were further analyzed by histochemistry. 

Results: Both tested probes could successfully visualize 

4T1-luc2 mouse breast cancer cells, both in 

vitro and in vivo. Our in vivo data showed that there 

was already a nice increase in the BLI signal 3 days 

after implanting 4T1-luc2 cells in the MFP of nude 

mice. We could also detect the tumor progression 

using the NIRF probes in intact animals. At the end 

of the experiment (18 days) the animals were surgically 

opened up, and we could clearly detect lung and 

axial LNs metastases, both by BLI and FLI. This was 

confirmed by ex vivo imaging and histochemistry. 

Conclusions: This preliminary study of 4T1-luc2 

mouse breast cancer indicates that the development 

and metastasis of cancer can not only be detected 

by BLI but also by FLI, using commercially available 

NIRF probes, especially after surgically opening 

up the animals. This should pave the way for realtime 

visualization of tumor tissues during operation, 

making radically removal of all tumor tissues and 

local metastases possible. 

Acknowledgement: This study is supported by the 

Dutch CTMM Project MUSIS 


P-012 

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P-013 Improved animal models to study tumor growth and spontaneous metastases: 

bioluminescence imaging characterization of the HT-29 human colorectal cancer cell line 

Fernández Y. , Miranda S. , López M.E. , Suárez L. , Céspedes M.V. , Mangues R. , Herance J. R. , Rojas S. , Abasolo I. , 

Schwartz Jr S. . 

CIBBIM-Nanomedicina. Edifici Hospital General. Bioquímica, Barcelona, Spain 

yofernan@ir.vhebron.net 

Introduction: The main problem in the treatment of 

colorectal cancer (CRC) is not so much the eradication 

of the primary tumor, but rather the formation 

of incurable metastases. To improve survival of 

these patients, there is an urgent need for new treatment 

strategies. For this purpose, the development 

of reliable, reproducible, clinically-relevant and 

inexpensive mouse models that fulfill tumor progression, 

invasion and metastasis of human CRC is 

needed. A new era of modeling cancer metastasis 

involves the use of optical imaging technologies to 

monitor tumor growth and colonization after introduction 

of cancer cells into the animals. The purpose 

of this study was to characterize the behavior 

of the human colon cancer cell line HT-29 in a subcutaneous, 

experimental metastases and orthotopic 

mouse models using non-invasive bioluminescence 

imaging (BLI) technologies, thereby providing an 

additional method to study CRC disease. 

Methods: Luciferase expressing HT-29 cells were injected 

subcutaneously, into the left ventricle and orthotopically 

into the cecal wall of immunodeficient 

nude mice. The tumor growth and metastatic dissemination 

patterns were quantitatively and continuously 

followed up via BLI. Non-invasive monitoring 

of tumorigenicity and metastasis was also compared 

to the traditional assays of tumor volume, weight or 

histology. Ex vivo BLI and histological analyses were 

performed to further identify the exact nature and location 

of lesions. BLI results were also validated using 

the positron emission tomography (PET) imaging. 

Results: We show that BLI has been successfully 

used to monitor colorectal tumor growth and metastases 

in vivo, and even helps to identify new 

metastatic sites. In the subcutaneous mouse model, 

BLI production and traditional tumor volume 

measurements show a strong correlation, demonstrating 

that BLI is an appropriate method to noninvasively 

quantify tumor burden. The left cardiac 

ventricle injection resulted in colonic tumor 

colonies in most organs, including the skeletal 

system of mice. In orthotopic implants, locoregional 

tumor growth and distant metastases occur 

spontaneously and rapidly, closely resembling 

imaging life 

clinical human disease progression that includes 

lymphatic, hematogenous and celomic dissemination. 

Ex vivo BLI confirmed the localization of 

metastases and dictated which tissues were going 

to be analyzed by histopathology reducing the 

number of histology rounds required to eventually 

detect all the small micrometastases identified 

by BLI. 

Conclusions: Our findings show that BLI improves 

upon and refines traditional animal cancer 

models by using fewer animals, offering a rapid, 

sensitive and less invasive monitoring of early 

neoplastic growth and metastases. Moreover, it 

provides an accurate and temporal assessment in 

the same animal over time increasing the statistical 

power of the model. In conclusion, BLI is a 

powerful tool for high-throughput longitudinal 

monitoring of tumor load in small animals and 

allows the implementation of more advanced orthotopic 

tumor models in therapy intervention 

studies with almost the same simplicity as when 

measuring traditional ectopic models. The availability 

of these bioluminescent models are powerful 

and reliable tools with which to investigate 

metastatic human CRC and novel therapeutic 

strategies directed against it. 

Acknowledgement: We are grateful to the Spanish 

Ministry of Science and Innovation for supporting 

laboratory technician and pre-doctoral personal, 

and CIBER-BBN for funding the project. 

References: 

1. Taketo, M. M. & Edelmann, W.; Gastroenterology 136, 

780-98 (2009) 

2. Cespedes, M. V.; et al. Am J Pathol 170, 1077-85 (2007) 

3. Weissleder, R. & Pittet, M.; J. Nature 452, 580-9 (2008) 

4. O’Neill, et al.; J Pathol 220, 317-327 (2009)


Development of dorsal skin-fold window chamber for the analysis of blood vessel 

modifications induced by electropermeabilization 

Golzio M. (1) , Bellard E. (1) , Markelc B. (2) , Cemazar M. (2) , Sersa G. (2) , Teissie J. (1) . 

(1) IPBS-CNRS, Toulouse, France 

(2) Institute of Oncology, Slovenia 

muriel.golzio@ipbs.fr 

Introduction: Recent developments in intravital microscopy 

(IVM) enable studies of tumour angiogenesis 

and microenvironment at the cellular level after 

different therapies. Preparation of skin fold chamber 

enables to follow fluorescent events on live animal. 

Electroporation/electropermeabilization, i.e. application 

of electric pulses to tissues, is a physical method 

for delivery of exogenous molecules. It is already used 

in clinical therapies of cancer, for electrochemotherapy 

of tumors (ECT). Its use was recently developed 

in electrogene therapy (EGT). In vivo, “electroporation” 

is associated with a blood -flow modifying effect 

resulting in decreased blood flow. 

The aim of our study was to observe directly on the 

living animal the effects of “electropermeabilization” 

on subcutaneous normal blood vessels by monitoring 

changes in morphology (diameter) and dynamics 

(vasomotricity, permeability and recovery). 

Methods: These parameters were measured using 

fluorescently labelled dextrans injected in the 

blood vessels observed via a dorsal skin fold window 

chamber, intravital digitized stereomicroscope, 

in vivo intravital biphoton microscopy and custom 

image analysis. A mathematical modelling gave access 

to the changes in permeability from the time 

lapse observation. Delivery of electric pulses was 

operated on the microscope stage directly on the 

animal under anaesthesia. 

Results: It resulted in immediate constriction of 

blood vessels that was more pronounced for arterioles 

(up to ~65%) compared to venules (up to ~20%). 

A rapid increase in vascular permeability was present 

that gradually decreased to basal (control) levels at 

1 h post-treatment. The decay of the high increase 

in vascular permeability was biphasic with an initial 

fast decrease, but was still present at 1h post-treatment. 

Furthermore, vasoconstriction of arterioles after 

“electropermeabilization” resulted in a “vascular 

lock” that remained for at least 6 minutes. This correlated 

approximately with the duration of decreased 

diameters of arterioles that lasted for 8 minutes. 

Conclusions: the results of our study provided 

direct in vivo monitoring of a vascular effect of 

electric pulses on normal vessels. The observed 

increase in permeability of vessels associated 

with delayed perfusion induced by electric pulses 

explains the improved delivery of molecules into 

tissues induced by this method after systemic 

delivery. 

Acknowledgement: CNRS, Region Midi Pyrenees, 

ARC, canceropole GSO, ANR “Cemirbio”, Slovenian 

French Proteus 

References: 

1. Cemažar M, Golzio M, et al. Current Pharmaceutical 

Design 12: 3817-3825. (2006). 

2. Marty M, et al. EJC 4(11): 3-13. (2006). 

3. Sersa G, et al. Br J Cancer; 98: 388-398 (2008). 

4. Golzio M., et al. Gene Therapy 11, S85-S91 (2004). 


P-014 

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P-015 Characterization and evaluation of a tumor specific RGD optical probe for time-domain 

near-infrared fluorescence imaging 

Mathejczyk J.E. (1) , Resch-Genger U. (2) , Pauli J. (2) , Dullin C. (3) , Napp J. (1) , Tietze L. F. (4) , Kessler H. (5) , Alves F. (1) . 

(1) Max-Planck Institute for Experimental Medicine, Göttingen, Germany 

(2) BAM Federal Institute for Materials Research and Testing, , Germany 

(3) University Medical Center Göttingen, Germany 

(4) University of Göttingen, Germany 

(5) Technical University of Munich, Germany 

jmathej@gwdg.de 

Introduction: RGD peptides provide useful tools 

for specific targeting of tumors overexpressing 

α ν β 3 integrins. We used a cyclic RGDfK peptide, 

coupled to the near-infrared fluorophore, Cy5.5, 

via an aminohexaonic spacer (RGD-Cy5.5) for 

functional imaging of α ν β 3 integrins on tumors in 

vivo. To assess the suitability and the achievable 

sensitivity of RGD-Cy5.5 for tumor imaging 

we characterized the spectroscopic properties 

of the optical probe and applied time-domain 

near-infrared fluorescence (NIRF) imaging for 

noninvasive and specific tumor targeting in vivo. 

Methods: Application-relevant properties like the 

fluorescence quantum yield, lifetime and the thermal 

stability of the cyclic RGDfK peptide coupled 

to Cy5.5 via an aminohexanoic acid spacer and its 

parent fluorophore, Cy5.5 were analyzed spectroscopically. 

For in vivo imaging, human α ν β 3 integrin-expressing 

glioblastoma cells, U87MG, were 

subcutaneously implanted into nude mice. Imaging 

was performed using the time-domain fluorescence 

imager, Optix MX2 (ART, Montreal, Canada). 

Results: RGD-Cy5.5 shows excellent spectroscopic 

properties making it a useful tool for in vivo NIRF 

imaging. Remarkable is the enhancement in fluorescence 

quantum yield of Cy5.5 in RGD-Cy5.5 increasing 

from 0.29 to 0.34. In vivo, the specificity 

of the RGD-Cy5.5-derived signals was confirmed 

by fluorescence lifetime measurements which were 

used to distinguish the probe signals from unspecific 

fluorescence. RGD-Cy5.5 binds selectively to 

glioblastoma with a maximum fluorescence intensity 

resulting 5 h after probe injection. The binding 

specificity was further confirmed by reduction 

of this fluorescence after application of an excess 

of unlabeled RGD peptides. 

Conclusions: Knowledge of the spectroscopic properties 

of fluorescent conjugates represents an important 

prerequisite for the successful application 

and sensitive detection of fluorescent probes in vivo. 

With RGD-Cy5.5, we developed a strongly emissive 

and stable optical probe for targeting α ν β 3 integrin 

receptors. Fluorescence lifetime measurements 

imaging life 

contributed to a further enhancement in detection 

sensitivity as compared to conventional steady state 

NIRF imaging in the intensity domain. Since the 

RGD probe contains an aminohexanoic acid spacer, 

that allows an easy and effective coupling with 

anti-cancer agents, this probe might be applied in 

oncology for therapeutic and diagnostic purposes.

18 F labeling of insulin via click chemistry 


Paris J. , Mercier F. , Thonon D. , Kaisin G. , Lemaire C. , Goblet D. , Luxen A. . 

University of Liege, Belgium 

J.Paris@ulg.ac.be 

Introduction: As a positron emission tomography 

probe, a new 18 F-bearing insulin derivative was prepared 

by an original labeling method. This tracer was 

required as a radiolabelled active principle model 

compound to perform biodistribution imaging studies 

of new pulmonary administrable formulations. 

Methods: A 1,3-dipolar azide/alkyne cycloaddition 

(click chemistry) approach was developed for the 

mild and efficient linking of the radioactive probe 

onto insulin (Fig1). 

Gly A1 Insulin 

H2N CO2H H2N Phe B1 

NH 2 

As an initial step, native insulin had to be derivatized 

in order to present an accessible alkyne 

group at an appropriate position. For this, two of 

the three amino functions available on the molecule 

(Glycine A1 and Lysine B29) were first protected 

as N-Boc derivatives. An alkyne bearing 

prosthetic group could then be selectively grafted 

on the phenylanine B1 residue which does not 

interfere in the insulin-receptor binding process 

[1]. The final [ 18 F] fluorine insertion step was carried 

out by reacting this insulin derivative with 

the radioactive azide synthon 1-(azidomethyl)- 

4-[ 18 F]-fluorobenzene[2]under Cu(I) catalysis 

conditions. 

Results: The clickable 18 F azide 1-(azidomethyl)-4- 

-[ 18 F]-fluorobenzene was obtained in 65 minutes 

with good radiochemical yield (40-45% decay-corrected) 

and radiochemical purity (>90%) thanks to 

a fully automated preparation method developped 

on remote-controlled commercial radiosynthesis 

CO 2H 

BocHN CO 2H 

H 2N 

Boc 2 O 

Lys B29 

CO2H NHBoc 

O 

O 

O 

N 

O 

BocHN CO 2H 

HN 

O 

unit. Subsequent copper catalyzed click reaction on 

the alkyne bearing insulin derivative was performed 

at room temperature in less than 20 minutes with 

current radiochemical yields of 70-80% (decaycorrected). 

Conclusions: A mild and efficient radiolabelling 

strategy of insulin was succesfully developped using 

click chemistry. This radiotracer can now be incorporated 

in new inhalable pharmaceutical formulations 

for biodistribution imaging studies. 

CO2H NHBoc 

Acknowledgement: NeoFor and Keymarker research 

platforms from the BioWin projects of the Walloon 

Region are acknowledged for their financial support. 

References: 

Click 

[ 18 F]AzidoFB 

18F + TFA (deprotection) 

1. Guenther K.J. ; Yoganathan, S.; Garofalo, R.; Kawabata, 

T.; Strack, T.; Labiris, R.; Dolovich, M.; Chirakal, R. and 

Valliant, J.F., J.Med.Chem., 49: 1466-1474 (2006) 

2. Thonon, D. ; Kech, C. ; Paris, J. ; Lemaire, C. and Luxen, 

A.; Bioconjugate Chem., 20(4):817-823, (2009) 


18 F 

N 3 

N 

N N 

HN 

O 

Insulin 

Fig1 

P-016 

poStEr 


130 


P-017 Whole-body distribution, pharmacokinetics and dosimetry of radioiodinated fully 

humanized anti-VAP-1 antibody – a PET imaging study of rabbits 

Roivainen A. (1) , Autio A. (2) , Suilamo S. (2) , Mali A. (2) , Vainio J. (2) , Saanijoki T. (2) , Oikonen V. (2) , Luoto P. (2) , Teräs M. (2) , Karhi T. (2) , 

Vainio P. (2) . 

(1) Academy of Finland/University of Turku, Finland 

(2) Turku PET Centre, Finland 

anne.roivainen@utu.fi 

Introduction: Vascular adhesion protein-1 (VAP-1) 

is an inflammation inducible endothelial glycoprotein 

[1]. It plays a key role in leukocyte trafficking 

and is a potential target for anti-inflammatory therapy. 

The antibody BTT-1023 is first-in-class, fully 

human monoclonal antibody to VAP-1. BTT-1023 

is potentially useful for the treatment of inflammatory 

diseases and in vivo imaging of inflammation. 

Positron emission tomography (PET) is powerful, 

non-invasive method particularly suitable for drug 

development because of its high sensitivity and ability 

to provide quantitative and kinetic data without 

sacrificing the animal. We assessed the usefulness 

of PET in evaluation of iodine-124 labeled antibody 

pharmacokinetics and distribution in rabbits. 

Methods: BTT-1023 was labeled with [ 124 I] using 

Chloramine-T method. Immunoreactivity of [ 124 I] 

BTT-1023 was verified in human VAP-1 transfected 

Chinese Hamster Ovary cells and by time-resolved 

immunofluorometric assay using human recombinant 

VAP-1. Ten rabbits were intravenously injected 

with [ 124 I]BTT-1023. PET/CT was obtained over the 

first 2 h after dosing and at 24, 48 and 72 h postinjection. 

Blood samples were collected during PET 

study to clarify in vivo stability and pharmacokinetics. 

As a final point, the human radiation dose estimates 

were extrapolated from rabbit information. 

Results: Binding of [ 124 I]BTT-1023 to hVAP-1 

transfected cells was app. 600-fold higher than 

in mock transfected controls. In rabbits, the radioactivity 

was distributed especially to liver and 

thyroid. Also heart and lungs showed some uptake 

whereas brain uptake was very low. Liver uptake 

is likely mediated, at least in a large part, by 

VAP-1, since the antigen is found on sinusoidal 

endothelia in the liver. Thyroid gland radioactivity 

is due to the de-iodination of [ 124 I] from the 

antibody. The plasma half-life of [ 124 I]BTT-1023 

was 58.3 h and clearance 7.8 mL/h/kg. [ 124 I]BTT- 

1023 showed good in vivo stability (80% of signal 

from intact antibody at 72 h after injection). 

The estimated human radiation dose resulting 

from [ 124 I]BTT-1023 was 3.66 mSv/MBq. Provided 

that thyroid gland uptake is blocked, the effective 

dose in a human adult of about 70 kg would 

imaging life 

decrease to 0.55 mSv/MBq, which is equivalent 

to 21 mSv from 35 MBq and 30 mSv from 50 MBq 

of [ 124 I]BTT-1023 PET. 

Conclusions: [ 124 I]BTT-1023 retained its biological 

activity to bind hVAP-1 also after radioiodination. 

This PET study of [ 124 I]-labeled VAP-1 targeting 

BTT-1023 antibody further elucidated whole-body 

distribution and pharmacokinetics of the therapeutical 

antibody, and supports clinical trials with [ 124 I] 

BTT-1023 at doses of 50 MBq or less. 

Acknowledgement: The study was conducted within 

the Finnish CoE in Molecular Imaging in Cardiovascular 

and Metabolic Research supported by the 

Academy of Finland, University of Turku, Turku 

University Hospital and Åbo Akademi University. 

Anu Autio is a PhD student supported by Drug Discovery 

Graduate School. 

References: 

1. Salmi M, Jalkanen S. Science. 257:1407-1409 (1992)


Pharmacological characterization of iodine labeled adenosine kinase inhibitors 

Sihver W. (1) , Schulze A. (1) , Kaufholz P. (1) , Meyer A. (1) , Grote M. (2) . 

(1) Research Center Jülich, Germany 

(2) Hannover Medical School, Germany 

w.sihver@fz-juelich.de 

Introduction: The intracellular enzyme adenosine 

kinase (AK) catalyzes the phosphorylation of adenosine 

to adenosine monophosphate thus controlling 

the adenosine concentration. The inhibition of 

AK enhances the amount of adenosine, which is suggested 

to have neuroprotective, anticonvulsant, and 

antinociceptive effects [1,2] . Years ago, the non-nucleoside 

AK inhibitor ABT-702 has been investigated in 

vivo and in vitro [3,4] . In the present study the distribution 

of the radioiodine labeled adenosine kinase 

inhibitors [ 131 I]iodotubercidine ([ 131 I]IT) and [ 131 I] 

ABT-702 as well as the pharmacological behaviour 

of other known AK inhibitors (e.g. A134974) versus 

[ 131 I]IT was compared in rat brain. Furthermore the 

AK inhibiting strength of other halogenated ABT- 

702 derivatives was determined. 

Methods: The syntheses of [ 131 I]IT and [ 131 I]ABT-702, 

as well as the different halogenated ABT-702 derivatives 

were performed in house. Autoradiography was 

conducted using frozen rat brain sections. Binding 

competition was done with IT and ABT-702 vs. [ 131 I] 

IT. AK inhibition assays were performed with [ 3 H] 

adenosine and ATP using pig hippocampus cytosol. 

Results: Autoradiography showed generally high 

binding all over the brain, but more distinct in the 

hippocampus, outer layers of the cortex, and gray 

matter of cerebellum using [ 131 I]IT (10nM) compared 

to [ 131 I]ABT-702 (7 nM). [ 131 I]IT Binding could 

be blocked more than 90% with 5-IT and A134974, 

about 70% with tubercidine and 60% with ABT-702. 

[ 131 I]ABT-702 Binding could be blocked not even 50 

% with ABT-702, and even less with the other AK inhibitors. 

K i s of IT and ABT-702 were 20 nM and 850 

nM, respectively, in rat hippocampus vs. [ 131 I]IT. 

In the AK assays, A134974 and IT had the highest 

AK inhibition strength in hippocampal cytosol, followed 

by ABT-702 > I- ABT-702 > Cl- ABT-702 > 

F- ABT-702 with 78%, 62%, 42%, 30% and 21% inhibition, 

respectively. 

Conclusions: As a result of this study it is suggested 

that [ 131 I]IT and [ 131 I]ABT-702 binding represent 

AK distribution in rat brain. [ 131 I]IT appears to be 

the preferred ligand for in vitro binding compared 

H 2N 

N 

NH 2 

N 

I 

N N 

N 

X = Br: ABT-702 

X = I: I-ABT-701 

X = Cl: Cl-ABT-702 

X = F: F-ABT-702 


N 

HO 

X 

O 

N N 

OH 

OH 

O 

5-Iodotubercidine (IT) 

to [ 131 I]ABT-702. ABT-702 Has recently shown 

beneficial therapeutic potential [3, 5] , but radioiodo-ABT-702 

seems to be unsuited as radiotracer. 

The AK inhibition strength was high both for IT 

and ABT-702, but low for the halogenated ABT-702 

derivatives. Since IT shows good in vitro behaviour 

as a radioligand as well as AK inhibitor, further experiments 

with [ 131 I]IT in vivo might be interesting. 

References: 

1. Kowaluk EA et al.; Expert Opin Investig Drugs.9:551-64 

(2000) 

2. Boison D; Drug News Perspect. 20:607-11 (2007) 

3. Jarvis MF et al.; J Pharmacol Exp Ther. 295:1156-64 

(2000) 

4. Kowaluk EA et al.; J Pharmacol Exp Ther. 295:1165-74 

(2000) 

5. McGaraughty S et al.; Curr Top Med Chem. 5:43-58 (2005) 

P-018 

poStEr 



P-019 Determining the nasal residence time of protein-polymer conjugates for nasal vaccination 

using a novel imaging technique 

132 

Slütter B. (1) , Que I. (2) , Soema P. (1) , Hennink W. (3) , Kaijzel E. (2) , Löwik C.W.G.M. (2) , Jiskoot W. (1) . 

(1) Amsterdam Center for Drug Research (LACDR), The Netherlands 

(2) Leiden University Medical Center, The Netherlands 

(3) Utrecht Institute for Pharmaceutical Sciences (UIPS), The Netherlands 

bslutter@lacdr.leidenuniv.nl 

Introduction: Nasal administration of vaccines 

holds great promise as a painless alternative for the 

use of needles. Nonetheless, only one nasal vaccine 

is currently on the market (Flumist®). A major hurdle 

for successful nasal vaccination is the relatively 

short residence time of the vaccine in the nasal cavity, 

which hampers efficient uptake of the antigen 

through the nasal epithelium. Increasing the nasal 

residence time of the antigen is therefore an interesting 

approach to improve the efficacy of nasal 

vaccines. Here we investigated the possibility of extending 

the nasal residence time of a small antigen 

(ovalbumin, OVA) with a mucoadhesive polymer, 

trimethyl chitosan (TMC), using a novel live imaging 

technique1. 

Methods: A near-IR fluorescent probe (IR dye 

CW800) was covalently linked to OVA. TMC was 

either mixed with or conjugated to OVA using the 

SPDP method2. After nasal administration of the 

antigen to hairless mice, the fluorescence intensity 

in the nasal cavity was assessed using an IVIS Spectrum® 

and followed in time (Fig1). 

Results: We observed an exponential decay of fluorescence 

intensity after administration of OVA alone, 

whereas after co-administration of OVA with TMC 

or TMC-OVA conjugate fluorescence decay was delayed 

substantially. 

Conclusions: Using a live imaging technique we successfully 

studied the nasal residence time of a model 

subunit antigen. TMC prolongs the nasal residence 

time of OVA, either by mixing it with or conjugating 

it to the antigen. 

imaging life 

Fig1: Nasal residence time of OVA determined using 

fluorescent detection of OVA-IRdye CW 800. Intensity of 

fluorescence signal from the nasal cavity was measured 

in time and normalized for time point 0. n=3+/- SEM. 

References: 

1. Hagenaars et al. Role of trimethylated chitosan (TMC) 

in nasal residence time, local distribution and toxicity 

of an intranasal influenza vaccine. J Control Release. 

2010, in press. 

2. Slütter et al. Conjugation of ovalbumin to trimethyl 

chitosan improves immunogenicity of the antigen. J 

Control Release. 2010, in press.


Nanotubes as mutli-modality vehicles for imaging and therapy of cancer 

Tworowska I. (1) , Mackeyev Y. (2) , Sims-Mourtada J. (1) , Wilson L. J. (2) . 

(1) RadioMedix, Houston, USA 

(2) Rice University, Houston, USA 

itworowska@radiomedix.com 

Introduction: There is a growing interest in development 

of new radiolabeled nanocarriers for targeted 

delivery of isotopes. Nanotubes have been actively 

explored as new systems for delivery of anticancer 

drugs [1], fluorescent probes [2], genes [3], and peptides 

[4] to the target tissues. Previously reported 

synthesis of Gd-loaded US-tubes [5] and I 2 -modified 

US-tubes [6] have opened new possibilities in 

designing of contracts agents for MRI and CT. Here, 

we report on the synthesis of radiolabeled SWNT 

that can serve as vehicles for PET/SPECT imaging 

and cancer therapy. 

Methods: HiPco single-wall nanotubes (SWNTs) 

were chemically cut into US-tubes, using the established 

procedure [7], sonicated at RT in 0.5M 

HNO for 30min-2h. US-tubes were loaded with 

3 

radioisotopes: 177LuCl , 3 99mTcCl , 3 68GaCl , and cold 

3 

Re compounds (ReCl 5 , NH 4 ReO 4 , [NBu 4 ] + ReClO 4 

manually or using automated module for labelling, 

SmarTrace TM . The radiochemical yield was determined 

by radio-TLC and ICP-OES (Inductively 

coupled plasma optical emission spectrometry). Stability 

tests of labelled US-tubes were performed at 

RT for 1- 24h using 1M PBS, 0.1% FBS and transchelator 

(0.1M EDTA) to determine the desorption 

half-life of the labelled US-tubes. 

Results: Loading of US-tubes with 68 Ga was performed 

in 0.5M NH 4 OAc buffer at 90°C for 10 

min using 68 GaCl 3 eluted from 68 Ge/ 68 Ga generator 

(iThemba). Yield of the synthesis was found to be 

pH dependent with over 70% loading at pH=3.6- 4.1 

and to decrease drastically to 1% at pH51%. Yield of the reaction decreased 

to 1% in the absence of SnCl 2 , or when reduction 

proceeded after completion of labelling. 

Best 177 Lu loading of US-tubes was performed in 

0.1 M NH 4 OAc at pH=5.1 at 90°C for 20 min. The 

final yield of this synthesis was found to be >55% 

after repeated dialysis with 0.1M EDTA, pH=4.7. 

- ) 

Loading of US-tubes with cold ReCl proceeded 

5 

in 0.6M NaOH at 90oC for 1h with final yield 

> 20%. Application of other Re compounds 

([NBu ] 4 + - ReClO and NH4ReO ) for modification 

4 

4 

of nanotubes gave products with yield higher than 

17% and approximately 0.4%, respectively. 

Conclusions: Our preliminary studies have shown 

that loading of US-tubes with imaging and therapeutic 

isotopes is feasible. Application of nanotubes 

in nuclear medicine may expand possibilities for 

development of new multi-modality agents for early 

detection and therapy of cancer. 

References: 

1. Tripisciano C., Kraemer K., Taylor A., Borowiak-Palen E., 

Chem. Phys. Lett., 2009, 478, 200-205 

2. Wong Shi Kam N., Liu Z., Dai H., Angew. Chem. Int. Ed., 

2006,45, 577-581 

3. Ramathan T, Fisher F.T., Ruoff R.S., Brinson L.C., Chem. 

Mater., 2005, 17, 1290-1295 

4. Kam N.W., Dai H., J. Am. Chem. Soc., 2005, 102, 6021- 

6026 

5. Mackeyev Y., Hartman K.B, Ananta J.S., Lee A.V., Wilson 

L.J., J. Am. Chem. Soc., 2009; 131(24): 8342–8343 

6. Ashcroft J.M, Hartman K.B., Kissel K.R., Mackeyev Y., 

Pheasant S., Young S., Van der Heide P.A., Mikos A., 

Wilson L.J., Advanced Materials, 2007, 19, 573-576 

7. Mickelson E.T., Huffman C.B., Rinzler A.G., Smalley R.E., 

Hauge R.E., Margrave J.L., Chem Phys Lett, 1998, 296, 

188-194. 


P-020 

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P-021 Early assessment of temozolomide treatment efficacy in glioblastoma using [ 18 F]FLT PET 

imaging 

134 

Viel T. (1) , Backes H. (1) , Hadamitzky M. (1) , Monfared P. (1) , Rapic S. (1) , Rudan D. (1) , Schneider G. (1) , Neumaier B. (1) , Jacobs A.H. (1,2) . 

(1) MPI for Neurological Research, Köln, Germany 

(1,2) European Institute for Molecular Imaging – EIMI, University Muenster, Germany 

thomas.viel@nf.mpg.de 

Introduction: Temozolomide chemotherapy to radiation 

therapy is now the standard therapy for 

glioblastomas [1] . However there is considerable uncertainty 

with regard to the indication for and the 

chances of success of chemotherapy in affected patients. 

Standard Temozolomide treatment in clinic 

consists of 5 days treatment with 150-200 mg/m2 per 

day, repeated every 28 days. Treatment efficacy assessment 

is performed using MRI Imaging before treatment 

and after 3 cycles of treatment (meaning after 

3 months). Due to the rapid evolution of the disease 

(median survival of patient bearing glioblastoma is 

around 15 months), methods to assess TMZ efficacy 

early during treatment is needed. In several clinical 

studies [ 18 F]FLT PET imaging has been validated to 

assess proliferation of different types of tumors in 

vivo [2] , and could therefore be of interest for evaluation 

of TMZ during treatment of glioblastoma. 

The purpose of this study was to monitor the metabolic 

effects of temozolomide (TMZ) chemotherapy 

in malignant gliomas by means of repeated Positron 

Emission Tomography (PET) with [ 18 F]FLT. 

Methods: A glioma cell line, displaying a low resistance 

to TMZ (Gli36) was treated with two low 

doses of TMZ (25 and 50 µM). Two stable cell lines 

were obtained and characterized regarding their 

resistance to TMZ by growth and clonogenic assay. 

Resistant and sensitive cells were xenografted into 

nude mice. Mice were treated during five days with 

daily intra-peritoneal injection of 50 mg/kg of TMZ. 

Efficacy of TMZ treatment is followed using FLT 

imaging, before treatment as well as two and seven 

days after beginning of treatment. 

Results: Two stable cell lines resistant to TMZ were 

established (Gli36-25TMZ and Gli36-50TMZ). The 

EC50 for the induction of cytotoxicity cell death (as 

determined by growth assay) were 10 µM (Gli36), 110 

µM (Gli36-25TMZ) and 460 µM (Gli36-50TMZ). The 

EC50 for the prevention of clonogenic growth (as determined 

by clonogenic assay) were 10 µM (Gli36), 50 

µM (Gli36-25TMZ) and 325 µM (Gli36-50TMZ). In 

vivo, the TMZ treatment induced a strong reduction 

of Gli36 tumor volume, while Gli36-50TMZ continued 

to grow. Correlation between diminution of SUV in 

imaging life 

the tumor after two days of treatment (with regards to 

SUV before treatment) and diminution of tumor size 

seems to be observed so far (n=3 for each tumor type). 

Conclusions: Our results so far indicate that [ 18 F]FLT 

PET scan could be appropriate for an early evaluation 

of the response of Glioblastoma to TMZ chemotherapy. 


by DiMI (LSHB-CT-2005-512146). 

References: 

1. Norden AD, Drappatz J, et al; Lancet Neurol. 7(12):1152- 

60 (2008). 

2. Ullrich RT, Zander T, et al; PLoS One. 3(12):e3908 

(2008).


Sensitive time-gated FRET microscopy of G-Protein coupled receptors using suicide enzymes, 

lanthanide cryptates and fluorescent ligands 

Zwier J. (1) , Laget M. (1) , Cottet M. (2) , Durroux T. (2) , Mathis G. (1) , Trinquet E. (1) . 

(1) Cisbio Bioassays, Bagnols sur Cèze, France 

(2) Insitut de Génomique fonctionelle, France 

jzwier@cisbio.com 

Introduction: Donor crosstalk and non-specific fluorescence 

in classical biological FRET experiments 

using fluorescent proteins remains a major concern 

in high content screening. Using a time-gated microscope 

equipped with an intensified CCD camera 

[1], lanthanide cryptates and fluorescent-ligands it 

is possible to visualize G-protein coupled receptors 

(GPCR) labeled with the SNAP-tag® suicide enzyme 

at the cell membrane. Oligomerization of GPCR’s can 

be studied using time gated FRET without significant 

background. Using fluorescent ligands, binding 

and receptor trafficking can be imaged without ratiometric 

treatment of data. 

Methods: A bright terbium-cryptate (lumi4®-Tb) 

coupled to 0 6 -Benzylguanine is used to covalently 

label GPCR’s tagged with the SNAP-tag suicide enzyme 

developed by the laboratory of Kai Johnsson 

[2]. The luminescence of this lanthanide compound, 

which has a lifetime of about 2 ms, can be time-gated 

in such a way that all background fluorescence from 

the cells has decayed and only specific lanthanide or 

sensitized acceptor emission to either red or green 

emitting dyes can be detected. An in-house developed 

microscope equipped with an intensified CCD 

camera can monitor this luminescence. HEK/COS/ 

CHO cells were either stably or transiently transfected 

with plasmids containing the SNAP-tagged GPCR 

under study and labeled with the benzylguanines of 

choice after which they are fixed. 

Results: Due to the significant Förster radius of 58 

A of the lumi4-Tb/red donor-acceptor couple, homodimerization 

of a GPCR can be monitored using 

a mix of the Tag-lite® reagent SNAP-Lumi4-Tb and 

SNAP-red wich results in a bright time gated sensitized 

emission image at 665 nm. These results confim 

the results obtained by dimerization assays [3] 

in standard plate readers. 

Due to the advantages of this technique it is also 

possible to visualize specific labeling of fluorescent 

ligands on the GPCR of interest. Using fluorescent 

ligands without the use of time gating gives significant 

contributions of non-specific binding. Since 

sensitized emission only occurs when donor and 

acceptor are in close proximity ligand receptor 

interactions were monitored for several GPCR’sligand 

couples including chemokine receptors. 

Conclusions: Time gated microscopy with terbiumcryptate 

is a convenient and novel way to avoid 

non-specific emission signals and to monitor FRET 

intensities showing genuine interactions between 

proteins and their ligands. This might become a 

powerful method to develop high content assays for 

drug evaluation. 

Acknowledgement: Tag-lite is a registered trademark 

of Cisbio bioassays. Lumi4 is a regis tered 

trademark of Lumiphore Inc. SNAP-tag is a registered 

trademark of New England Biolabs. 

References: 

1. Ghose S et al, J. Alloys and cmpds 451:35-37 (2008) 

2. Keppler A et al, Nature Biotechnology 21:86-89 (2003) 

3. Maurel D, Comps-Agrar L et al, Nature Methods 5:561- 

567 (2008) 


P-022 

poStEr 


136 


P-023 Selection of a nanobody scaffold with low renal retention 

D’huyvetter M. , Vaneycken I. , Tchouate Gainkam O. , Hernot S. , Caveliers V. , Xavier C. , Devoogdt N. , Lahoutte T. . 

Vrije Universiteit Brussel, Jette, Belgium 

mdhuyvet@vub.ac.be 

Introduction: Nanobodies are small (15kDa) antibody 

fragments, derived from heavy chain-only 

antibodies present in Camelidae. The structure 

consists of a scaffold and three CDR loops. The 

CDR loops determine the specificity of the nanobody. 

Nanobodies, labelled with a therapeutic 

radionuclide, could be used for the treatment of 

cancer. Unfortunately, the in vivo biodistribution 

shows high renal retention of radio-labeled nanobodies 

in the kidney cortex and this could result 

in an important kidney toxicity. However, during 

the in vivo screening of a large set of nanobodies 

we noticed that there is a wide variety in this kidney 

retention. This variability could be partially 

related to the amino acid sequence of the nanobody 

scaffold. In this study we aim to identify the 

nanobody scaffold with the lowest renal retention. 

Methods: The renal retention of 8 Nanobodies was 

evaluated in healthy Wistar rats. 99mTc labeling 

was performed using 99mTc-tricarbonyl (Isolink, 

Covedien). Dynamic planar imaging with a gamma 

camera was performed immediately after injection 

(100 frames of 30s). Three hours post injection an 

additional static image was acquired. Time activity 

curves of the kidney were generated using AMIDE. 

imaging life 

Results: We measured two distinct kinetic profiles: 

one showing a steadily increase in function of time 

and the other showing an initial peak (5 min p.i.) 

followed by a decrease. The highest renal activity 

measured at 1h p.i. was 46.8 % IA (range 41.6-53.3 

%IA), while the lowest was 11.3 % IA (range 10.1- 

13.2 %IA). At 3h p.i. we measured upto 69.3 %IA 

in the kidney for the group with a steadily increasing 

profile, while for the other group it remained 

low at 14.8 %IA. 

Conclusions: We identified a scaffold with low 

renal retention. The use of this scaffold for nanobody 

based targeted radionuclide therapy could 

significantly reduce kidney toxicity. 

Acknowledgement: The research at ICMI is funded 

by the Interuniversity Attraction Poles Program – 

Belgian State – Belgian Science Policy. Matthias 

D’huyvetter is funded by SCK-CEN/VUB.


In vitro assessment of androgen mediated uptake of 18 F-FDG, 11 C-choline and 11 C-acetate in 

prostate cancer 

Emonds K. (1) , Swinnen J. (1) , Van Weerden W. (2) , Nuyts J. (1) , Mortelmans L. (1) , Mottaghy F. (3) . 

(1) KULeuven, Belgium 

(2) Josephine Nefkens Institute, The Netherlands 

(3) University Hospital Aachen, Germany 

Kimy.Emonds@med.kuleuven.be 

Introduction: Androgen deprivation is one of the 

first line palliative treatment approaches in recurrent 

prostate cancer. Although 18 F-FDG is mostly applied 

in oncological PET imaging, a low diagnostic performance 

has been shown for prostate cancer detection. 

Otherwise, both 11 C-choline and 11 C-acetate offer a 

higher diagnostic efficiency for PET detection of relapsing 

patients. With this study we aimed to evaluate the 

androgen dependency of the uptake of metabolic PET 

tracers ( 18 F-FDG, 11 C-choline and 11 C-acetate) in five 

different human prostate cancer cell lines, distinctive 

considering their androgen responsiveness, in order to 

define the most reliable tracer for treatment response 

assessment. 

Methods: Cell uptake experiments were performed with 

the prostate cancer cell lines LNCaP, PC346C, 22Rv1, 

PC346DCC and PC3, and the benign prostatic hyperplasia 

cell line BPH-1. Prior to the uptake experiments, 

cells were cultured in the presence of charcoal treated 

serum (native). In parallel cell cultures 10 -8 M R1881, 10 - 

10 M R1881, 10 -6 M bicalutamide or the combination of 

10 -10 M R1881 and 10 -6 M bicalutamide was added to the 

medium. The significant influence of androgens on the 

uptake of 18 F-FDG, 11 C-choline and 11 C-acetate in each 

prostate cancer cell line was evaluated using ANOVA 

and Tukey-HSD post-hoc analysis. The relative tracer 

uptake was evaluated in all prostate cancer cell lines 

with respect to the tracer uptake in BPH-1. 

Results: A significant increased 11 C-choline uptake is 

observed in the androgen responsive and unresponsive 

cell line, respectively PC346C and 22Rv1, grown in the 

presence of androgens (10 -8 M R1881). The same androgen 

concentration also caused a significant increase 

in 18 F-FDG uptake in LNCaP (androgen dependent) 

and 22Rv1. In both androgen unresponsive cell lines 

(PC3, PC346DCC) androgens did not significantly affect 

the uptake of these metabolic PET tracers. Unlike 

11 C-choline and 18 F-FDG, 11 C-acetate uptake was not 

influenced by androgen supplementation in any prostate 

cancer cell line. 

Compared to the tracer uptake in BPH-1, all prostate 

cancer cell lines showed a significant higher 11 C-acetate 

and 11 C-choline uptake. In contrast, a relative increased 

18 F-FDG uptake was only observed in PC346C and -DCC. 

Conclusions: 11 C-acetate uptake is androgen 

independent in every prostate cancer cell line, 

whereas a significant higher 11 C-choline and 

18 F-FDG uptake was established by androgens 

in respectively two (PC346C, 22Rv1) and three 

(LNCaP, PC346C, 22Rv1) human prostate cancer 

cell lines. Also, 11 C-acetate has the most 

clearly elevated uptake in all prostate cancer 

cell lines with respect to the benign prostatic 

hyperplasia cell line. 

The absent influence of androgens on 11 C-acetate 

uptake and the better differentiation of 

cancer from benign prostatic hyperplasia with 

this PET tracer compared to 11 C-choline and 

18 F-FDG, suggests a higher efficiency of 11 C-acetate 

PET for evaluation of treatment response. 


P-024 

poStEr 

CANCER from BENCH to BEDSIDE

138 


P-025 Comparison of two 68 Gallium-labeled octreotide analogues for molecular PET(CT) imaging 

of neuroendocrine tumours 

Garcia C. , Woff E. , Muylle K. , Ghanem G. , Van der Linden B. , Vandormael S. , Rutten E. , Flamen P. . 

Institut Jules Bordet. Université Libre de Bruxelles, Brussels, Belgium 

camilo.garcia@bordet.be 

Introduction: PET(CT) using 68 Ga-labeled octreotide 

analogues is able to show the expression of 

somatostatin receptors (SSR) on gastroenteropancreatic 

neuro-endocrine tumours (GEP NET). The 

study aim was to compare the biodistribution and 

the tumour uptake intensity of two radiolabeled 

octreotide analogues, 68 Ga-DOTA-TOC ([DOTA- 

DPhe1,Tyr3]-octreotide) and 68 Ga-DOTA-TATE 

([DOTA-DPhe1,Tyr3]-octreotate). 

Methods: Octreo-PET(CT) was performed on 58 

patients: 29 patients using 68Ga-DOTA-TOC and 

29 patients using 68 Ga-DOTA-TATE. Standard Uptake 

Values (SUV) was measured in normal organs 

and in the lesions (SUVmax). Patient preparation, 

acquisition and image processing was standardized 

and identical in both subgroups. None of the 

patients had received any peptide therapy or radiolabeled 

peptide therapy before the study. The 

time delay between tracer injection and PET(CT) 

acquisition for respectively DOTA-TOC vs. DOTA- 

TATE patients was 95 min ± 0.01 vs. 100 min ± 0.01 

(p=NS); the mean injected tracer activity was 90.28 

MBq ± 46.62 vs. 88.8 MBq ± 21.09 (2.44 mCi ± 1.26 

vs. 2.40 mCi ± 0.57) (p=NS), and BMI 25.7 ± 3.71 

vs. 25.2 ± 3.6 (p=NS). 

imaging life 

Results: PET(CT) identified 107 tumor lesions: 52 

(49%) in the DOTA-TOC, and 55 lesions (51%) in 

the DOTA-TATE group. No significant differences 

of SUV(max) values were found in GEP NET lesions 

between DOTA-TATE and DOTA-TOC groups. 

Physiologic uptake in organs did not significantly 

differ between the 2 radiopharmaceuticals, except 

for the uptake of 68 Ga DOTA-TOC which was significantly 

lower in the head of pancreas than the 

uptake of 68 Ga DOTA-TATE (3.4 ± 1.5 vs. 4.6 ± 1.6) 

(P


Real time per operative optical imaging for the improvement of tumour surgery in an in vivo 

micro-metastases model 

Keramidas M. , Josserand V. , Righini C. , Coll J. L. 

INSERM U823, Grenoble, France 

michelle.keramidas@ujf-grenoble.fr 

Introduction: In a wide range of cancer cases, 

surgery is the first therapeutic indication before 

radiotherapy and chemotherapy. In that 

way the prognostic strongly depends on the tumour 

removal exhaustiveness and in particular 

on metastases elimination. 

We developed a couple near-infrared fluorescent 

tracer / per operative detection system in 

order to improve tumour surgery efficacy. 

RAFT-c(RGD)4-Alexa 700 (Angiostamp®, Fluoptics) 

specifically bind to integrin αvß3, a 

receptor strongly expressed in angiogenesis 

and in many tumour types. The per-operative 

detection system (Fluobeam 700, Fluoptics) is 

a portative 2D fluorescent imager that can be 

used in white light environment and so, could 

be used directly during surgery to help the surgeon 

for tumour and metastases excision. 

We already demonstrated in animal models 

the very significant improvement in primary 

tumours resection: higher number of tumour 

nodules removed, sane margins on the removed 

fragments and surgery time divided by 2 (Keramidas 

M., British Journal of Surgery 2010). 

In the clinical situation, recurrence of cancer 

by metastases invasion after primary tumour 

surgery is a critical point. The aim of the present 

study is to evaluate the metastases resection 

impact on the survey. In order to proceed we 

first need to establish a suitable animal model 

of micro-metastases following primary tumour 

surgery. After calibration of the metastatic 

in vivo model, we will evaluate the survey of 

the twice-operated animals (metastases resection 

after primary tumour removal) versus the 

once-operated animals (only primary tumour 

removal). 

Methods: Luciferase positive tumour cells (TS/ 

Apc-luc) are injected in the kidney capsule of 

nude mice and the primary tumour growth is 

followed by in vivo bioluminescence imaging. 

7 days after tumour cells implantation, the tumoral 

kidney is removed and the metastases 

development is followed by in vivo bioluminescence 

imaging. Then RAFT-c(RGD)4-Alexa 

700 is injected intravenously and twenty-four 

hours later the portative fluorescence detection 

system is used to assist the metastases excision. 

The possible recurrence of cancer is followed 

by in vivo bioluminescence imaging. Mice are 

sacrificed when they loose 10% of their weight. 

The mice survey is compared with the one of 

two control groups: in one group the mice don’t 

undergo the second surgery for metastases resection, 

and in a second group the mice don’t 

undergo any surgery and keep the primary kidney 

tumour. 

Results: Micro-metastases appear about 7 days 

after the primary tumour removal and soar up 

to 20 days after the first surgery. The metastases 

removal assisted by the RAFT-c(RGD)4-Alexa 

700 and the per operative fluorescence detection 

system significantly improved the mice 

survey (36 days). 

Conclusions: We developed an in vivo model of 

micro-metastases invasion following primary 

tumour surgery which is very relevant regarding 

to the clinical situation of head and neck 

or prostate cancer. The use of RAFT-c(RGD)4- 

Alexa 700 coupled with the portative device allows 

micro-metastases detection, significantly 

improve their resection and increase the mice 

survey. 

References: 

1. Intraoperative near-infrared image guided surgery for 

peritoneal carcinomatosis in a preclinical experimental 

model. M. KERAMDAS, V. JOSSERAND, RIGHINI C.A., WENK 

C., FAURE C. And COLL J.L. British Journal of Surgery,2010. 


P-026 

poStEr 


140 


P-027 Fluorescence imaging modalities for imaging gastrointestinal tumor models 

Schulz P. , Cordula D. , Rexin A. , Wiedenmann B. , Groetzinger C. . 

Charite, Universitätsmedizin Berlin, Germany 

petra.schulz@charite.de 

Introduction: A number of peptide receptors have 

been found to be overexpressed in tumors of the GI 

tract. The overexpression of somatostatin receptors 

in neuroendocrine gastroenteropancreatic tumors 

has long been utilized for molecular imaging using 

radiolabelled somatostatin analogs in scintigrafic 

procedures. Other peptide GPCRs may therefore 

represent attractive new targets. The ongoing project 

is occupied in particular with the design of GPCR 

peptide ligands, which bind with high specificity to 

surface receptors of tumor cells. Pharmacologically 

optimized peptides could be used as vehicles for 

transport of contrast agents as well as therapeutics. 

Methods: The ongoing project follows four main 

goals: (1) The validation of GPCR peptides as tumor 

targets for the gastrointestinal tract (2) The identification 

of new peptide binding targets (3) The Optimization 

of peptide structures for imaging and targeted 

therapy and (4) the development of NIRF-probes 

and –techniques for a better diagnosis of tumors. 

NIRF probes are validated in established in vivo tumor 

models, including subcutaneous tumor models 

and in a number of orthotopic tumor models using 

planar and tomografic fluorescent imaging, micro 

CT and endoscopy for small animals. For monitoring 

and characterization of tumor growth of human cancer 

cells in nu/nu mice the bioluminescence imaging 

approach is utilized. For this purpose several stable 

cancer lines (colorectal, pancreatic, neuroendocrine), 

expressing the luciferase gene were established. 

Results: Currently, a number of peptide, antibody 

and small-molecule contrast agents are validated. 

Each tracer is characterized for its potency as a contrast 

agent by imaging with a 2-D fluorescence imaging 

system, which reveals target-to background 

ratios of the contrast agents, characteristic for target 

tissue specificity. Limitations of planar imaging 

systems are still the lack of depth resolution and 

difficult quantification. Currently we are therefore 

evaluating a fluorescence tomographic system with 

regard to deep tissue imaging and signal quantification. 

Tomograpic images also allow fusion with 

other imaging mmodalities such as CT to confirm 

the anatomical localization of the fluorescent signal. 

imaging life 

Conclusions: Although near-infrared radiation has 

a higher penetration depth than the visual light, 

scattering and absorption still prevent whole-body 

imaging. One potential application for the use of 

near-infrared optical agents in humans is therefore 

fluorescence-guided endoscopy. We are currently establishing 

a protocol for endoscopic imaging in mice 

with colorectal cancer with a rigid endoscopic device 

in combination with fluorescence-guided detection 

using a fiber endoscope. 

Acknowledgement: This work was supported by grant 

03IP614 from German ministry of research (BMBF)


Assessment of effectiveness and toxicity of the therapy with somatostatin analogue labelled 

90Y-DOTATATE in patients with non-functional pancreatic neuroendocrine tumours (PNT) 

Sowa-Staszczak A. . 

Nuclear Medicin Unit, Krakow, Poland 

sowiana@gmail.com 

Introduction: Therapy with labelled somatostatin 

analogues is the modern approach to the patients 

with disseminated or unresectable NETs expressing 

somatostatin receptors (SSTR). Octreotate is 

the somatostatin analogue with very high affinity 

to the SSTR type 2, most commonly present in 

neuroendocrine tumours. In non-functional PNT, 

grading and systemic metastases have a significant 

impact on survival. Overall, the 5-year survival 

rate is about 33%. The chemotherapy is the most 

common treatment approach. The aim of the study 

was to assess the efficacy and toxicity of peptide 

receptor radionuclide therapy (PRRT) with the use 

of the high affinity somatostatin receptor subtype 

2 analogue, 90Y labelled Tyr3-octreotate, (90Y- 

DOTATATE) in non-functional pancreatic neuroendocrine 

tumours. 

Methods: 19 patients with metastatic NET were diagnosed 

in the Department of Endocrinology UJCM. 

5 patients with high proliferation index (Ki >20%, 4- 

negative SRS, tumour size 3-7cm) were directed to 

chemotherapy. 14 patients with positive SRS scan due 

to disseminated and/or unoperable PNTs (2 patients) 

were qualified to PRRT (6 men, 8 women, mean age 54,7 

years old, Karnofsky’s index > 70-100%). The size of the 

tumour was 2,3-12cm, Ki-67 was

142 


P-029 Intra operative near-infrared fluorescent imaging of colorectal liver metastases using 

clinically available Indocyanine Green in a syngene rat model 

Van Der Vorst J. , Hutteman M. , Mieog J. , De Rooij K. , Kuppen P. , Kaijzel E. , Löwik C.W.G.M. , Van De Velde C. , 

Vahrmeijer A. . 

Leiden University Medical Centre, The Netherlands 

j.r.van_der_vorst@lumc.nl 

Introduction: The survival of patients with colorectal 

carcinoma is mostly determined by the occurrence 

of distance metastases. When liver metastases occur, 

surgical resection can offer a 5-year survival of 35- 

40%. However, during resection, an adequate assessment 

of the extent of disease is limited, resulting in 

a high percentage of recurrence (40-50%). Ishizawa 

et al. reported that liver cancer could be identified 

using near-infrared (NIR) fluorescence and the clinically 

available indocyanine green (ICG) (1). However, 

the optimal dose of ICG and the interval between the 

administration of ICG and surgery is unclear. 

Methods: In the current study, the NIR probe ICG 

and the Mini-FLARE (Dr. J.V. Frangioni, Boston, 

USA) camera system were used. In 6 rats, 125,000 

CC531 cells were inoculated subcapsularly in 

three different lobes of the liver. After four weeks, 

tumors of approximately 3-5 mm diameter were 

present. In each rat, fluorescence was measured at 

24 and 48 hours post-injection of 0.04 mg (n = 3) 

or 0.08 mg (n = 3) ICG. 

Color 

Fig1: Intraoperative NIRF identification of colorectal liver metastases 

imaging life 

Results: In 6 rats, all colorectal liver metastases 

(n = 10) were intraoperatively identified using 

ICG and the Mini-FLARE camera system (Figure 

1). The fluorescent signal of the colorectal liver 

metastases was significantly higher than the signal 

of surrounding normal liver tissue (P < 0.001). 

No significant difference in mean signal to background 

ratio was found between imaging at 24 and 

48 hours post-injection. Furthermore, no significant 

difference in mean signal to background ratio 

was found between injection of 0.04 and 0.08 mg 

of ICG. 

Conclusions: This study demonstrated that colorectal 

liver metastases can be clearly identified during 

surgery using the clinically available NIRF probe 

ICG and the Mini-FLARE camera system. When 

the current intraoperative identification can be improved 

using this technique, resections can be performed 

more accurately and preoperatively missed 

metastases can be involved in surgical decision 

making during surgery. 

NIR Merge 

References: 

1. Ishizawa et al.; Cancer. 1;115(11):2491-504. (2009)


Molecular imaging of resistance to EGFR tyrosine kinase inhibitors by 18F-FLT PET/CT and its 

reversal in non small cell lung cancer 

Zannetti A. (1) , Iommelli F. (1) , Lettieri A. (1) , Pirozzi G. (2) , Salvatore M. (3) , Del Vecchio S. (3) . 

(1) Institute of Biostructures and Bioimages, National Research Council, Naples, Italy 

(2) Department of Experimental Oncology, National Cancer Institute, Naples, Italy 

(3) Institute of Biostructures and Bioimages, National Research Council; Department of Biomorphological and Functional Sciences, University of Naples “Federico II”, Naples, Italy 

antonella.zannetti@ibb.cnr.it 

Introduction: Multiple molecular mechanisms 

may underlie the resistance to EGFR tyrosine kinase 

inhibitors (TKIs), including the occurrence 

of secondary mutations such as T790M in the kinase 

domain of EGFR, redundant lateral signalling 

or alterations of apoptotic program mainly due to 

dysregulation of Bcl-2 family members. The aim 

of our study was to test whether 18F-Fluorothymidine 

(18F-FLT) could detect EGFR TKI refractory 

tumors and identify the mechanisms underlying 

such resistance so that specific therapeutic strategies 

can be adopted in patients with non-small cell 

lung cancer (NSCLC). 

Methods: EGFR TKI sensitive and resistant 

NSCLC cells were evaluated for drug-induced 

apoptosis and growth arrest. Cells were also 

tested for inhibition of downstream signalling 

and expression or drug-induced upregulation 

of Bcl-2 family members. Nude mice bearing 

sensitive and resistant NSCLC were i.v. injected 

with 7.4 MBq of 18F-FLT and then subjected to 

microPET/CT (eXplore Vista Pre-Clinical PET 

Scanner GE Healthcare) before and after treatment 

with reversible or irreversible EGFR TKIs. 

Results: We found that NSCLC bearing T790M 

mutations showed a persistent high uptake 

of 18 F-FLT after treatment with reversible inhibitors 

and lack of growth arrest. Treatment 

of the same animals with irreversible inhibitors 

caused a reduction of 18F-FLT uptake in 

tumors indicating the reversal of T790M mutation-dependent 

resistance. Conversely, NSCLC 

cells that were resistant due to dysregulation of 

Bcl-2 family members became sensitive to EGFR 

TKIs when treatment included Bcl-2 inhibitors. 

Conclusions: Resistance to EGFR TKI may 

be caused by multiple mechanisms. Molecular 

imaging with 18F-FLT may contribute to the 

selection of patients that may benefit from 

treatment with irreversible EGFR TKI inhibitors. 


P-030 

poStEr 


P-031 

144 


Synchronised Cardiac and Lung CT in Rodents Using the Mobile CT Scanner LaTheta 

Glowalla A. . 

Zinsser Analytic GmbH, Frankfurt, Germany 

a.glowalla@zinsser-analytic.com 

Introduction: Historically, in vivo CT images of 

the chest region of small animals were blurred and 

streaked due to movement artefacts caused by the 

heartbeat and breathing during scanning. However, 

synchronised Cardiac and Lung CT in rodents has the 

enormous potential to contribute greatly to research in 

heart and lung diseases. Using the mobile CT Scanner 

LaTheta, scanning artefacts are greatly reduced. Here 

we present scanning results of the chest region of mice 

and discuss their relevance in pre-clinical research. 

Methods: Manufacturers standard protocols for synchronised 

CT scanning were used to scan the chest 

region of anaesthetised mice with respiratory rates 

below 75bpm, as below: 

Synchronised CT scanning results for A) heart in systole phase 

and B) lung in mouse 

A) Cardiac Sync. Scan (systole + diastole) 

- scan range axial: heart, 15mm length 

- scan time: 13.1min 

B) Respiratory Sync. Scan 

- scan range axial: heart, 10mm length 

- scan time: 4.5min 

X-ray tube voltage: 50kV at 0.5mA; Pixel resolution: 

48um; Slice thickness: 192um; Contrast media: Iopamiron 

300, 1.5ml/h in 30g mouse given 10min. 

before scanning. 

imaging life 

A 

CT images were constructed using methods for reduction 

of motion artefacts and with focus on soft 

tissues. 

Results: CT images of heart (diastole and systole 

phases) and lung were acquired and reconstructed 

without common blurring and streaking effects. 

Conclusions: Synchronised scanning modes with 

LaTheta LCT -200 produce high-resolution CT 

cross-sections of heart and lung in living mice 

within a short time period. Similar methods are 

applicable also for rats. Since no additional sensor 

hardware is necessary, handling of the animal is as 

simple as for standard CT. 

The potential for the detection of lung diseases such 

as pulmonary fibrosis, pulmonary emphysema and 

also lung tumours has already been validated by 

manufacturer. With this technology LaTheta LCT 

-200 can also contribute to the studies of cardiovascular 

diseases in rodents. 

Acknowledgement: 

1. Manufacturer of LaTheta(TM): Aloka Co., Ltd., 6-22-1 

Mure, Mitaka-shi, Tokyo, 181-8622, Japan 

2. Distributor: Zinsser Analytic GmbH, Eschborner 

Landstr. 135, 60489 Frankfurt am Main, Germany 

B


Molecular imaging of neurovascular inflammation in a mouse model of focal cerebral 

ischemia using Ultra small Superparamagnetic Particles of Iron Oxide (USPIOs) targeted to 

vascular cell adhesion molecule-1 (VCAM-1) 

Montagne A. (1) , Gauberti M. (1) , Orset C. (1) , Macrez R. (1) , Rubio M. (1) , Raynaud J. S. (2) , Vivien D. (1) , Maubert E. (1) . 

(1) Inserm U919 UMR 6232 CNRS Ci-Naps, Caen, France 

(2) Experimental Imaging, MRI unit. Research Division. GUERBET, Roissy CdG, France 

amontagne@cyceron.fr 

Introduction: Among several mechanisms, brain 

inflammation is thought to have critical functions 

in lesion progression during acute and 

subacute stages of ischemic stroke. Accordingly, 

anti-inflammatory strategies are neuroprotective 

in preclinical studies [1]. However, to date, all 

clinical trials with anti-inflammatory agents have 

failed to improve clinical outcome in acute ischemic 

stroke patients. New tools for non-invasive 

monitoring of inflammation following stroke are 

needed to select patients who are more susceptible 

to benefit from anti-inflammatory treatment. 

Vascular cell adhesion molecule 1 (VCAM-1), an 

endothelial adhesion molecule, is overexpressed 

in the injured brain and is therefore thought to be 

a good target for the molecular imaging of inflammatory 

processes [2]. The aim of this study was 

to investigate the use of targeted USPIOs against 

VCAM-1 to follow neurovascular inflammation in 

a mouse model of in situ thromboembolic stroke 

with recombinant tissue-type plasminogen activator 

(rt-PA) induced reperfusion [3]. 

Results: Our present data confirm an increase of 

vascular cell adhesion molecule-1 (VCAM-1) immunoreactivity 

(-ir) in the ischemic brain with 

a peak at 24 hours post-ischemia. Interestingly, 

V-CAM-1-ir was significantly increased in late 

rt-PA-thrombolyzed animals (4 hours after clot 

formation) compared to early (20 minutes) or 

unthrombolyzed mice. These data suggest that 

inflammation could be involved in the deleterious 

clinical outcome reported with late rt-PA 

mediated thrombolysis. Targeted USPIOs against 

VCAM-1 showed numerous signal voids in the ipsilateral 

side by 7T magnetic resonance imaging 

(MRI) at 24 hours post-ischemia and the use of 

an antibody directed against polyethylene-glycol 

(USPIOs coating) confirmed histologically our 

MRI analysis. 

Conclusions: Our data indicate that non invasive 

imaging of VCAM-1 with targeted USPIOs allows 

reliable imaging of brain inflammation after 

stroke. Such molecular MRI approaches could be 

used to evaluate inflammatory processes in stroke 

patients and thus to adapt therapy on an individual 

basis. Furthermore, molecular imaging of 

VCAM-1 could also provide valuable clinical information 

in other diseases involving inflammatory 

processes (atherosclerosis, multiple sclerosis …). 

References: 

1. Machado L et al; Stroke. 40;3028-3033 (2009). 

2. Hoyte L et al; J Cereb Blood Flow Metab.1-10 (2010). 

3. Orset C et al; Stroke. 38;2771-2778 (2007). 


P-032 

poStEr 

MI in CARDIOVASCULAR DISEASE


P-033 Scintigraphy with the use of 123 I-IL-2: a new promising tool for cardiovascular risk assessment 

in patients with high cardiovascular risk 

146 

Opalinska M. (1) , Hubalewska-Dydejczyk A. (1) , Stompor T. (2) , Krzanowski M. (3) , Mikołajczak R. (4) , Sowa-Staszczak A. (1) , 

Karczmarczyk U. (5) , Glowa B. (1) , Kuśnierz-Cabala B. (6) , Pach D. (1) , Sułowicz W. (3) . 

(1) Nuclear Medicine Unit, Department of Endocrinology, Jagiellonian University Medical School, Krakow, Poland (2) Chair and Department of Nephrology, 

Hypertesiology and Internal Medicine, University of Warmia and Mazury , Olsztyn, Poland (3) Chair and Department of Nephrology, Jagiellonian University 

Medical School, Krakow, Poland (4) Radioisotope Center POLATOM, Otwock-Swierk, Poland (5) Departament of Radiopharmaceuticals, National Medicines 

Institute, Warsaw, Poland (6) Chair of Clinical Biochemistry, Jagiellonian University Medical School, Krakow, Poland 

mkal@vp.pl 

Introduction: Cardiovascular diseases are the 

main cause of deaths in general population 

(0,28%/year) whereas in selected population like 

in end-stage renal disease patients, mortality 

reaches even 20%. More than half of the cardiovascular 

events occur among the patients without 

any previous symptoms of cardiovascular disease, 

when atherosclerotic plaques obliterate less than 

50% of arterial lumen. 

The majority of widely available methods of cardiovascular 

risk estimation are based on arterial 

lumen assessment, estimation of atherosclerotic 

plaque size or on intima-media complex measurements. 

It means that those methods may by imprecise 

in the identification of plaques of the highest 

risk of rupture. For that reason, the methods 

which enable the visualization and estimation of 

the intensity of inflammatory process within atherosclerotic 

lesions, seem to play the most promising 

diagnostic role. 

The histopathological studies of the atherosclerotic 

plaque have revealed that activated lymphocytes 

T, which contain IL-2 receptors on their surfaces, 

comprise at least 20 % of inflammatory cells in unstable 

plaques. Those receptors can be identified 

with the use of scintigraphy with labeled IL-2. 

Methods: 10 patients (5 women, 5 men, aged 62,4 

± 10,4), with the highest cardiovascular risk, were 

chosen from 67 peritoneal dialysis cohort patients. 

All of them underwent 123 I-IL2 scintigraphy, coronary 

calcium scoring by CT and common carotid 

artery intima-media thickness assessment by USG. 

The levels of some atherogenic, inflammatory and 

calcium-phosphate indicators were measured. Target/non-target 

ratio of 123 I-IL-2 uptake in atherosclerotic 

plaque confirmed by carotid artery USG 

with IMT, calcium score result and concentration 

measured agents were compared. 

Results: In the performed scintigraphies increased 

focal 123 I-IL-2 uptake in 16/16 (100%) atherosclerotic 

plaques previously visualized by neck ultrasound 

was detected. Mean T/nT ratio of focal 123 I- 

imaging life 

IL-2 uptake within atherosclerotic plaques was 

3,15 ± 0,54 (median 3,22, range 2,0 – 3,6). The 

levels of the inflammatory and atherogenic factors 

and IMT (mean 0,975 ± 0,337 mm) were increased 

in the majority of patients. High positive correlation 

between 123I-IL-2 uptake within atherosclerotic 

plaques and IMT in corresponding artery 

was observed (R = 0,92, p = 0,01). Significantly 

higher 123 I-IL-2 uptake on scintigraphy in patients 

who developed cardiovascular event during observation 

period (average 36 months), compared 

to patients without cardiovascular event, was revealed 

(3,45 ± 0,22 vs 2,91 ± 0,54, p = 0,046). No 

statistically significant association was found between 

123 I-IL-2 uptake and the levels of measured 

agents, calcium score or classical cardiovascular 

risk factors. 

Conclusions: Scintigraphy with the use of labeled 

123 I-IL-2 enables the visualization of inflamed 

atherosclerotic (vulnerable) plaque within common 

carotid arteries in end-stage renal disease 

patients. 

Quantitative results of the carotid arteries scintigraphy 

with 123 I-IL-2 correlate with the results of 

IMT and the risk of cardiovascular event during 3 

years of follow-up. 


Polish Committee for Scientific Research (KBN) 

within Research Project 2 P05B 003 28 

References: 

1. Annovazzi A et al; 99mTc-interleukin-2 scintgraphy for 

the in vivo imaging of vulnerable atheroslerotic plaque. 

Eur J Nucl Med Mol Imaging 2006; 33: 117 – 126. 

2. Buyukhatipoglu H et al; Inflammation as a risk factor 

for carotid intimal-medial thickening, a measure of 

subclinical atherosclerosis in haemodialysis patients: 

the role of chlamydia and cytomegalovirus infection. 

Nephrology 2007; 12: 25 – 32.


MEMRI-DTI study of focal transient ischemia in immature rat brain 

Dupont D. , Bogaert-Buchmann A. , Sebrie C. , Gillet B. . 

IR4M , UMR 8081 CNRS-Univ. Paris-Sud, ORSAY, France 

damien.dupont1@u-psud.fr 

Introduction: The aim of the study was to investigate 

anatomo-functional changes in immature rat brain 

after focal transient cerebral ischemia occurred 

when they were 7-day-old. The MRI study was carried 

out 14 days after ischemia by serial MEMRI 

combined with DTI. 

Methods: Focal ischemia was induced in P7 rats as 

previously described [1], and the size of the lesion 

was pointed out by DWI 3h after ischemia. MRI 

studies were carried out at 7T on P21 rats (n=5), 14 

days after focal transient ischemia and compared to 

normal P21 rats (n=5). DTI sequence used was DTI- 

EPI (2 b-values=500/1000s/mm2; 30 directions; 

resolution=0.156*0.156*1 mm3). For the MEMRI 

experiments, 160nl of a 50mM isotonic manganese 

chloride aqueous solution (pH=7.3) was injected 

in S1 at 1.5mm below the dura, at a rate of 20nl/ 

min. T1-weighted experiments were achieved at 3, 

5, 7 and 24h post-injection, using 3D MP-RAGE 

sequence (resolution=0.156*0.156*0.5mm3; TR/ 

TE=15/4.5ms, α=20°; Ti=1s). The manganese containing 

voxels were defined as hyper intense voxels 

and selected as those with a significant higher signal 

(p

148 


P-035 A clinically relevant model of in situ embolic stroke in the anesthetized monkey (macaca 

mulatta): long-term electrophysiological and mri analyses 

Gauberti M. (1) , Guedin P. (1) , Etard O. (2) , Diependaele A.S. (2) , Chazalviel L. (3) , Lamberton F. (3) , Vivien D. (1) , Young A. (1) , 

Agin V. (1) , Orset C. (1) . 

(1) INSERM, Caen, France 

(2) CHU Caen, France 

(3) CNRS, France 

gauberti@cyceron.fr 

Introduction: The lack of relevant stroke models in 

large animals is a limitation for the development of 

innovative therapeutic/diagnostic approaches. The 

aim of the present study was to develop an original 

and clinically relevant pre-clinical model of embolic 

stroke in the monkey. 

Methods: During full physiological and biochemical 

monitoring, six monkeys underwent enucleation, 

the right MCA was exposed and alpha-thrombin 

was injected into the MCA. The monkeys were subjected 

to somatosensory evoked potentials (SEPs) 

and MRI studies (T2, FLAIR, DWI, PWI and MRA) 

prior to, and following the acute (2 h) and chronic 

stages (24 h to 3 months) of stroke. 

imaging life 

Results: This feasibility study showed that, it is possible 

to induce ischemic lesions in the MCA territory 

of the monkey following the direct injection of 

thrombin into the lumen of the M1 segment of the 

MCA. This procedure leads to cortical or subcortical 

ischemic lesions and to a persistent impairment 

of somatosensory responses as evidenced by MRI 

and SEPs data, respectively. 

Conclusions: In situ induction of an endogenous fibrin 

clot together with the lack of mortality make 

this original model of stroke in large non-human 

primates, highly relevant to determine the effectiveness 

of drug administration including thrombolytic 

therapy and to validate new imaging procedures.


Automated radiosynthesis of [ 18 F]MPPF derivatives for imaging 5-HT 1A receptors 

Goblet D. (1) , Thonon D. (1) , Plenevaux A. (1) , Defraiteur C. (2) , Wouters L. (2) , Franci X. (2) , Luxen A. (1) . 

(1) University of Liege, Liege, Belgium 

Z2) GE Healthcare Diagnostic Imaging, Belgium 

david.goblet@ulg.ac.be 

Introduction: Dysfunction of the central serotoninergic 

system is implicated in numerous neurodegenerative 

disorders such as Alzheimer’s disease, 

dementia, depression, anxiety, schizophrenia, and 

Parkinson’s disease. 5-HT 1A receptors are involved 

in several physiological functions including sleep, 

mood, neurogenesis and learning [1]. Consequently, 

there have been huge efforts to find ligands for this 

receptor. [ 11 C]WAY-100635 is a high affinity radioligand 

used for quantifying 5-HT 1A receptors with 

positron emission tomography. An 18 F-labeled radioligand 

would be advantageous because of higher 

specific activity and physical/nuclear properties 

(t 1/2 = 109 min, 97% of positron decay and positron 

energy of 635 keV maximum). [ 18 F]MPPF, a selective 

5-HT 1A antagonist derived from WAY-100635, 

is currently one of the most successful PET ligands 

used for 5-HT 1A receptor imaging [2]. However the 

affinity is lower than WAY-100635 and the amount 

of [ 18 F]MPPF reaching the brain is relatively low, 

as MPPF is a substrate for P-glycoprotein [3]. 

Methods: In order to improve the brain uptake of 

the radiotracer, a desmethylated analog has been 

developed in our lab and preliminary in vitro 

studies show positive results [4]. Nevertheless, 

the radiosynthesis takes place in two steps, as the 

removal of a protecting group is needed. A one 

step procedure with a MPPF derivative could be 

of very great interest. We have synthesized many 

MPPF derivatives in our lab (modification on the 

phenylpiperazine moiety) and developed an automated 

radiosynthesis procedure for the production 

of these radiotracers. [ 18 F]MPPF was chosen 

as the model compound. We used a GE Healthcare 

FASTlab TM module and made modifications to the 

[ 18 F]FDG synthesis sequence and cassette. [ 18 F] 

MPPF was synthesized by coupling of [ 18 F]FBA 

with the corresponding amine. After coupling, the 

crude solution was diluted with water and passed 

through a tC 18 cartridge for prepurification. After 

elution, the [ 18 F]MPPF was purified by semi-preparative 

HPLC. 

Results: Total synthesis time, including purification 

was approximately 100 min. [ 18 F]FBA and [ 18 F]MPPF 

were obtained at a corrected yield of 60% (n=20) 

and 45% (n=5) respectively. The radiochemical purity, 

checked by radio-TLC and UPLC, was >95%. 

Conclusions: We have developed an automated 

method for [ 18 F]MPPF (and derivatives) production 

using a commercial synthesizer (FASTlab TM 

from GE Healthcare) and a conventional HPLC 

system resulting in good yields and high (radio) 

chemical purity. By simply switching the vial containing 

the modified amine, an 18 F-labeled MPPF 

derivative could be obtained. Radiosynthesis is 

still under optimization and the radiotracers 

synthesized need to be tested as suitable 5-HT 1A 

radioligands. 

Acknowledgement: This work was supported by 

the Fondation Rahier of the University of Liege. 

This work was supported by a GE Healthcare Diagnostic 

Imaging grant. 

References: 

1. Filip M., Bader M. et Al, Pharmacol Rep. 2009 Sep-Oct; 

61(5):761-77 

2. Aznavour N, Zimmer L. Et Al, Neuropharmacology. 

2007 Mar; 52(3):695-707 

3. Laćan G., Plenevaux A. et Al, Eur J Nucl Med Mol 

Imaging. 2008 Dec;35(12):2256-66 

4. Defraiteur C., Plenevaux A. et Al., Br J Pharmacol. 2007 

Nov; 152(6):952-8 


P-036 

poStEr 

NEUROIMAGING from BENCH to BEDSIDE

P-037 

150 


The vitamine E analogue CR6 protects against the long-term microstructure damage induced 

by MCA occlusion: a longitudinal Diffusion Tensor Imaging study 

Justicia C. (1) , Soria G. (1) , Tudela R. (2) , Van Der Linden A. (3) , Messeguer A. (4) , Planas A. (5) . 

(1) Investigaciones Científicas (CSIC), Institut d‘Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 

(2) CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Group of Biomedical Imaging of the University of Barcelona, Spain 

(3) University of Antwerp, Antwerp, Belgium 

(4) Institut de Química Avançada de Catalunya (IQAC), CSIC, ,Spain 

(5) Institut d‘Investigacions Biomèdiques de Barcelona (IIBB)-Consejo Superior de Investigaciones Científicas (CSIC), Institut d‘Investigacions Biomèdiques 

August Pi i Sunyer (IDIBAPS), Spain 

cjmfat@iibb.csic.es 

Introduction: Oxidative and nitrosative stress are 

targets for intervention after ischemia/reperfusion. 

CR-6 is a synthetic analogue of vitamin-E, with the 

additional capacity to scavenge nitrogen-reactive 

species. Recently it has been demonstrated that 

CR-6 exerts a protective action against cerebral ischemia/reperfusion 

injury. The aim of this study was 

to investigate whether CR-6 can protect the brain 

microstructure after brain ischemia as assessed by 

longitudinal Diffusion Tensor Imaging (DTI). 

Methods: Sprague–Dawley rats had the middle 

cerebral artery occluded for 90 mins to induce brain 

ischemia. CR-6 (100 mg/kg) or vehicle (oliva oil) 

was orally administered at 2 and 8 h after ischemia 

onset. Longitudinal MRI scans were performed under 

isofluorane anaesthesia in a BioSpec 70/30 horizontal 

animal scanner (Bruker BioSpin, Ettlingen, 

Germany), equipped with a 12 cm inner diameter 

actively shielded gradient system (400 mT/m). Receiver 

coil was a phased-array surface coil for rat 

brain. Animals were scanned before surgery and 

1, 3 and 5 weeks later. The lesion was monitored 

by T2 mapping of coronal slices acquired with 

a MSME sequence by applying 16 different echo 

times (TE), repetition time (TR) = 4764 ms, Field of 

view (FOV) = 40 x 40 x 21 mm3, matrix size = 256 

x 256 x 21 pixels, resulting in a spatial resolution of 

0.156 x 0.156 mm in 1.00 mm slice thickness. DTI 

images were acquired by using a EPI DTI sequence 

applying TR = 3800 ms, TE = 30.85 ms, 4 segments, 

b-value = 1000, 30 different diffusion directions, 5 

A0 images, slice thickness = 1 mm, number of slices 

= 14, FOV = 2.0 x 2.0 x 1.4 mm3, matrix size = 96 

x 96 x 14 pixels, resulting in a spatial resolution of 

0.21 × 0.21 mm in 1 mm slice thickness. T2 maps 

were analysed using ImageJ. DTI maps of the tensor 

diffusivities, fractional anisotropy (FA), apparent 

diffusion coefficient (ADC), axial diffusivity (λ = 

λ1) and radial diffusivity (λ = [λ2+ λ3/2]), were 

calculated using Paravision 5.0 software (Bruker 

Biospin, Etlingen, Germany) and custom programs 

written in Matlab (The MathWorks, Inc., Natick, 

MA, USA). ROIs were individually drawn for each 

imaging life 

subject and hemisphere, by an experienced neurobiologist 

blinded to the treatments, overlaying the 

MRI images with the digital Paxinos and Watson 

rat brain atlas. 

Results: DTI diffusion indices, particularly axial and 

radial diffusivities, demonstrated that microstructure 

was better preserved after brain ischemia in 

CR6-treated animals than in animals receiving the 

vehicle. The latter showed a significant increase of 

MD, axial and radial diffusivities in the ipsilateral 

anteroventral thalamus, caudate putamen, globus 

pallidus and internal capsula 3 and 5 weeks after 

ischemia versus pre-scan. However, CR6 treated 

animals did not show such increase of DTI indexes 

in any of the ROIs analysed revealing preserved microstructure 

of the brain tissue. 

Conclusions: This work demonstrates that transient 

cerebral ischemia induces long-term microstructure 

alterations of brain tissue that are detected by DTI 

magnetic resonance imaging technique, and that the 

vitamin-E analogue CR6 attenuates these alterations. 

Acknowledgement: S and RT are supported by CSIC 

(JaeDoc) and CIBER-BBN, respectively. We acknowledge 

the European Erasmus program supporting 

Sofie de Prins and Stephan Cools that helped in this 

study. Work supported by national grants (SAF2008- 

04515-C02-01, FIS PI081880), European Network 

of Excellence DiMI (LSHB-CT-2005-512146), and 

FP7/2007-2013 project (grant agreement n°201024).


Design and synthesis of fluorescent probes for serotonin 5-HT1A receptors 

Leopoldo M. (1) , Lacivita E. (1) , Berardi F. (1) , Perrone R. (1) , Jafurulla M. (2) , Saxena R. (2) , Rangaraj N. (2) , Chattopadhyay A. (2) . 

(1) Università degli studi di Bari “A. Moro”, Bari, Italy 

(2) Council of Scientific and Industrial Research, Hyderabad, India 

leopoldo@farmchim.uniba.it 

Introduction: The serotonin1A 

receptor subtype (5-HT1A) 

OCH3 belongs to the large family of 

5-HT receptors that comprises 

at least 14 receptor subtypes. 

5-HT1A receptor is likely the 

most extensively studied serotonin 

receptor. The 5-HT1A 

receptor has been initially 

N 

N 

implicated in anxiety and de- 

O 

pression. Recent studies have 

O 

evidenced its implication in 

neuroprotection, cognitive 

N 

impairment, and pain [1]. 

Many information are avail- 

1 

able about the molecular pharmacology of 5-HT1A 

receptor. However, the study of 5-HT1A pharmacology 

at the single cell and single molecule level 

by fluorescence-based techniques has not been possible 

due to the lack of an effective 5-HT1A fluorescent 

ligands. We here describe design, synthesis 

and preliminar pharmacological evaluation of two 

fluorescent probes for 5-HT1A receptor. 

Methods: The new fluorescent probes have been 

designed following a classic approach [2]: a pharmacophore 

moiety, selected from the literature, has 

been conjugated through a linker to a fluorescent 

dye. One red-emitting and one near infrared dye 

were selected because such excitation wavelengths 

cause reduced light scattering and do not cause cell 

damage. The synthesis and purification of the target 

compounds was accomplished by standard methods. 

The compounds 1 and 2 underwent binding assays to 

test their ability to displace [3H]-8-OH-DPAT from 

5-HT1A receptors overexpressed in HEK-298 cell 

membranes. The fluorescent ligand 1 was evaluated 

in visualization experiments of 5-HT1A receptor on 

CHO cells by confocal laser scanning microscopy. 

Results: Compounds were obtained in good yields 

and in good quantities to allow further biological 

studies. Both compounds 1 and 2 possessed good 

5-HT1A receptor affinities (Ki = 35 nM and 68.9 

O 

N + 

Br- O 

OCH 3 

N N + 

Cl- NaO 3S SO 3Na 


N 

N 

nM, respectively). The target compounds displayed 

the same fluorescent properties as the corresponding 

fluorophores. In visualization experiments by confocal 

laser scanning microscopy compound 1 was able 

to label 5-HT1A receptors on CHO cells overexpressing 

the receptor. This interaction revealed to be specific 

since the compound was unable to bind wildtype 

CHO cells and also because 5-HT significantly 

displaced competitively 1 from the binding site. 

Conclusions: This study indicates that fluorescent 

probes for 5-HT1A receptor can be successfully 

designed on the basis of knowledge of structure- 

activity relationships of 5-HT1A receptor agents. 

The 5-HT1A fluorescent ligand that have been identified 

possess suitable characteristic for visualization 

of 5-HT1A receptors. 

References: 

1. Lacivita E et al; Curr Top Med Chem. 8:1024-1034 

(2008) 

2. Leopoldo M et al: Drug Disc Today 14:706-712 (2009) 

H 

N 

2 

S 

O 

P-038 

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152 


P-039 Comparative evaluation of cerebral blood flow in a rat model of cerebral ischemia using 15 O- 

H 2 0 positron emission tomography and 99m Tc-HMPAO single-photon emission tomography 

Martin A. (1) , Boisgard R. (1) , Gervais P. (2) , Thézé B. (1) , Vuillemard C. (2) , Tavitian B. (1) . 

(1) CEA, DSV, I²BM, SHFJ, Laboratoire Imagerie Moléculaire Expérimentale; INSERM U803, Orsay, France 

(2) CEA, DSV, I²BM, SHFJ, France 

abraham.martin-munoz@cea.fr 

Introduction: Ischemic stroke occurs when cerebral 

blood flow is interrupted in a specific region of the 

brain. After ischemic stroke, cerebral parenchyma 

suffers perfusion changes over time which have been 

correlated with biological processes as compensatory 

growth of blood vessels to supply metabolic demand 

[1], underlying inflammation [2] and angiogenesis 

[3].Thereafter, a precise understanding of the cerebral 

blood flow evolution after ischemic stroke is essential 

for the comprehension of cerebral ischemia physiopathology. 

Here, a comparative imaging evaluation 

of cerebral blood flow using PET and SPECT during 

and after cerebral ischemia was performed in rats. 

Methods: [ 15 O] H20 and [ 99m Tc] HMPAO were used 

in a rat model of 2 hours transient middle cerebral 

artery occlusion (tMCAO) during occlusion, during 

early reperfusion and later on at 1, 2, 4 and 7 days. 

The tissue was analized ex vivo using histological 

(HE) and immunohistochemistry (CD31). 

Results: In vivo PET imaging showed a significant 

decrease of the ipsilateral or lesioned area versus 

contralateral ratios during the occlusion in 

relation to control, days 4 and 7 after reperfusion 

(P


Comparison of dopamine transporter density in Parkinson’s disease patients with and 

without autonomic dysfunction using F-18 FP-CIT PET/CT 

Park E. , Park K.W. , Hwang Y.M. , Oh S.Y. , Choe J.G. . 

Korea University Anam Hospital, Korea University College of Medicine, Seoul 

angela_ekpark@yahoo.co.kr 

Introduction: Autonomic dysfunction is a common 

non-motor feature of Parkinson’s disease (PD) 

which can severely impair the life quality of PD patients. 

However, evaluation of striatal presynaptic 

dopaminergic function with SPECT or PET imaging 

using various radiotracers has been limited in 

the differential diagnosis of parkinsonism and in 

the severity assessment of Parkinson’s disease. We 

compared the dopamine transporter (DAT) density 

of PD patients with and without autonomic dysfunction 

using F-18 FP-CIT PET/CT, which has been 

approved recently by Korean FDA for clinical uses. 

Methods: Twenty clinically diagnosed PD patients 

(mean age 68.6±8.1 y, M:F=5:15) and 8 age-matched 

healthy normal controls (65.1±4.7 y, M:F=2:6) were 

studied with F-18-FP-CIT PET/CT. Among 20 PD 

patients, 10 had no significant autonomic dysfunction 

(PD-AD) while the other 10 had autonomic dysfunction 

(PD+AD). F-18-FP-CIT PET/CT images were 

obtained 120 minutes after injection of 185 MBq 

F-18-FP-CIT. Semi-quantitative analysis was performed 

using manual ROI method. A DAT parameter 

V3”, a measure directly related to the density of DAT, 

was calculated in striatal regions as (striatal ROI– 

cerebellar ROI mean radioactivity)/cerebellar ROI 

mean radioactivity on F-18-FP-CIT PET/CT images. 

Results: PD patients demonstrated significantly 

lower DAT V3” in the striatum (2.29±0.69), caudate 

nucleus (2.56±0.67), and putamen (1.99±0.77) 

than those of healthy normal controls (4.02±0.55, 

3.88±0.54 and 4.16±0.61, respectively) (p

154 


P-041 Synthesis of tosylate and mesylate precursors for one-step radiosynthesis 

of [ 18 F]FECNT 

Pijarowska J. , Jaron A. , Mikolajczak R. . 

Insitute of Atomic Energy POLATOM, Otwock, Poland 

j.pijarowska@polatom.pl 

Introduction: Dopamine transporter (DAT) is critical 

to the regulation of dopamine neurotransmission and 

is decreased by Parkinson’s disease. Several tropane 

analogues of cocaine have been developed and used in 

PET studies to evaluate the physiology and pharmacology 

of the dopamine transporter (DAT). However, 

low selectivity and unfavourable kinetics of most of the 

compounds limit their use in quantitive PET studies. 

The fluorine-18 labelled ligand 2-beta-carbomethoxy-3- 

-beta-(4-chlorophenyl)-8-(2-fluoroethyl)-nortropane 

(FECNT) has promising properties and appears to be 

an excellent imaiging PET agent. The development of 

automated [18F]FECNT synthesis system is a crucial 

because high amounts of radioactivity and availability 

of radiotracer for multiple PET study is necessary. A 

semi-automated synthesis of [18F]FECNT based on 

the two-steps has been developed at 16% decay corrected 

yield [1]. We hypothesize that N-[18F]fluoroalkylnortropane 

analogs could be synthesized at hight 

yield by direct [18F]fluorination from appropriate precursors: 

N-tosylate and N-mesylate derivatives. This 

compounds and non-radioactive FECNT as a standard 

in order to characterize [ 18 F]FECNT on HPLC are 

synthesized in accordance with requirements for Investigational 

Medicinal Product (IMP). 

Methods: The synthetic approach which we adopted 

based upon the published procedures [2,3] 

with some modifications. The essential feature 

of this route was the reaction of Grignard reagent 

with the critical intermediate anhydroecognine 

methyl ester, which was obtained from 

cocaine hydrochloride by hydrolysis in HCl and 

esterification with methanol. 3-β-substituted tropane 

derivative obtained in Grignard reaction 

was subjected to demethylation, as described [4]. 

Non-radioactive FECNT was prepared by direct 

N-(2- fluoroethyl) alkylation of analytically pure 

3-β-substituted nortropane precursor. The alkylating 

agent 2-fluoroethyl brosylate was prepared 

from 2-fluoroethanol and 4-bromobenzenesulfonoyl 

chloride. The crude product was purified 

by recrystallization. The tosylate (TsOECNT) 

and mesylate (MsOECNT) precursors were 

imaging life 

synthetised from 3-β-substituted nortropane precursor 

in two steps by N-hydroxyethylation with 

2-bromoethanol and subsequent tosylation of the 

obtained alcohol with appropriate anhydride. The 

crude products were purified by preparative HPLC. 

Results: In the present study we investigated and 

optimized synthetic route of non-radioactive 

FECNT, tosylate and mesylate analogs. Overall 

production yield were 74% for FECNT, 58% 

for TsOECNT and 81% for MsOECNT synthesis 

and a purity of this products were over 

99% measured by the analytical HPLC(UV, 220 

nm). The 1 H NMR and MS analysis confirmed 

a structure of this compounds. 

Conclusions: The synthesis method of tosylate 

and mesylate precursors were established and an 

automated radiosynthesis of [18F]FECNT will be 

evaluated. We expect that the new one–step method 

will provide a facile and reliable procedure 

for [18F]FECNT preparation in routine clinical 

applications. 

Acknowledgement: This work is supported by DiMI, 

LSHB-CT-2005-512146 

References: 

1. Voll R.J. et al. Appl. Rad. Isot. 2005 (63) 353 

2. Zirkle C. L et al. J. Org. Chem. 1962 (34) 1269 

3. Clarke R. L. et al. J. Med. Chem. 1973 (16) 1260 

4. Meegalla S. K. et al. J. Med. Chem. 1997 (40) 9


Sex differences inDopamine D 2 Receptor Occupancy in the Amygdala using AMPT with PET 

and [ 18 F]fallypride 

Riccardi P. (1) , Park S. (2) , Carroll X. (1) , Anderson S. (3) , Benoit D. (2) , Li R. (2) , Bauernfeind A. (4) , Schmidt D. (2) . 

(1) Mercer University, Macon, United States 

(2) Vanderbilt university, United States 

(3) Georgia State University ,United States 

(4) George Washington university, United States 

riccardip@aol.com 

Introduction: Dopaminergic neurotransmission 

plays an important role in many psychiatric disorders 

which show sex differences in incidence, clinical 

course, and treatment outcomes. We examined 

whether PET studies using [ 18 F]fallypride performed 

prior to and following alphamethylparatyrosine 

(AMPT) administration could be used to estimate 

sex differences in baseline Dopamine D 2 receptors 

(DAD2r) occupancy. 

Methods: Four females and four males normal subjects 

were recruited with no history of psychiatric, 

neurological or medical illness. PET studies were 

performed using a GE Discovery LS PET scanner 

with 3-D emission acquisition and transmission 

attenuation correction. [ 18 F]fallypride PET scans 

(5.0 mCi, specific activity > 2,000 Ci/mmol) were 

performed prior to and following AMPT administration 

over 26 hours. Serial scans were obtained 

for 3.5 hours. Blood samples for HVA plasma levels 

were collected. 

Results: Analysis of variance of the ROI data with 

treatment status, region, sex and laterality as factors 

revealed significant effects of sex in the right 

(F = 9.403, P = 0.002) and left amygdala (F = 3.486, 

P = 0.05). No difference in b.p was found in the 

left amygdala at baseline, but significant differences 

were determined at baseline and post treatment in 

the right amygdala (p=0.005 and p=0.002) and post 

treatment in the left amygdala (p=0.019) with females 

demonstrating lower levels of D 2 /D 3 receptors 

compare to males. Effect of treatment was observed 

in females in the left amygdala with a percentage of 

change of 6.7%. 

Conclusions: The amygdala has been shown to be 

functionally asymmetrical in animals and humans, 

is involved in stress and emotion processing, and is 

importantly modulated by dopamine. We report here 

for the first time a sex differences in the occupancy 

of DA D 2 r by dopamine in vivo in the amygdala. Previous 

AMPT administration and [18F]fallypride PET 

studies to assess the baseline occupancy of DAD 2r by 

endogenous dopamine in-vivo reported no significant 

effects of treatment in the amygdala when males and 

females were grouped together. The results of this 

study underscore the importance of considering sex 

differences in the context of DAD 2r availability. 

Acknowledgement: Funding for this reaserch 

was provided by a NIH grant entltled “PET imaging 

of Extrastriatal Dopamine levels” NIMH 5RO1 

MH6898-03 

References: 

1. Verhoeff NP, Kapur S, Hussey D et al (2001). A simple 

method to measure baseline occupancy of neostriatal 

dopamine D2 receptors by dopamine in vivo in healthy 

subjects. Neuropsychopharmacology 25; 213-223 

2. Riccardi P., Baldwin R, Salomon R., et al. (2008). 

Estimation of baseline Dopamine D2 receptor 

occupancy in Striatum and extrastriatal regions in 

humans with Positron emission tomography with 

[18F]fallypride. 


P-042 

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156 


P-043 Manual versus automatic delineation of VOIs for analysis of nuclear medicine images 

Svarer C. (1) , Hammers A. (2) , Heckemann R. A. (2) , Knudsen G.M. (1) . 

(1) Neurobiology Research Unit, Rigshospitalet,, Copenhagen, Denmark 

(2) The Neurodis Foundation, Lyon, France 

csvarer@nru.dk 

Introduction: Quantification and analysis of nuclear 

medicine images is performed by visual inspection in 

most cases. More objective and less observer-dependent 

analysis often requires automatic identification 

of volumes of interest (VOIs) in the images. In this 

study we investigated the levels of precision achievable 

with manual delineation on high resolution MR 

images and compared them to the precision of a fully 

automatic method. 

Methods: The method described in Svarer et al(1) has 

been refined to handle neighborhood voxels in labeled 

template volumes using a maximum-probability algorithm. 

We applied it to 19 MR data sets, each with 67 

manually delineated VOIs(2). To test the precision of 

the automatic algorithm, we performed a leave-oneout 

cross-validation-comparison: using each data set 

in turn as the target, we automatically generated two 

independent VOI sets by randomly bisecting the template 

pool, leading to two independent segmentations 

of each target by nine atlases each, and calculated the 

average VOI voxel overlap (intersection divided by 

average labeled volume). To test the reproducibility/ 

precision of the manually delineated VOI sets, a third 

VOI set was generated for each target using the entire 

template pool but itself, and its overlap with the 

manual VOI set were calculated. 

Results: The voxel overlap for the two automatically 

generated VOI sets was 88.3±5.2%, whereas the voxel 

overlap between the automatically generated and the 

manually delineated VOI set was 76.3±9.8%. 

Conclusions: Of the 24% non-overlap between manual 

and automatic methods approx. 12 percentage points 

may be attributable to variation in the automatic method 

using different VOI template sets whereas precision 

in manual delineation of the VOIs may largely underlie 

the other half of the difference between manual and 

automatically generated VOI sets. This variation will 

be removed using an automatic approach based on 

multiple template sets. 

Acknowledgement: Supported by The Lundbeck Foundation, 

Rigshospitalet, and the Danish Medical Research 

Council. These studies were funded in part by 

the EC - FP6-project DiMI, LSHB-CT-2005-512146. 

imaging life 

References: 

1. Svarer C, Madsen K, Hasselbalch SG, Pinborg LH, 

Haugbøl S, Frøkjaer VG, Holm S, Paulson OB, and 

Knudsen GM (2005) MR-based automatic delineation 

of volumes of interest in human brain PET images 

using probability maps. Neuroimage 24: 969-979. 

2. Hammers A, Allom R, Koepp MJ, Free SL, Myers R, 

Lemieux L, Mitchell TN, Brooks DJ, and Duncan JS 

(2003) Three-dimensional maximum probability atlas 

of the human brain, with particular reference to the 

temporal lobe. Hum Brain Mapp 19: 224-247.


PET Amyloid and Tau Ligand [18F]FDDNP uptake in early Alzheimer disease 

Tauber C. (1) , Beaufils E. (1) , Kepe V. (2) , Vercouillie J. (1) , Venel Y. (1) , Baulieu J.L. (1) , Barrio J. (2) , Hommet C. (1) , Camus V. (1) , 

Guilloteau D. (1) . 

(1) UMRS INSERM U930 - CNRS ERL3106 - University of Tours, France 

(2) Dpt of Molecular and Medical Pharmacology, D. Geffen School of Medecine, UCLA, United States 

clovis.tauber@univ-tours.fr 

Introduction: The PET tracer [18F]FDDNP specifically 

binds amyloid-beta plaques and tau neurofibrillary 

tangles, which are typical in the development 

of the Alzheimer disease (AD). We evaluated 

the feasibility of a non-invasive quantification of 

[18F]FDDNP to detect these proteins and discriminate 

early AD patients from healthy controls (HC). 

This abstract presents the preliminary results on the 

currently included cohort of 4 AD and 2 controls. 

Methods: All the potential subjects underwent Neuropsychological 

Assessment (MMSE, Trail Making 

Test, Semantic Fluency Task, Free and Cued 

Recall Test, 80 item Boston Naming Test). Patients 

meeting research criteria for AD and cognitively 

normal controls underwent PET imaging with 

2-(1-{6-[(2-[F-18]fluoroethyl) (methyl)amino]-2naphthyl}ethylidene)malononitrile 

(FDDNP) and 

[18F]FDG. All subjects underwent MR 3D axial 

T1 weighted imaging. FDDNP distribution volume 

ratios (DVR) parametric images were generated using 

Logan graphical analysis with the cerebellum 

grey matter as a reference region. Regions of interest 

(ROIs) were defined in the Posterior Cingulate, 

Parietal, Frontal and Temporal regions. The DVR 

scores were measured as the mean DVR values of 

each of these regions. A global score was calculated 

as the mean DVR scores of all these regions. 

Results: All the AD patients had positive FDDNP 

scans by visual inspection, while all the HC had negative 

scans. Global values of the FDDNP-PET binding 

were significantly higher (p

158 


P-045 Cryogenic brain injury as a model of brain trauma: Use of GFAP-luc mice to assess GFAP 

expression as an indication of neural injury 

Van Beek E., Blankevoort V., Snoeks T., Kaijzel E., Löwik C.W.G.M. . 

Leiden University Medical Center, The Netherlands 

e.r.van_beek@lumc.nl 

Introduction: Applying a cryogenic lesion of the 

cerebral cortex provides a simple and highly reproducible 

model of brain trauma without entering the 

intracranial cavity. In the present study we assessed 

and mapped the sequence of events that occur following 

induction of a cryogenic brain lesion using 

transgenic glial fibrillary acidic protein (GFAP)-luc 

reporter expressing mice. In these mice, the GFAP 

reporter is inducible following injury to the CNS 

and this model provides an easy model feasible for 

the study of transcriptional regulation of the GFAP 

gene and indications of possible neural injury. 

Methods: A brain lesion was induced by placing a 

liquid nitrogen cooled cone shaped copper device 

with a tip diameter of 1 mm for 1 min. onto the skinfree 

surface of the cranium of the cerebral cortex of 

a mouse. At different time points after lesion induction 

GFAP-luc activity was measured, mice were injected 

with Evans blue and brains were stained with 

2,3,5-Triphenyltetrazolium chloride (TTC). 

Results: 24h after induction of the cryogenic lesion, BLI 

measurement showed that, at the lesion site, GFAP-luc 

expression was increased 2-3 times over control level 

and gradually returned to basal after 72h. Evaluation 

imaging life 

Gfap-luc expression (Relative Light Units) 

Control GF AP–luc 

Control 

GFAP-luc expression expression 

24h 

24h after after freeze freeze injury injury 

6.0E+06 

5.0E+06 

4.0E+06 

3.0E+06 

2.0E+06 

1.0E+06 

0.0E+00 

of tissue viability using TTC staining showed that 24h 

after lesion, the primary lesioned cortex appeared as 

an unstained white area (dead tissue), whereas, the 

rest of the brain appeared red (living tissue). Moreover, 

at the lesion site, i.v. injected Evans blue leaked 

into the brain tissue, indicative for disruption of the 

blood brain barrier. This was also visualised in intact 

isolated brains by Fluorescence Imaging (FLI). 

Conclusions: Cryogenic lesion in the GFAP-luc mouse 

model showed a transient induction of GFAP expression, 

indicative for astrocyte activation in reaction 

Base 1 Base 2 24h 48h 72h 

to neural injury. Brain injury was confirmed by TTC 

staining and leakage of Evans blue over the blood 

brain barrier. GFAP-luc mice provide an easy and convenient 

model to study cryogenic brain trauma. 

Acknowledgement: This project was supported by 

EU-FP7 ENCITE (HEALTH-F5-2008-201842) and 

Volkswagen Stiftung.


Plaque burden in the APPPS1 mouse picked up with diffusion kurtosis magnetic resonance 

imaging 

Vanhoutte G. , Geys R. , Pereson S. , Veraart J. , Van Broeckhoven C. , Kumar-Singh S. , Sijbers J. , Van Der Linden A. . 

University of Antwerp, Belgium 

greetje.vanhoutte@ua.ac.be 

Introduction:Transgenic mouse models are essential 

in understanding the pathogenic role of the 

β-amyloidogenic pathway in Alzheimer(AD).In vivo 

detection of the amyloid deposits in the brain would 

be beneficial in terms of diagnosis and therapy follow-up.Therefore 

we investigated the sensitivity of 

a new method, diffusion kurtosis imaging (DKI), to 

detect amyloid burden, a correlate for brain damage 

in AD patients and transgenic APP (swe) -PS1 (L166p) 

mice. The use of this model was carefully chosen 

for its effective amyloidosis in the brain without the 

occurrence of neurodegeneration, tau-pathology or 

behavioural changes.All mice of 17months manifested 

amyloid burden in all brain regions(1).Since 

previous studies could show different ex vivo patterns 

for mean kurtosis in an APPPS1 mouse model, 

we hypothesize that the microstructural changes in 

the brain, due to extracellular amyloid deposits,can 

be measured in vivo by DKI(2). We investigated the 

neocortical and hippocampal regions linked to the 

cognitive impairment in AD. Further studies will be 

conducted to determine the longitudinal effects. 

Methods:Experiments were conducted on a 9,4T 

MR system(Bruker Biospec, Ettlingen Germany). 

DKI scans were performed on APP (swe) -PS1 (L166p) 

mice(n=5) and control WT mice(n=5). The mice 

were anaesthetized with isoflurane and monitored to 

maintain physiological parameters. The DKI protocol 

included 7 b 0 -images and 30 gradient directions 

with 7 b-values (400 to 2800s/mm²) acquired with 

multi-slice 2-shot SE-EPI(TR/TE=7500/24ms,δ=5ms, 

Δ=12ms,acquisition=96*64, FOV=19,2*12.8mm², 

slice thickness=0,40mm, NEX=4). Realignment, 

was carried out by the vision lab (Univ. Of Antwerp) 

and diffusion kurtosis tensor and diffusion tensor 

derived parametric maps were computed (Matlab). 

These include mean kurtosis (MK), radial kurtosis 

(RK), axial kurtosis (AK), fractional kurtosis anisotropy 

(KA), fractional anisotropy (FA), mean diffusion 

(MD), axial and radial diffusion (AD, RD) 

maps. Regions of interest (cortex and hippocampus) 

were chosen based on the AD pathology and delineated 

on grey values of FA, MD and magnitude maps 

in AMIRA (Mercury Computer systems, San Diego, 

USA). Differences of diffusion parameters between 

WT and APPPS1 mice were computed by means of 

the Mann-Withney non-parametric statistical test in 

SPSS 16.0 (SPSS Inc. Chicago, USA). 

Results: This is the first in vivo study on AD mouse 

models using DKI.Already with a small set of 

number of animals, we could detect differences according 

to genotype. MK(p

160 


P-047 In vivo Imaging of Rat Glioma using the TSPO-ligand [ 18 F]DPA-714 

Winkeler A. (1) , Boisgard R. (1) , Dubois A. (1) , Awde A. (1) , Zheng J. (1) , Ciobanu L. (2) , Siquier-Pernet K. (1) , Jego B. (1) , Dollé F. (1) , 

Tavitian B. (1) . 

(1) CEA\DSV\I2BM\SHFJ, Orsay, France 

(2) roSpinCEA\DSV\I2BM\NeuroSpin,France 

alexandra.winkeler@cea.fr 

Introduction: In the last years there has been an 

enormous increase in the development of radioligands 

targeted against the translocator protein 

TSPO (18 kDa). TSPO expression is nearly absent 

in the intact CNS parenchyma but increases rapidly 

upon inflammation in activated microglia and 

serves as a biomarker for imaging cerebral inflammation 

(1). In addition, TSPO has also been reported 

to be over-expressed in a number of cancer cell 

lines (2, 3) and human tumours including glioma 

(4). Here, we investigated the use of the PET-radioligand 

[ 18 F]DPA-714 (5) as new marker to image 

glioma in vivo. 

Methods: 9L rat glioma cells have been stereotactically 

implanted in the striatum of Fisher, Wistar and 

Sprague Dawley rats. Dynamic [ 18 F]DPA-714 PET 

imaging was performed 11-14 days after implantation. 

The injected dosed was 1.24 + 0.30 mCi (mean 

+ std). T2w-MRI and/or [ 11 C]Methionine PET were 

acquired prior to the [ 18 F]DPA-714 PET imaging 

session in order to monitor tumor growth. The [ 18 F] 

DPA-714 PET images were then co-registered to 

the corresponding MRI. For quantitative analysis 

a volume-of-interest (VOI) analysis was performed 

on both the kinetic and summed image data sets. In 

addition, the expression of TSPO 9L rat glioma cells 

was investigated using Western Blot. 

Results: 9L glioma tumors grown in Fisher (n=5), 

Wistar (n=4) and Sprague Dawley (n=6) rats were imaged 

by [ 18 F]DPA-714 PET. Tumors grown in Fisher 

and Wistar rats were also monitored by MRI. All rats 

showed significant [ 18 F]DPA-714 PET accumulation 

at the site of tumor implantation compared to the 

contralateral site. Tthe %ID/cc in Fisher, Wistar and 

Sprague Dawley rats is listed in the following table: 

imaging life 

control (mean+std) tumor (mean+std) 

Fisher 0.15 + 0.02% 0.49 + 0.05% 

Wistar 0.13 + 0.06% 0.35 + 0.09% 

Sprague Dawley 0.11 + 0.05% 0.26 + 0.06% 

TSPO expression was confirmed by Western Blot in 

9L cells in vitro and by immunohistochemistry ex vivo. 

Conclusions: This study demonstrated the feasibility 

of using the TSPO-radioligand [ 18 F]DPA-714 to 

characterize 9L glioma in vivo in different rat models 

with PET imaging. [ 18 F]DPA-714 therefore has 

the potential to become a promising radiotracer to 

image human glioma. 

Acknowledgement: This work has been supported 

by the 6th FW EU grant EMIL (LSHC-CT-2004- 

503569) and DiMI (LSHB-CT-2005-512146). 

References: 

1. Winkeler A, Boisgard R, Martin A, & Tavitian B (2009) J 

Nucl Med 51, 1-4. 

2. Hardwick M, Fertikh D, Culty M, Li H, Vidic B, & 

Papadopoulos V (1999) Cancer Res 59, 831-842. 

3. Starosta-Rubinstein S, Ciliax BJ, Penney JB, McKeever 

P, & Young AB (1987) Proc Natl Acad Sci U S A 84, 891- 

895. 

4. Black KL, Ikezaki K, Santori E, Becker DP, & Vinters HV 

(1990) Cancer 65, 93-97. 

5. Damont A, Hinnen F, Kuhnast B, Schollhorn- 

Peyronneau MA, James M, Luus C, Tavitian B, Kassiou 

M, & Dolle F (2008) Journal of Labelled Compounds & 

Radiopharmaceuticals 51, 286-292.


A novel 19F MRI-based migration assay: application to primary human dendritic cells 

Bonetto F. (1) , Srinivas M. (1) , Weigelin B. (1) , Cruz Ricondo L.J. (1) , Heerschap A. (2) , Figdor C. (1) , De Vries I.J. (1) . 

(1) Nijmegen Center for Molecular Life Science, Radboud University Nijmegen Medical Centre, The Netherlands 

(2) Radboud University Nijmegen Medical Centre, Nijmegen, , Netherlands 

bonetto.fernando@gmail.com 

Introduction: The decisive role of dendritic cells 

(DCs) in inducing immunity formed the rationale 

for DC immunotherapy: DCs loaded with tumor 

antigens are injected into cancer patients to stimulate 

T cells to eradicate tumors. However, success 

in clinical trials has been limited mainly due to 

an inefficient migration rate post-vaccination. As 

DC migration can be affected by several factors, a 

suitable in vitro assay is required to reproduce in 

vivo conditions. Here we present a novel 19 F MRIbased, 

quantitative assay to measure cell migration 

in varying chemokine environments in a 3D 

scaffold specially designed to mimic biological tissue. 

We obtained migration rates comparable with 

clinical results (using scintigraphy for quantification) 

[1] showing the potential application of this 

assay to simulate different in vivo migration conditions. 

Moreover, we observed that the percentage 

of migrating cells strongly depends on the initial 

cell density. 

Methods: Primary human DCs were cultured, as 

per standard protocols for DC vaccination trials 

[2]. For 19 F-labeling, a biocompatible polymer 

currently in clinical use, poly(D,L-lactide-co-glycolide) 

(PLGA) was used to entrap a perfluorcarbon 

tracer. After maturation, cells were harvested, 

and viability and maturation marker expression 

determined. The presence of label within the cells 

was checked by optical microscopy. For migration 

assays, a variable number of cells were embedded 

in a fixed volume of a scaffold matrix (cell layer) 

and a chemokine gradient was created above it. A 

chemokine-free gel set below the cell layer served 

as a control. The sample was placed vertically in 

the MRI scanner and only upward migration was 

considered to exclude the effects of gravity. Temperature 

was maintained at 37 o . All experiments 

were performed on a 7T horizontal bore MR system 

with a 1 H/ 19 F volume coil. 1 H 2D spin echo images 

and chemical shift spectroscopy (CSI) were used 

to track and quantify the migration of 1-15x10 6 

cells. Nine 19 F-CSI experiments (1.1 hours each) 

were sequentially performed in order to keep track 

of the migrating cells. 

Results: Cell migration was assessed by measuring 

cell movement relative to their initial position above 

and below the cell-gel layer. Minimal migration was 

observed in samples with 10-15×10 6 cells. However, 

with smaller cell numbers, 5×10 4 from 5×10 6 cells 

(1%) and 2.5×10 4 from 1×10 6 cells (2%) were found 

to migrate up. Migration was found to stop 10 hrs 

after the start of the experiment. No cell migration 

was found below the initial cell layer during the 

whole study for all cell layer numbers, indicating 

that the upward cell movement was truly caused by 

the chemokine gradient. 

Conclusions: In the present study we showed that 

19 F-CSI and 19 F-MRI can be used to track and quantify 

cell migration in 3D collagen scaffold assays. 

The number of cells in the initial cell layer was in the 

order of the typical cell bolus injection in patients 

[1] and the migration rates were measured to be in 

the order of 2%, similar to that obtained in clinical 

results. The study showed that the initial cell density 

plays a decisive role in DCs migration, which 

is important information to optimize cell migration 

in a clinical setting. Thus, this assay is suitably to 

simulate in vivo clinical conditions, giving the potential 

chance for detailed analysis of DC migration 

by MRI. 

Acknowledgement: This work was partially supported 

by NWO (VISTA grant) and ZONMW (911-06- 

021) for investments. 

References: 

1. P. Verdijk, T. W. Scheenen, et al.. Int J Cancer 120(5): 

978-84 (2007). 

2. I. J. de Vries, W. J. Lesterhuis et. al.. Nat. Biotechnol. 

23(11), 1407-1412 (2005). 


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GENE and CELL based THERAPIES

P-049 

162 


In vivo magnetic resonance imaging reveals altered migration of endogenous neural 

progenitor cells following cuprizone-induced central nervous system demyelination 

Guglielmetti C. , Praet J. , Vreys R. , Ponsaerts P. , Van Der Linden A. . 

University of Antwerp, Belgium 

carolineguglielmetti@hotmail.com 

Introduction: The Cuprizone mouse model represents 

a highly reproducible tool to study in vivo demyelination 

and remyelination processes. The time 

course of demyelination-remyelination during Cuprizone 

administration has been well characterized 

and it has been suggested histologically that endogenous 

subventricular zone neural stem/progenitors 

cells (NSPC) are recruited during the remyelination 

process [1] . We tested whether the recently validated 

in situ NSPC labelling technique with micron-sized 

iron oxide particles in combination with the transfection 

agent Poly-L-lysine [2] (MPIOs-PLL) is suitable 

to visualize NSPC migration towards demyelinated 

areas. 

Methods: 20 C57BL/6 mice (8 weeks old) were used. 

All mice were injected in the right lateral ventricle 

with 2 µl MPIOs-PLL (9.1 10 5 particles, 0.67 mg Fe/ 

ml). 10 mice were fed a diet containing Cuprizone 

(0.2%) during a 4-week period post-injection (p.i). 

In vivo T 2 *-weighted 3D-MGE (78 µm isotropic resolution) 

was performed at 4 weeks (peak of inflammation), 

6 weeks (peak of proliferation of NSPC) 

and 12 weeks (complete remyelination) pi. All images 

were acquired on a 9.4T Bruker console. 5 animals 

of each group received an injection of 5-bromo-2’-deoxyuridine 

(BrdU; 50 mg/kg i.p.) twice a 

day over a period of 5 days before either the first or 

the second imaging time point. 

Results: MRI revealed hypointense voxels along the 

rostral migratory stream (RMS) and in the olfactory 

bulb (OB) for controls. In contrast, for the Cuprizone 

treated mice, hypointense areas towards the 

OB were detected only in 2 mice at week 4. From 

week 6 onward they were detected in both groups. 

Hypointense voxels are also found in the region of 

the external capsule (EC) at the ipsilateral side of 

injection from 4 weeks onward in the Cuprizone 

treated group. Each MR image is a compilation of 

5 consecutive MR slices. Histological analyses are 

being performed in order to confirm whether the 

hypointense voxels are MPIO particles in oligodendrocyte-differentiated 

NSPC. 

imaging life 

Conclusions: In situ labelling of endogenous NSPC 

by direct injection of MPIOs-PLL in the lateral ventricle 

revealed NSPC migration impairment towards 

the OB, as well as the presence of MPIOs in the EC 

in the Cuprizone mouse model. Histological analysis 

is ongoing in order to further characterise migrated 

MPIO-labelled cells. 

Acknowledgement: This work is supported by IWT 

(SBO/030238), EC-FP6-project DiMI (LSHB-CT- 

2005-512146) and IUAP-NIMI-P6/38 

References: 

1. Matsushima G K et al; Brain Pathology, 11: 107-116 

(2001) 

2. Vreys R et al; NeuroImage 49, 2094-2103 (2010)


Evolution pathways of compartmentalization and distribution of labeling iron-oxide 

particles in tumor tissue 

Kotek G. , Van Tiel S. , Wielopolski P. , Krestin G. , Bernsen M. . 

Erasmus University Medical Center Department of Radiology, Rotterdam, Netherlands 

g.kotek@erasmus.nl 

Introduction: In our study we addressed the effects 

and the evolution of SPIO distribution on R2 and 

R2* values according to specific pathways, such as 

cell death, cell mixing and cell division. Also we 

investigated the effects of technical variations in 

labeling efficacy, cell density, imaging resolution 

and aggregation of labeling particles. We identify 

areas for improvement in cell labeling technique 

and imaging for quantitative cell tracking. 

Methods: Brown Norway 175 (BN175) sarcoma 

cells were labeled with iron-oxide (SPIO) particles. 

Various intravoxel SPIO distributions were 

prepared by methods mimicking biologically relevant 

processes (compartmentalization, mixing, 

division). R2* and R2 relaxometry was performed 

at high resolution at 3.0T, iron concentration 

was measured by optical emission spectrometry. 

Effects of spatial distribution and compartmentalization 

of SPIO on relaxivity (dR/dcFe) was 

analyzed. 

Results: We showed that relaxivity is sensitive to 

variations of cell labeling, cell density and imaging 

resolution. Our data suggest that intracellular 

variance of cell division rate potentially lead to 

breakdown of unique relaxation rate vs. iron concentration 

relationship. Our results indicate that 

cell death can be identified by parallel monitoring 

of R2 and R2* values, and we confirm that R2’ differentiates 

between intracellular and extra-cellular 

SPIO. We found a unique relaxation rate vs. iron 

concentration relationship in case of dominance 

of cell division. Our results suggest that tighter 

control on labeling variance put in place for stem 

cell labeling. 

Conclusions: We challenge labeled cell quantification 

by identifying sensitivity of the relaxivity 

to imperfections of labeling technique and imaging 

method; besides the numerous complications 

in quantification, with thorough consideration 

of limitations and monitoring of biologically relevant 

factors, quantification is feasible. 

In case of mixing labeled and non-labeled cells 

(likely process in stem cell tracking) a strong 

control on labeling efficiency is required, since 

R2*(cFe) and R2(cFe) curves are sensitive to initial 

intracellular iron content and cell density. 

References: 

1. Bowen CV, Zhang X, Saab G, et al. 

2. Application of the static dephasing regime theory to 

superparamagnetic iron-oxide loaded cells. Magn 

Reson Med 2002;48:52–61. 

3. Rad AM, Arbab AS, Iskander AS, et al. Quantification 

of superparamagnetic iron oxide (SPIO)-labeled cells 

using MRI. J Magn Reson Imaging 2007;26:366–74. 

4. Kuhlpeter R, Dahnke H, Matuszewski L, et al.R2 and R2* 

mapping for sensing cell-bound superparamagnetic 

nanoparticles: in vitro and murine in vivo testing. 

Radiology. 2007 Nov;245(2):449-57. Epub 2007 Sep 11. 


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P-051 Acupuncture Works on Endorphins via Activating Stretch-Activated Cation Channels 

164 

Liang J. (1) , Zhong P. (2) , Yang X. (3) , Li G. (3) , Yang E. (3) , Cheung P. (1) . 

(1) The University of Hong Kong, Hong Kong, Hong Kong 

(2) First Affiliated Hospital of Sun Yat-Sen University, 

(3) Hong Kong Applied Science and Technology Research Institute Company Limited, Hong Kong 

cmleung@eee.hku.hk 

Introduction: Acupuncture has been in use for three 

thousand years in China but was not practiced in the 

West until the last three decades. Among acupuncture 

therapies, the acupuncture-induced analgesic effect 

has been used widely to alleviate diverse pains, particularly 

chronic pain. To shed light on the 2500-yearold 

acupuncture analgesia, pain control by acupuncture 

through Ca2+-regulated release of opioid peptides 

is proposed. 

Methods: 4-week-old C57BL/6N mice were kept in 

a specially designed holder, with their hind legs and 

tails exposed during acupuncture. An acupuncture 

needle (Hwato) 0.4 mm in diameter, 50 mm in length 

was inserted into the right hind leg, between the 

tibia and fibula, approximately 5 mm lateral to the 

anterior tubercle of the tibia. The acupuncture needle 

was driven by a piezoelectric bending element at 1 

Hz. After receiving acupuncture treatment for 40 

minutes, animals were sacrificed. Blood samples were 

harvested from the left ventricle of the heart. Plasma 

was separated by centrifuging samples at 1000 × g 

for 15 minutes at 4 ˚C and diluted 1:10 in a sample diluent 

before assay. The levels of plasmaβ-endorphin 

were measured using a Mouse β-Endorphin ELISA kit 

from USCN Life Science. Method for in vivo monitoring 

of Ca2+ excitation in mouse skeletal muscle during 

acupuncture was described in [1]. 

Results: Acupuncture was initiated by the stimulation 

of needle driver describe above in the hind-limb muscle 

of the mouse. Plasma β-endorphin was measured 

40 min after the acupuncture treatment by immunoassay. 

Elevation of β-endorphin levels is observed 

(Fig.1b) along with cytosolic Ca2+ activation in muscle 

fibers in vivo (Fig.1a) 40 min after the stimulation 

imaging life 

is applied. The acupuncture induced β-endorphin 

secretion is blocked by the intraperitoneal injection 

of Gd3+, thestretch-activated Ca2+ channels blocker. 

Conclusions: These findings suggest that acupuncture 

analgesia depends on the physiological afferent signal 

elicited in the mechanosensation pathway. The acupuncture 

needle manipulation induces a Ca2+-dependent 

secretion of β-endorphin via interactions with 

stretch-activated Ca2+ channels. The depolarizationevoked 

opioid peptide β-endorphin then penetrates 

the fenestrated capil ary vessels in the pituitary by diffusion, 

and so enters the general circulation. Our results 

are in agreement with the studies on endogenous 

neuropeptide release during electroacupuncture [2] 

and may lead to new directions for acupuncture analgesia 

research by unveiling its cellular signal transduction 

pathways. 

References: 

Fig1: Acupuncture stimulates 

ß -endorphin release via 

activating stretch-activated 

cation channels. 

1. Liang J.M., Li G., Yang E.S., Cheung P.Y.S. In Vivo Monitoring 

of Ca2+ Excitation in Mouse Skeletal Muscle during 

Acupuncture. WMIC 2009, p.527. [2] Han J.S. Acupuncture: 

neuropeptide release produced by electrical stimulation 

of different frequencies. Trends Neurosci. 26, 17-22 (2003).


Labeling protocols for MRI and optical imaging of human muscle cells precursors 

Libani I.V. (1) , Lui R. (1) , Martelli C. (1) , Clerici M. (1) , Fiorini C. (2) , Lucignani G. (1) , Ottobrini L. (1) . 

(1) University of Milan, Rescaldina, Italy 

(2) Politecnico of Milan, Italy 

ilaria.libani@unimi.it 

Introduction: The interest about cell-mediated 

therapy finalized to tissues regeneration research 

is increased in the last few years. Although numerous 

protocols which include extraction of stem cells 

from healthy animals and implantation in diseased 

models were set up, important parameters such as 

the distribution and localization of the injected cells, 

cell survival, proliferation and differentiation cannot 

be evaluated in vivo. Here we refined and tested specific 

labelling protocols for in vivo visualization by 

MRI, SPECT and Optical Imaging of a human muscle 

cells precursor cell line as a proof of principle for 

the application of these procedure to stem cells in the 

evauation of muscle stem cell mediated treatments. 

Methods: Human Skeletal Muscle Cells (HSkMC) 

were labelled for 24 or 48h with different amounts 

of Endorem® (0-100–200 μg Fe/mL) in presence or 

not of Poly-L-Lysine (PLL), Protamine Sulfate (PrS), 

Polybrene (PB) or infected, with a lentiviral vector 

[1] carrying Luciferase gene under control of the 

muscle specific Myogenin promoter (pGZ.Myo.L 

vector). Labelled HSkMC were analized for viability, 

iron content (Perl’s Staining and spectrophotometer 

analysis [2]), morphology, differentiation capability 

or intrarterially (i.a.) injected into NUDE mice for 

in-vivo imaging by means of MRI, FLI or BLI. Initial 

cell distribution was also followed with scintigraphy 

after cell labelling with 111Indium-oxime (60 

μCi/106 cells) and gamma counting of explanted organ 

was performed to validate imaging data ex-vivo. 

Results: HSkMC incubated for 24 or 48h with 0-100- 

200 μg Fe/mL did not show significant differences, 

in terms of viability, between labelled/non-labelled 

cells in the presence or absence of PLL, PB or PrS 

(n=3) remaining higher than 84+8% after 24h and 

higher than 80,6+4,6% after 48h. The percentage of 

Iron+ cells increased in proportion to the iron content 

in the medium even if there are not significat 

differences between CTRL and PLL. In particular we 

obtained more than 90% Iron+ cells in the samples 

incubated with 200 μg Fe/mL for 24 or 48h. Intracellular 

iron content reached the higest levels in the 

samples loaded with 200ug/ml+PLL: 61,5+2,8 pg/ 

cells after 24h and 114,9+9,6 pg/cells after 48h. For 

this reason 200 ug/mL endorem+PLL was deemed as 

the ideal condition for cell labeling for in vivo visualisation 

by MRI. Loaded HSkMC were injected i.a. into 

NUDE murine model of muscle inflammation. MRI 

permitted to follow over time HSkMC distribution 

into the injured muscle. SPECT and BLI of HSkMC 

not only confirmed celldistribution to muscle but also 

revealed an early localisation into the lung. HSkMC infected 

with pGZ.Myo.L, i.a. injected in the same mouse 

model, were detectable in muscle up to 5 weeks after 

injection. Interestingly, at this timepoint, the signal 

is still present only near the lesion area while disappeared 

in the rest of the body suggesting that this construct 

could be used to evaluate not only localisation 

but also the differentiation in vivo by means of BLI. 

Conclusions: We set up protocols to efficiently 

visualize human muscle cells precursors localization 

and differentiation by MRI, SPECT or in vivo 

optical imaging. These protocols will be useful to 

study the fate of cells once injected into recipient 

NUDE mice with different techniques and will 

make it possible to study their behaviour in vivo 

over time. Furthermore, it will be possible to use 

these instruments for the in vivo study of muscular 

stem cells in restoring skeletal muscles after 

damage. 

Acknowledgement: This work was supported by a 

Cariplo Foundation grant, the FP6 Hi-CAM project 

(LSHC-CT-2006-037737) and from the Doctorate 

School of Molecular Medicine, University of Milan. 

References: 

1. Naldini L. et al; Science 272(5259):263-7 (1996) 

2. Boutry N. et al; Contrast Media Mol Imaging 3(6):223- 

32 (2008) 


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P-053 Studying molecular processes in-vivo: A framework for quantifying variability in molecular 

MRI 

166 

Plenge E. (1) , Kotek G. (1) , Guenoun J. (1) , Doeswijk G. (1) , Krestin G. (1) , Niessen W. (2) , Meijering E. (1) , Bernsen M. (1) . 

(1) Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands 

(2) Erasmus University Medical Center, Rotterdam and Faculty of Applied Sciences, Delft University of Technology, The Netherlands 

e.plenge@erasmusmc.nl 

Introduction: Quantitative characterization of cellular 

and molecular processes in-vivo is dependent 

on consistent image data. To justify conclusions regarding 

observed biological changes over time, the 

changes should exceed those attributed to imaging 

artifacts and inconsistencies. The aim of this work 

is to establish a framework for quantifying the variability 

inherent to molecular magnetic resonance 

imaging (mMRI) protocols. 

Methods: Our framework consists of variance maps, 

variance histograms, visualization and quantification 

of the precision of expert contouring. To demonstrate 

the efficacy of our framework we have compared the 

variability of two different acquisition protocols in 

the challenging environment of a rodent heart. SPIO 

labeled stem cells were injected into the myocardium 

of a rat and cardiac MRI performed. Without moving 

the anesthetized rat at any point, seven 3D nontriggered 

images and seven 2D cine sequences were 

acquired intermittently to capture inevitable changes 

due to e.g. breathing, coil-heating etc. in both datasets. 

The assumption was made that no biological changes 

occur within the acquisition time. After rigid registration 

of the images in each dataset, the per-voxel variance 

over each dataset was calculated. Variance maps 

were generated for visualization, variance distributions 

for quantification. Three experts manually segmented 

the cell cluster in each slice/frame of each 3D/cine sequence. 

The inter- and intra-observer variability was 

visualized and quantified by a similarity index (SI) [1]. 

imaging life 

Results: The created framework allows objective 

quantification of the variability inherent to mMRI 

protocols. Fig. 1 and table 1 visualize and quantify 

the variance distribution of the datasets due to imaging 

inconsistencies, and the inter- and intra-observer 

variability of the expert segmentation. Through 

such a quantitative assessment a lower limit on the 

longitudinal changes in the biological process to be 

studied can be defined. 

Conclusions: We have proposed a framework for 

evaluating mMRI protocols in terms of variability. 

The proposed framework allows anyone to objectively 

assess how well-suited an mMRI protocol is 

for a longitudinal study of a given biological process. 

Acknowledgement: Supported by ENCITE, funded 

by the European Community under FP7. 

Inter-obs. 

variability, SI 

Intra-obs. 

variability, SI 

3D 2D cine 

0.70 0.79 

0.56 0.64 

Fig1: Top and bottom row shows visualizations related to the 3D 

and 2D cine datasets, respectively. Left: Slice/frame of each dataset. 

Middle: Histograms of per-voxel variance of each dataset. Right: Variability 

among three expert’s segmentations of cell cluster (colors represent 

the number of experts including the voxel in the cell cluster). 

References: 

1. Pohl KM et al; Med.Img.Analysis, 11:5:465-477 (2007)


Clinically applicable cell tracking by MRI in cartilage repair using Superparamagnetic Iron 

Oxide (SPIO) 

Van Buul G. , Kotek G. , Wielopolski P. , Farrell E. , Uijtterdijk A. , Bos P. , Weinans H. , Verhaar J. , Krestin G. , Van Osch G. , 

Bernsen M. . 

Erasmus MC, Rotterdam, The Netherlands 

g.vanbuul@erasmusmc.nl 

Introduction: Cell tracking is a useful tool for 

optimizing cell-based cartilage repair. Cell labeling 

using superparamagnetic iron oxides (SPIOs) 

enables non-invasive in vivo cell tracking by MRI, 

and has already been used experimentally in a 

clinical setting[1]. We investigated the clinical, 

intra-articular applicability of this cell tracking 

technique regarding safety, MRI traceability and 

label re-uptake. 

Methods: Part 1: Human bone marrow stromal 

cells (hBMSCs) were labeled with SPIO (ferumoxides-protamine 

sulphate complexes) in a range 

of 0 - 250 μg/ml. Cell viability was asses-sed and 

metabolic cell activity was quantified up to seven 

days. Part 2: SPIO-labeled hBMSCs (100,000 to 

5,000,000 cells) were injected ex vivo in pig knees, 

to mimic a clinically relevant sized model. Furthermore, 

SPIO-labeled cells (10,000 - 1,000,000 

per 75 μl) were seeded in cartilage defects in vitro. 

Scanning was performed on a clinical 3.0 T MRI 

scanner. Part 3: To show possible SPIO re-uptake 

by synovial cells, viable and dead GFP-SPIO double-labeled 

chondrocytes were seeded on human 

synovium explants for five days. Samples were 

analyzed using fluorescence- and light microscopy. 

Results: Part 1 Cell labeling 

and -behaviour: 

SPIO labeling resulted 

in labeling efficiencies 

of approximately 90% 

and did not negatively 

affect cell viability or 

cell proliferation for 

dosages up to 250 μg/ml. 

Part 3 SPIO re-uptake: GFP+-SPIO+ cells, indicating 

originally seeded cells, were seen in synovium 

samples containing living cells. GFP – -SPIO+ cells, 

indicating SPIO re-uptake by synovial cells, were 

found in samples containing dead cells. 

Conclusions: Although possible SPIO re-uptake 

by host cells might limit duration of accurate cell 

tracking, we showed promising results for the use 

of SPIO labeling for cell tracking in clinical cartilage 

repair procedures. This approach provides the 

extra advantage to simultaneously track cells and 

evaluate cartilage repair in one MRI session. 

Acknowledgement: This work is supp. by the Smart- 

Mix Prog. of the Neth. Min. of Econ. Aff. & the 

Neth. Min. of Educ., Cult. & Science; and the EC- 

FP7 project ENCITE (HEALTH-F5-2008-201842). 

Part 2 MRI traceability: Fig1: Intra-articular injected SPIO-labeled cells (1A) and cells seeded in circular cartilage defects in a volume of 75 

All SPIO-labeled cell dosages, 

both intra-articular 

µl (1B) were accurately visualized by MRI. The amount of intensity of signal voids was related to used cell number. 

injected or seeded in cartilage defects, were visualized 

by MRI (Fig. 1). The amount of signal voids 

was related to the used cell number. SPIO-labeled 

cells seeded in cartilage defects in vitro could be visualized 

and quantified using a T2* mapping MRI 

References: 

technique. 

1. Bulte JW; AJR Am J Roentgenol. 193(2):314-25 (2009) 


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168 


P-055 Labeling of HUVEC with different iron oxide particles: An in vitro study about incorporation, 

distribution, retention and toxicity 

Van Tiel S. , Wielopolski P. , Houston G. , Krestin G. , Bernsen M. . 

ErasmusMC, Rotterdam, The Netherlands 

s.vantiel@erasmusmc.nl 

Introduction: For in vivo cell tracking it is essential 

that the cells have incorporated a label so that 

they can be distinguished from their surroundings 

in Magnetic Resonance Imaging (MRI)[1-3]. A vast 

amount of studies have been published dealing with 

labeling of various cell types with iron oxide nanoparticles. 

In these studies a large variety of labeling 

protocols have been described. While for every cell 

type tested efficient labeling and subsequent detection 

by MRI[4] has been reported, it is not clear 

how different labeling protocols may influence labeling 

efficiency. The purpose of this study was to 

systematically investigate the effect of variations in 

dose and duration of labeling on label incorporation, 

distribution, retention and toxicity using two commonly 

used types of iron oxide nanoparticles; the 

so-called SPIO and MPIO particles. 

Methods: Primary culture Human Umbilical Vein 

Endothelial Cells (HUVECs) were grown to 80- 

90% confluence and labeled with SPIO or MPIO 

at concentrations ranging from 0 - 100 µg Fe and 

incubation times of 4-48hrs. Cellular iron load was 

measured by Inductible Coupled Plasma- Optical 

Emission Spectrometry (ICP-OES). Cellular localization 

and retention over time of iron oxide complexes 

was assessed on cytospin slides. Cell functionality of 

labeled cells was tested by a tube forming assay. MRI 

traceability of labelled cells was tested using a 3D- 

SPGR sequence with TR/TE 41.1/10.5 ms, and a flip 

angle (α) of 50º with a resolution of 38 μm x 38 μm x 

100 μm and a FOV of 2 cm x 2 cm. 

Results: Under the conditions tested a maximal 

iron load of 18.2 pg per cell was obtained for SPIO. 

A much higher iron load of 661 pg per cell was 

achieved with MPIO. Inter and intra cellular distribution 

of label are both strongly dependent on 

the labeling protocol used. Labeling with high doses 

and short incubation times may result in large 

intra-cellular vesicles with multiple iron-oxide 

complexes. Labeling with low doses and long incubation 

times may result in small intra-cellular 

vesicles with just one iron-oxide complex. A better 

retention of label was observed after short incubation 

times and high labeling doses. For both MPIO 

imaging life 

and SPIO higher doses were tolerated at shorter incubation 

times. At equal incubation times, MPIO 

was better tolerated than SPIO. In FACS studies, 

clear changes in forward scatter and side scatter 

plots, corresponding to changes in cell size and cell 

granularity respectively were observed following 

labeling. Both effects were more pronounced after 

labeling with MPIO than with SPIO. At the highest 

doses of MPIO tested cell sizes increased 4-7 

times in cell volume compared to unlabeled control 

cells. The effects of SPIO and MPIO labeling 

on tube forming capacity of HUVECS was tested at 

all doses that did not significantly affect cell survival. 

For all these conditions tested HUVECs still 

displayed tube forming capacity. Sensitive imaging 

by MRI at the single cell level in vitro was possible 

for all conditions tested. 

Conclusions: HUVECs can be labeled efficiently with 

SPIO and MPIO, but dose and duration of exposure 

of cells to the particles strongly influence label incorporation, 

distribution, retention and toxicity. 

Acknowledgement: This research has been done in 

part through support from ENCITE - funded by the 

European Community under the 7th Framework 

program. 

References: 

1. Bulte JW. Methods Mol Med.124:419-439 (2006) 

2. Modo M et al. Mol Imaging. 4:143-164 (2005) 

3. Hoehn M et al. J Physiol. 584:25-30. (2007) 

4. van Buul GM et al. Contrast Media Mol Imaging. 4:230- 

236 (2009)


Visualization of aberrant migration in the YAC 128 mouse model for Huntington’s disease by 

in situ labelling of neural progenitor cells with iron oxide particles 

Vreys R. (1) , Blockx I. (1) , Kandasamy M. (2) , Nguyen H.H.P. (3) , Verhoye M. (1) , Aigner L. (2) , Van Der Linden A. (1) . 

(1) University of Antwerp, Belgium 

(2) Paracelsus Medical University Salzburg, Austria 

(3) University of Tübingen, , Germany 

ruth.vreys@ua.ac.be 

Introduction: The outcome of a recent validation 

study of various in situ cell labelling strategies is 

that intraventricular injection of a small number of 

micron-sized iron oxide particles (MPIOs) in combination 

with the transfection agent Poly-L-lysine 

(PLL) is a successful method for the MRI visualization 

of endogenous neural progenitor cell (NPC) 

migration in the adult mouse brain [1] . As it was 

recently reported that NPC migration is impaired 

in a mouse model of Huntington’s disease (HD) [2] , 

we tested if the in situ labelling technique using 

MPIOs-PLL is suitable to visualize aberrant NPC 

migration in YAC128 mice, a mouse model of HD. 

Methods: YAC128 transgenic mice (n=5; 66-69 

weeks old) maintained on a FVB/N background 

and age-matched non-transgenic littermates (n=5; 

wild-types) were used. Mice were injected intraventricular 

with 1.5 µl MPIOs-PLL (9.1 x 10 5 particles, 

0.67 mg Fe/ml). In vivo T 2 *-weighted 3D-GE 

(78 µm isotropic resolution; 9.4T) was performed 

at 1 week up to 13 weeks post injection. Ex vivo 

T 2 *-weighted 3D-GE (66 µm isotropic resolution; 

9.4T) was performed on perfused and fixed brains 

in their skulls. Four days before perfusion, mice 

received 5 times (every 3 h) an injection of 5-bromo-2’-deoxyuridine 

(BrdU; 50 mg/kg i.p). Immunohistochemistry 

was performed for BrdU on 25 

µm sagittal sections. 

Results: MRI revealed hypointense pixels along the 

rostral migratory stream (RMS) and in the olfactory 

bulb (OB) for all wild-types (WT). In contrast, for the 

YAC 128 mice (YAC HD), hypointense areas towards 

the OB were detected in only two mice. As neurogenesis 

is reduced upon ageing, the fraction of MPIOlabelled 

NPCs was very limited. The first column in 

the figure shows for both groups minimum intensity 

projection (mIP) compositions of ex vivo MR images 

comprising the RMS of five animals. The mIP of YAC 

HD shows less hypointense areas in the RMS and OB 

compared to the WT. Histological staining for BrdU 

shows that there are less BrdU + cells present in the 

RMS and OB of the YAC HD compared to the WT 

(figure, second and third column). 

Conclusions: In situ labelling of endogenous NPCs 

by intraventricular injection of MPIOs-PLL revealed 

aberrant NPC migration towards the OB in 

the YAC128 mouse model for HD. 

Acknowledgement: Supported in part by: IWT (Ph.D. 

grant; SBO/030238), EC-FP6-project DiMI (LSHB- 

CT-2005-512146) and IUAP-NIMI-P6/38 

References: 

1. Vreys R et al; NeuroImage 49, 2094-2103 (2010) 

2. Moraes L et al; Neuropathology 29, 140-147 (2009) 


P-056 

poStEr 



P-057 Optimization of in vitro radiolabeling of mesenchymal stem cells with 18 Ffluorodeoxyglucose 

170 

Wolfs E. (1) , Bormans G. (2) , Van Santvoort A. (3) , Vermaelen P. (3) , Mortelmans L. (3) , Verfaillie C. (4) , Van Laere K. (3) , Deroose C. (3) . 

(1) K.U.Leuven, Leuven, Belgium 

(2) Laboratory for Radiopharmacy, K.U.Leuven, Belgium 

(3) Division of Nuclear Medicine, K.U.Leuven, Belgium 

(4) Stem Cell Institute, K.U.Leuven, Belgium 

Esther.Wolfs@med.kuleuven.be 

Introduction: Because of their great differentiation 

potential and immunomodulatory properties, mesenchymal 

stem cells (MSCs) are considered to be a 

potential source for tissue regeneration and immunomodulatory 

therapy [1-4]. For the optimization 

of this type of stem cell therapy, novel methods are 

required to follow the in vivo fate of stem cells after 

injection [5]. Stem cells can be labeled with radioisotopes 

in vitro, such as 18 F-fluorodeoxyglucose 

( 18 F-FDG), a positron emitting glucose-analogue 

which is taken up by cells in a similar manner as 

glucose. After phosphorylation, 18 F-FDG is trapped 

intracellularly, since it misses the 2’-hydroxyl group 

blocking further steps of the glycolysis pathway. The 

molecule stays in the cell until its decay [6]. The aim 

of this study is to optimize the radioactive labeling 

of MSCs in vitro with 18 F-FDG. 

Methods: Mouse MSCs were obtained from the 

Stem Cell Institute Leuven, and were seeded in 24 

well dishes for uptake experiments. 18 F-FDG (5µCi) 

was added to each well, in the presence of different 

insulin concentrations, and for uptake kinetics for 

up to 6 hours. In addition, 18 F-FDG washout after 

labeling was assessed. Additionally, 3 mice were 

injected with 500 000 18 F-FDG-labeled MSCs, and 

stem cell biodistribution was investigated with a 

µPET scanner 10 minutes after injection. Furthermore, 

a µCT image was acquired for coregistration 

with the µPET images. 

Results: 18 F-FDG uptake by MSCs was slightly higher 

with the addition of insulin (0,01 IU) but with only 

limited additional effect of higher concentrations. 

Furthermore, experiments also showed significantly 

higher 18 F-FDG uptake with longer incubation periods, 

slowly reaching a plateau after 6h of incubation. 

However, a significant tracer washout could be 

observed after 1 hour. In addition, in vivo µPET experiments 

with radiolabeled MSCs in mice showed 

almost exclusive pulmonary activity, confirming the 

intracellular location of the tracer. 

Conclusions: This study demonstrates that mouse 

MSCs can be successfully labeled with 18 F-FDG 

in vitro. Labeling efficiency increases even when 

very low insulin concentrations are added. Uptake 

imaging life 

kinetics assessment showed a significantly higher 

uptake with increasing incubation time for up to 6 

hours of incubation. Despite the tracer washout after 

one hour in vitro, in vivo µPET studies confirmed 

the intracellular location of the tracer through an 

almost exclusive pulmonary signal. Furthermore, 

in vitro labeling with the given dose (5µCi) permits 

solid imaging of stem cells injected in mice. 

References: 

1. Caplan A. Adult mesenchymal stem cells for tissue 

engineering versus regenerative medicine. J Cell 

Physiol 2007;213(2):341-7. 

2. Jackson L, Jones D, Scotting P, Scottile V. Adult 

mesenchymal stem cells: differentiation potential 

and therapeutic applications. J Postgrad Med 

2007;54(2):121-8. 

3. Caplan A. Why are MSCs therapeutic? New data: new 

insight. J Pathol 2009;217(2):318-24. 

4. Jiang Y, et al. Pluripotency of mesenchymal 

stem cells derived from adult marrow. Nature 

2002;418(6893):41-9. 

5. Lauwers E, et al. Non-invasive imaging of 

neuropathology in a rat model of a-synuclein 

overexpression. Neurobiol Aging 2007;28(2):248-57. 

6. Caracó C, et al. Cellular release of [18F]2-fluoro- 

2-deoxyglucose as a function of the Glucose-6phosphatase 

Enzyme system. J Biol Chem 2000; 275 

(24): 18489-2000


Responsive MRI contrast agent for specific cell imaging of inhibitory, GABAergic neurons 

Aswendt M. (1) , Gianolio E. (2) , Kruttwig K. (1) , Brüggemann C. (1) , Aime S. (2) , Himmelreich U. (3) , Hoehn M. (1) . 

(1) Max Planck Institute for Neurological Research, Cologne, Germany 

(2) University of Turin, Italy 

(3) Katholieke Universiteit Leuven, Belgium 

Markus.Aswendt@nf.mpg.de 

Introduction: γ-Aminobutyric acid (GABA) is the 

major known inhibitory neurotransmitter. The ratelimiting 

step in the synthesis of GABA is the decarboxylation 

of glutamate by the enzyme glutamic acid 

decarboxylase (GAD), which exists in two isoforms: 

GAD65 and GAD67 [1]. 

We exploited the feasibitity of cell specific contrast 

generation with the new smart contrast agent Gd- 

DOTAgad responsive to GAD activity. It consists of 

the chelate Gd-DOTA coupled to a long hydrocarbon 

backbone with glutamic acid residues. Upon cleavage 

of the glutamate moieties the hydration of the 

paramagnetic metal ion increases, 

leading to a higher relaxivity. 

Methods: Postnatal day 14, 

Wistar rat tissues were mechanically 

homogenized, and 

lysed with cell lysis reagent 

M-PER (Pierce, USA). After 

protein determination, same 

amounts were incubated at 

37°C for 24 hours with 1 mM 

Gd-DOTAgad together with 

the GAD activator pyridoxal- 

5’-phosphate. MR measurements 

were made at 4.7T (Bruker, 

Germany) using a custom 

made coil system. The MR phantoms consist of 

plastic tubes embedded in 1.5% agarose (Fluka, 

Switzerland). RAREVTR scans (TE=12.0ms, 

TR=30–250 ms, matrix=128x128; FOV=30.0 mm, 

resolution=0.234x0.234 mm; slice thickness=2.0 

mm). CGR8 murine embryonic stem cells were differentiated 

in a 3 step protocol using chemically 

defined media. 

Results: Tissue lysates of cerebellum, cortex and 

liver (as a negative control) were incubated with 

Gd-DOTAgad. Corresponding controls were Gd- 

DOTAgad in PBS and tissue lysates without contrast 

agent. T1 maps calculated from RAREVTR with an 

in-house software were used to evaluate the change 

in relaxation rate ΔR1 (Fig. 1A) identifying the highest 

changes with brain lysates. CGR8 cells were successfully 

differentiated in a 10 day-lasting protocol 

resulting in a high proportion of GAD65/67 positive 

cells, proven by immunocytochemistry (Fig. 1B). 

Conclusions: Gd-DOTAgad incubated with tissue 

lysates leads to enhanced ΔR1 rates in highly expressing 

GAD65/67 brain regions. A differentiation 

protocol to obtain GABAergic neurons from ES cells 

was successfully established. Efficient labelling with 

Gd-DOTAgad of murine ES and neuronal precursor 

cells is in progress for evaluation of differentiation 

induced contrast. With this approach it will be possible 

to label cells prior to transplantation and to 

follow their differentiation fate in vivo with MRI. 


by grants from the European Union under FP6 program 

(StemStroke, LSHB-CT-2006-037526) (DiMI, 

LSHB-CT-2005-512146), and under FP7 program 

(ENCITE, HEALTH-F5-2008-201842). 

References: 

1. Erlander, M.G., et al., Two genes encode distinct 

glutamate decarboxylases. Neuron, 1991. 7(1): p. 91-100. 


P-058 

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172 


P-059 Novel ultrasound contrast agents for drug delivery 

Berti R.P. (1) , Freret L. (1) , Haiat G. (2) , Pisani E. (3) , Díaz-Lópeza R. (3) , Tsapis N. (3) , Pucci B. (4) , Fattal E. (4) , Taulier N. (1) , Urbach W. (1) . 

(1) Laboratoire d’Imagerie Paramétrique, PARIS, France 

(2) Laboratoire de Biomécanique et Biomatériaux Ostéo-Articulaires, France 

(3) Laboratoire de Pharmacie Galénique, France 

(4) Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, France 

romain.berti@upmc.fr 

Introduction: We are working on novel Ultrasound 

Contrast Agents (UCA) to improve the signal 

for echographic diagnosis and at the same time 

to extend their applications as possible drug carriers. 

Methods: We have investigated two types of agents:The 

first one is made of polymer PLGA (Poly Lactic Glycolic 

Acid) that encapsulates a liquid PFOB (PerFluoro- 

Octyl Bromide) core. The agent radius can be varied 

from 150 nm to 10 µm and it remains stable for weeks [1] . 

The second type is made of a telomer FTAC (fluorinated 

polymer) encapsulating a fluorinated liquid or gas core. 

It forms agent with a radius of about 100 nm and are 

stable over days. The size of both types of agent is weakly 

polydisperse. In addition, for a better understanding 

of the relationship between mechanical and ultrasound 

properties of the above contrast agents, we performed 

2D and 3D time domain simulations to compute the 

acoustic wave propagation in an our UCA solution. 

Results: We have measured in vitro the signal-to-noise 

ratio, backscattered signal, and destruction by ultrasound 

waves of the two nano-size ultrasound contrast 

agents, where the applied ultrasound signal has a frequency 

of either 5 or 50 MHz. In addition, the comparison 

between 2D numerical and experimental results 

shows a good agreement [3] , the 3D simulations are still 

under ways and should permit an improvement of our 

predictions. Our first tests performed in vivo showed 

that our agents induce a significant enhancement in the 

backscattered signal. For targeting purpose, the surface 

chemistry of the first UCA particles type was modified 

by incorporating phospholipids (fluorescent, pegylated, 

and biotinylated phospholipids) in the organic phase 

before emulsification [2] . Microscopy shows that phospholipids 

are present within the shell and that the core/ 

shell structure is preserved. The functionalization did 

not modify the echographic signal arising from capsules. 

Conclusions: These results demonstrates that these 

nano and polymeric capsules are suitable ultrasound 

contrast agent and can be easily modified 

for targeted therapy or molecular imaging purpose. 

Acknowledgement: This work is supported by a 

grant from ANR (n° NT05-3-42548) and EC- 

FP6-project DiMI (LSHB-CT-2005-512146) 

imaging life 

References: 

1. Pisani et al. Adv. Funct. Mater. 18 (2008) 1-9 

2. Diaz-Lopez et al. Biomaterials 30 (2009) 1462-1472 

3. Haiat & Al, J. Acoust. Soc. Am. 127 1, (2010)


Methods to study the interaction between aptamer probes and cell surface biomarkers 

Cibiel A. , Pestourie C. , Gombert K. , Janssens I. , Thézé B. , Tavitian B. , Ducongé F. . 

CEA,DSV,I²BM,SHFJ,INSERMU1023, Orsay, France 

agnes.cibiel@cea.fr 

Introduction: Aptamers are specific nucleic acidsbased 

ligands selected by an in vitro molecular evolution 

process (named SELEX). Recently, SELEX 

has been performed against living cells and allows 

for successful selection of aptamers against a specific 

transmembrane protein [1] or against cell surface 

biomarkers without prior knowledge of the target [2] . 

Here, we compared different techniques to study 

the interaction between aptamers and their target. 

Methods: For the binding experiments, 10 5 cells were 

first incubated with 5’- 32 P-labeled aptamers or control 

sequences for 15 minutes at 37°C and then washed. 

Bound sequences were recovered in 400µL of lysis 

buffer and the amount of radioactivity was counted. 

To optimise this protocol, we evaluated the number 

of washings and different combinations of unspecific 

competitors (tRNA, ssDNA and Polyinosine) at different 

concentrations. This allowed us to determine the 

protocol that yielded the highest signal/background 

ratio and to measure the affinity and the Cmax of different 

aptamers. In addition, an automation of the 

protocol has been performed. For microscopy analysis, 

aptamers were first labelled using Ulisys Alexa fluor 

546. Then, they were imaged using epifluorescence 

microscopy (Axio Observer Z1 Zeiss) at different time 

points during incubation (up to 40 minutes). 

Results:We observed that 5 washing cycles during the 

binding experiments were enough to decrease the 

background. In addition, we observed that the best 

combination of competitors seemed to be dependent 

on the cell type. In contrast, the optimal concentration 

of competitors was always around 100µg/ml. The use 

of higher concentrations did not have any effect suggesting 

that unspecific targets were saturated at this 

concentration. Finally, the use of competitor during 

pre-incubation and/or incubation of aptamers had the 

same impact on the signal/background ratio. Labelling 

aptamers with different amounts of Ulysis Alexa 

546 showed that when the ratio of Alexa/aptamer was 

increased, fluorescence intensity decreased, due to 

self-quenching phenomenon. This labelling method 

has been optimised to study the cellular localisation 

of aptamers by microscopy. In addition to providing 

information on the membrane or intracellular location 

of aptamers (after internalisation), microscopy 

allowed for evaluation of the kinetics of association 

between the aptamer and the target. 

Conclusions: The use of the above described two 

methods allowed us to obtain complementary results. 

Binding experiments led to the quantification 

of the kD or the number of targets on the cell surface 

whereas microscopy provided the localisation 

of the aptamer on the cell surface and its evolution 

over time. Aptamers selected against cell surface 

biomarkers could have many applications in the biomedical 

field (e.g. cell sorting, biomarker discovery, 

imaging, drugs design, etc.). Our methods can be 

used to rapidly assess their potential. 

Acknowledgements: This work was supported by 

grants from the European Molecular Imaging Laboratory 

(EMIL) network (EU contract LSH-2004- 

503569), the FMT-XCT program (EU contract 

201792) and l’Agence Nationale pour la Recherche 

(project ANR-Emergence ARTIC and ANR-TecSan 

DOT-IMAGER). AC was supported by a fellowship 

(Irtélis) from the CEA. 

References: 

1. Cerchia L et al. (2005) PLoS Biol 3(4) 

2. Pestourie C, Tavitian B, Duconge F (2005) Biochimie 

87(9-10): 921-930 


P-060 

poStEr 

PROBE DESIGN


P-061 MRI intelligent contrast agents as enzyme responsive nanosystems 

174 

Figueiredo S. (1) , Cittadino E. (1) , Terreno E. (1) , Moreira J.N. (2) , Geraldes C. F. (3) , Aime S. (1) . 

(1) Department of Chemistry IFM and Molecular Imaging Center, Torino, Italy 

(2) cLaboratory of Pharmaceutical Technology, Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, , Portugal 

(3) Department of Life Sciences, Faculty of Sciences and Technology, and Center for Neurosciences and Cell Biology, University of Coimbra, Portugal 

Saharic@gmail.com 

Introduction: When Magnetic Resonance is the imaging 

modality of choice it is necessary to design 

highly sensitive systems in order to overcome MRI 

relatively low sensitivity. We have envisaged an approach 

to enzyme-responsive agents based on the 

use of liposomes loaded in the aqueous cavity with a 

high number of paramagnetic complexes. Liposomes 

are self-assembled vesicles formed by saturated and 

unsaturated phospholipids commonly used in drug 

delivery. The overall relaxation enhancement of solvent 

water protons depends upon the permeability/ 

disruption of the liposome membrane to water molecules. 

Our work has addressed the objective of i) 

modifying the permeability of liposome membrane 

thus pursuing an enhancement of the observed proton 

relaxation rate upon the enzymatic cleavage of 

peptides covalently bound to the phospholipid moieties 

or ii)promoting the disruption of low relaxivity 

aggregates formed by the binding capabilities of a 

macromolecular substrate that is selectively cleaved 

by the enzyme. 

Methods: Paramagnetic liposomes were prepared using 

the proper membrane by the thin film hydration 

method followed by extrusion. The lipidic film was 

hydrated with a 200mM solution of GdHPDO3A, 

followed by dialysis to remove non-entrapped material. 

The T1 measurements in vitro were carried 

out at 0.5T on a Stelar Spinmaster, whereas the in 

vivo measurements were acquired at 7T on a Bruker 

Avance 300 spectrometer. The temporal evolution of 

T1 contrast was determined in vivo after intratumor 

injection to mice bearing xenografted B16 melanoma. 

Results: i)The relaxometric properties of the liposomes 

loaded with the lipopeptide was tested in vitro 

measuring the r1 over time of three samples: a) the 

suspension of liposomes incorporating the lipopeptide 

in the presence (I) and absence (II) of MMP1 

and b) liposomes incorporating the stearic acid in 

the presence of MMP1. The results reported indicated 

that sample I showed a slight increase in the relaxivity 

likely due to partial instability of the liposome. 

In the presence of MMP, a more pronounced enhancement 

was observed. Importantly, the liposomal 

sample in which the incorporated lipopeptide was 

replaced by stearic acid did not show any significant 

imaging life 

enhancement, confirming that the enhancement observed 

for sample a-I) is related to the MMP1 activity. 

This different behaviour can be well appreciated 

by looking at the different contrast exhibited in the 

corresponding T1weighted MR image. In vivo kinetic 

experiment following the intratumor injection of 

the lipopeptide-based paramagnetic liposome indicated 

a rapid washout of the imaging probe, consistent 

with a relevant release of the Gd(III) complex in 

the extracellular fluid, where MMPs accumulates. ii) 

The interaction of anionic liposomes and protamine 

yields supramolecular adduct with low relaxivity. The 

action of trypsin causes the digestion of protamine 

and the consequent de-assembly of the adduct. The 

process is accompanied by an overall relaxation enhancement 

as consequence of the recovering of the 

original permeability of the liposome membrane to 

water molecules. An illustrative example of the utilization 

of this responsive probe consists in the entrapment 

of the supramolecular assembly in alginate 

vesicles. The detected change in r1 due to trypsin is 

correlated to the one in the absence of alginate. Thus, 

an in vivo exploitation relies on the entrapment of 

micron-sized particles into an alginate matrix that 

has often been considered as a bio-compatible device 

in cell-based therapies. 

Conclusions: We have now envisaged a molecular 

probe that work as an enzymatic substrate in a particular 

microenvironment, hence enhancing the MRI signal 

as a function of enzymatic activity. As further goal, 

an anti-tumoral drug will be co-encapsulated with the 

MRI probe, allowing supervise the tumoral therapy.


Standardization of molecular PBCA-microbubbles for routine use 

Fokong S. , Liu Z. , Gätjens J. , Kiessling F. . 

Helmholtz Institute, Aachen, Germany 

sfokong@ukaachen.de 

Introduction: To develop standardized highly monodisperse 

targeted polybutylcyanoacrylate (PBCA)microbubbles 

for contrast enhanced molecular 

ultrasound imaging. 

Methods: PBCA-microbubbles were produced by 

mechanical agitation and size isolated by centrifugation. 

Physical parameters of the size optimized 

microbubbles and regular (Sonovist) microbubbles 

were compared. Targeting of the microbubbles for 

molecular imaging was achieved by covalently binding 

streptavidin molecules onto the shell of surface 

activated PBCA-microbubbles and the subsequent 

attachment of biotinylated markers. The amount of 

targeting ligands on the surface of the microbubbles 

was quantified using a fluorescence activated cell 

sorter (FACS). The suitability of these microbubbles 

for destructive imaging methods like power Doppler 

ultrasound, used for the evaluation of the degree of 

angiogenesis and the effect of antiangiogenic therapy, 

was investigated in gelatin phantoms. 

Results: Following a new protocol for the synthesis 

of PBCA microbubbles, highly monodisperse populations 

could be isolated. Curves obtained from 

particle sizer measurements showed size isolated 

microbubbles which are normally distributed with 

a narrower standard deviation (> 97% ± 1.0-3.0 µm) 

in comparison to regular Sonovist microbubbles 

(> 97% ± 0.2–10 µm). The high monodispersity was 

also confirmed by SEM (scanning electron microscopy) 

pictures. The population distribution remained 

constant even with prolonged storage in solution 

(over 4 months), indicating high stability. Comparison 

of the resonance frequencies and the persistence 

in an ultrasound field showed a significantly lower 

resonance frequency and a lower persistence in the 

ultrasound field for the monodispersed microbubbles 

compared to the polydispersed microbubbles. 

The high monodispersity was maintained even with 

covalent coupling of streptavidin molecules onto the 

shell of the microbubbles. A quantification of the 

number of streptavidin molecules on the surface by 

the use of a FACS gave approximately 2 x 10 4 streptavidin 

molecules per microbubble. This number can 

be tuned according to the degree of surface activation 

of the microbubbles. Destructive imaging using 

SPAQ (Sensitive Particle Acoustic Quantification) 

[1] was also possible with these monodispersed microbubbles 

showing a highly reproducible destruction 

with high mechanical index ultrasound waves. 

Conclusions: Size optimized non modified PBCAmicrobubbles 

are more suitable for use as ultrasound 

contrast agents in comparison to regular Sonovist 

microbubbles. The simplified synthetic protocol allows 

for a speed up in the synthesis and purification 

process (6 days vs. 3 h). The low resonance frequency 

and persistence lead to more efficient detection using 

power Doppler techniques. Also, given the relative 

high number of targeting ligands which can be 

easily attached to their surface, and the possibility to 

efficiently quantify their amounts in a region of interest, 

they are very suitable for molecular imaging of 

intravascular targets. In summary, the tuned properties 

of the microbubbles presented in this study, will 

make them very interesting as building blocks for 

tailoring of specific contrast agents for ultrasound. 


German Federal Ministry of Education and Research 

(BMBF-0315017). 

References: 

1. M. Reinhardt, P. Hauff, A. Briel, V. Uhlendorf, M. Schirner, 

Sensitive particle acoustic quantification (SPAQ), 

Investigative Radiology 40 (2005) 2-7. 


P-062 

poStEr 

PROBE DESIGN


P-063 Novel Gd(III)-based probes for MR molecular imaging of matrix metalloproteinases 

176 

Gringeri C. (1) , Catanzaro V. (2) , Menchise V. (3) , Digilio G. (1) , Aime S. (2) . 

(1) Department of Environmental and Life Sciences, University of Eastern Piedmont “A. Avogadro”, Alessandria, Italy 

(2) Department of Chemisty IFM & Center for Molecular Imaging, University of Turin, Italy 

(3) Institute for Biostructures and Bioimages (CNR) c/o Molecular Biotechnology Center (University of Turin), Italy 

cgringeri@gmail.com 

Introduction: The assessment of the activity of a 

selected panel of MMPs in a given tissue or anatomical 

district is very important for the typization 

and staging of cancer and autoimmune diseases, 

and for the evaluation of the efficacy of therapies. 

[1] “Molecular Imaging” is a relatively new and rapidly 

growing branch of diagnostic imaging devoted 

to visualize tissue specific patho-physiological processes 

on the basis of their cellular/biochemical molecular 

signatures. MRI is one of the most clinically 

relevant imaging techniques, because of its great 

spatial resolution (


Functionalization of nanoparticles for molecular imaging; a covalent approach 

Herranz F. (1) , Salinas B. (1) , Rosell Y. (1) , Desco M. (2) , Ruiz-Cabello J. (1) . 

(1) Universidad Complutense de Madrid- CIBERES, Madrid, Spain 

(2) Hospital Gregorio Marañón, Spain 

fherranz@pdi.ucm.es 

Introduction: The key point in the development 

of new nanoparticles (NPs) for imaging is the 

surface functionalization. By the attachment of 

new molecules one should, ideally, provide stability 

in physiological conditions, with a narrow 

size distribution and a functional group for the 

binding of biomolecules. These changes on the 

NPs surface can be made by weak non-specific 

interactions or by strong specific covalent bonds. 

Methods: Iron oxide nanoparticles were synthesized 

by the decomposition of organic precursors redendering 

hydrophobic Fe 3 O 4 NPs. These nanoparticles 

have oleic acid as surfactant, providing excellent size 

distribution, crystallinity and stability. To transfer 

the NPs to water the chemical structure of the oleic 

acid was modified by chemical methods, obtaining 

water stable NPs ready for further modification. 

Results: Fe 3 O 4 NPs were synthesized (7 ± 1 nm) by 

the decomposition of organic precursors. The surfactant 

of the NPs (oleic acid) was oxidized by means 

of potassium permanganate in a two phase system. 

This way we obtained water stable, carboxylic acid 

functionalized NPs, that were fully characterized 

(47 ± 4 nm, Zeta potential -46 mV, r 1 =4 s -1 mM -1 

and r 2 = 115 s -1 mM -1 , FTIR and 1 H-NMR). 1 These 

NPs are stable in buffer conditions and are being 

used for in vivo MRI. Besides, we took advantage of 

the carboxylic acid on the surface to covalently funcionalize 

the NPs with different molecules and dyes, 

like glucose, EDANS, 2 and streptavidin-alexa647. 

Conclusions: Here we report a new approach for the 

covalent functionalization of superparamagnetic 

nanoparticles. We demonstrate the usefulness of 

the method with several examples of covalent functionalization 

with different molecules and in vivo 

MRI use. 


by MAT2008-01489 and SAF2008-0512-C02-01. 

References: 

1. Herranz, F.; Morales, M. P.; Roca, A. G.; Desco, M.; Ruiz- 

Cabello, J., Chemistry A European Journal 2008, 14, 

(30), 9126-30. 

2. Herranz, F.; Morales, M. P.; Roca, A. G.; Vilar, R.; Ruiz- 

Cabello, J., Contrast Media Mol. Imaging 2008, 3, (6), 

215-22. 


P-064 

poStEr 

PROBE DESIGN

178 


P-065 Comparison of different chelating systems for the synthesis of Ga-68 labelled peptides for 

molecular imaging using RGD-peptides as model compound 

Knetsch P. (1) , Petrik M. (1) , Rangger C. (1) , Griessinger C. (2) , Fani M. (3) , Wester H.J. (4) , Pichler B. (2) , Pietzsch H.J. (5) , 

Decristoforo C. (1) , Haubner R. (1) . 

(1) Medical University Innsbruck, Innsbruck, Austria 

(2) University of Tuebingen, Germany 

(3) University of Freiburg, Germany 

(4) Technische Universitaet Muenchen, Germany 

(5) Research Center Dresden-Rossendorf, Germany 

peter.knetsch@i-med.ac.at 

Introduction: Due to its increasing availability 

Ga-68 attracts increasing interest in molecular imaging 

with PET. Moreover, due to the straightforward 

labelling protocols especially for labelling of 

peptides this is an interesting alternative to F-18 

labelling strategies. Here the imaging properties 

of c(RGDfK) conjugated to different chelating systems 

are compared. 

Methods: Peptides were synthesised using standard 

SPPS protocols. After cyclisation in solution 

and selective deprotection of the amino function 

of the lysine the chelating moieties were conjugated 

via in situ activation. The chelating systems 

include 1,4,7,10-tetraazacyclododecane-1,4,7,10acetic 

acid (DOTA), a 1,4,7–triaaza-10-oxocyclododecane-1,4,7-acetic 

acid derivative (B505), 

1,4,7-triaazacyclononane-4,7-acetic acid-1-2-glutaric 

acid (NODAGA), and a tris(2-mercaptoethyl) 

amine derivative (NS3). Labelling was carried out 

using the fractionated elution method in sodium 

acetate or phosphate buffer, respectively. In vitro 

evaluation included determination of the partition 

coefficient, protein binding properties, metabolic 

stability, binding affinity, and cell uptake characteristics. 

In vivo evaluation was carried out using nude 

mice bearing alpha(v)beta3-positive and alpha(v) 

beta3–negative tumours. For all tracer biodistribution 

data were collected. For the most promising 

also small animal PET imaging was carried out. 

Results: All peptides could be labelled with Ga-68 

in good radiochemical yields. Labelling of NOD- 

AGA-RGD could be achieved even at room temperature. 

Whereas labelling of NS3-RGD has to be 

followed by Seppak separation to obtain the product 

in high radiochemical purity. The compounds 

showed comparable partition coefficients, binding 

affinity for the alpha(v)beta3 integrin as well as receptor 

specific uptake. However, great differences 

were found in the protein binding properties. Out 

of the four peptides tested only NODAGA-RGD 

showed low protein binding. This is also reflected 

in the biodistribution data. Lowest activity concentration 

in blood and best tumour/background ratios 

imaging life 

were found for NODAGA-RGD. Subsequent small 

animal imaging showed best imaging properties 

for NODAGA-RGD, which seems to be comparable 

with F-18-Galacto-RGD. 

Conclusions: In this series NODAGA-RGD revealed 

most promising properties for molecular 

imaging applications. Easy radiolabelling at room 

temperature, low amount of protein bound activity 

and the resulting lower activity concentration 

found in blood compared to the other compounds 

makes it to an interesting alternative to F-18-Galacto-RGD 

for imaging alpha(v)beta3 expression. 

However, the general advantage of NOTA derivatives 

for imaging purposes have to be confirmed by 

additional studies using other peptide structures.


GdDOTA-PIB: a potential MRI marker for Alzheimer’s disease 

Martins A. (1) , Morfin J.F. (1) , Hamplova A. (1) , Kubicek V. (1) , Suzenet F. (2) , Salerno M. (3) , Lazar A. (4) , Duyckaerts C. (4) , 

Geraldes C. (5) , Toth E. (1) . 

(1) Centre de Biophysique Moléculaire,CNRS, Orléans, France 

(2) Université d’Orléans, France 

(3) Université Paris 13, France 

(4) Hôpital de la Pitié-Salpêtrière, France 

(5) University of Coimbra, Portugal 

andre.martins@cnrs-orleans.fr 

Introduction: Alzheimer’s disease (AD) is the most 

frequent form of intellectual deterioration in elderly 

individuals, characterized by the brain deposition of 

amyloid plaques and neurofibrillary tangles. Early detection 

of the β-amyloid (Aβ) deposits in vivo is very 

difficult. Recently 11 C-radiolabeled small-molecules 

have been developed, capable of entering the brain 

and specifically targeting amyloid plaques for imaging 

with PET, such as several Thioflavin T derivatives 

[1-2]. In particular, the uncharged analogue 6-OH- 

BTA-1 (Pittsburgh compound B - PIB) is highly efficient 

both in crossing the BBB and in selective binding 

to AD amyloid aggregates. A major limitation of PET 

is the requirement for the markers to be labelled with 

short-lived isotopes. The use of Aβ marker linked to 

a MRI CA would constitute an attractive noninvasive 

in vivo imaging approach. Recently, Poduslo used CA 

aided MRI to image AD plaques with Gd(III)DTPA 

conjugated to a putrescine-modified human Aβ peptide 

able to cross the BBB and selectively target individual 

amyloid plaques in the brain of Alzheimer’s 

disease transgenic mice. Nevertheless, due to its large 

size, several days (weeks) of incubation with the CA 

are necessary to obtain the labeling of amyloid plaques 

in transgenic mouse brain in vivo. In an attempt to label 

Aβ plaques using small metal complexes for the 

diagnostics of Alzheimer disease, we synthesized a 

PIB-derivative of DOTA. 

Methods: DOTA-PIB was synthesized using a new, 

versatile strategy. The Gd 3+ complex has been characterized 

by relaxometric methods. The compound 

forms micelles in aqueous solution; the critical micellar 

concentration has been measured. Experiments 

on human brain slices have been carried out to assess 

binding of the compound to the β-amyloid deposits. 

HOOC 

N 

N 

O 

N 

H 

N N 

HOOC COOH 

DOTA-PIB 

S 

N 

O 

Results: The 1 H NMRD profiles evidence aggregation 

of the GdDOTA-PIB complex in aqueous solution, 

with a cmc of ~1.5 mM. The preliminary experiments 

on human brain slices show good binding affinity of 

the LnDOTA-PIB compound towards the amyloid 

plaques. 

Conclusions: We synthesized and investigated a 

novel Gd 3+ complex with good binding properties to 

β-amyloid deposits. 

Acknowledgement: We thank the support from the 

F.C.T. Portugal (project SFRH / BD / 46370 / 2008 and 

COST D38. 

References: 

1. Mathis CA, Bacskai BJ, Kajdasz ST, McLellan ME, Frosch 

MP, Hyman BT, Holt DP, Wang Y, Huang GF, Debnath ML, 

Klunk WE. (2002) Bioorg Med Chem Lett.12(3):295-8. 

2. Nesterov EE, Skoch J, Hyman BT, Klunk WE, Bacskai 

BJ, Swager TM. (2005) Angew Chem Int Ed Engl. 

26(34):5452-6. 


P-066 

poStEr 

PROBE DESIGN


P-067 A comparative study of the self-elimination of para-aminobenzyl alcohol and hemithioaminalbased 

linkers. Application to the design of Caspase-3 sensitive pro-fluorescent probes 

180 

Meyer Y. (1) , Richard J. A. (1) , Delest B. (2) , Noack P. (2) , Renard P. Y. (1) , Romieu A. (1) . 

(1) University of Rouen, Mont Saint Aignan, France 

(2) Quidd: smart molecular imaging, Mont Saint Aignan, France 

yvesmeyer@estvideo.fr 

Introduction: This study focuses on the disassembly-behavior 

of self-immolative pro-fluorescent 

linkers under physiological conditions occurring 

via an enzyme-initiated domino reaction. The targeted 

linkers are based on para-aminobenzylalcohol 

(PABA) or hemithioaminal derivatives of para-carboxybenzaldehyde 

or glyoxilic acid. We found that a 

fine tuning of the kinetic properties can be obtained 

through the modulation of the linker structure, giving 

either a fast signal response or customizable 

systems suitable for the design of protease-sensitive 

fluorogenic probes or prodrug systems. 

Methods: Five model compounds bearing specific 

self-immolative linkers were synthesised and all 

integrate both: (1) a phenylacetamide moiety to be 

cleaved by Penicillin G Acylase (PGA) functionning 

as the triggering agent, and (2) a masked umbelliferone 

(i.e., 7-hydroxycoumarin) unit to be released as 

the final product and to elicit turn-on fluorescence. 

All these fluorogenic probes were incubated at the 

same concentration (3 µM) at 37°C with PGA (0.12 

U) in phosphate buffered saline (PBS, pH 7.5) and 

the corresponding fluorescence time-courses were 

recorded at λ = 460 nm. 

Results: Comparison of the resulting fluorescence 

recovery curves clearly indicates that the decomposition 

of PABA-based self-immolative linkers was 

faster than with the hemithioaminal counterparts. 

No nonspecific cleavage of these pro-fluorescent 

probes was detected in control reactions when incubated 

only in PBS. These results demonstrate that 

the use of PABA or hemithioaminal based linkers 

affords enzyme-reactive pro-fluorophores with 

high chemical stability. Despite the slower release 

of umbelliferone from the hemithioaminal probe 

derived from glyoxylic acid, this linker presents a 

peptide-like structure able to be readily and widely 

functionalised. 

Conclusions: PABA derivatives will be used for getting 

fast-response probes whereas the hemithioaminal 

spacers will be preferred for the construction of 

finely tunable (bio)functionalised probes. A first 

generation of Caspase-3 (apoptosis process) sensitive 

pro-fluorescent probes using PABA linkers has 

imaging life 

been obtained (AcDEVD-PABA-Umbelliferone) and 

in vitro fluorescence assay was performed with the 

corresponding recombinant human protease: 10fold 

increase of the enzyme velocity was obtained as 

compared to the commercially available profluorophore 

AcDEVD-AMC. 

Acknowledgement: This work is supported by CNRS, 

Quidd: Smart Molecular Imaging and La Région 

Haute-Normandie. 

References: 

1. R. Erez and D. Shabat, Org. & Biomol. Chem., 2008, 15, 

2669-2672. H. Y. Lee, X. Jiang and D. Lee, Org. Lett., 

2009, 11, 2065-2068. 

2. C. Fossey, A.-H. Vu, A. Vidu, I. Zarafu, D. Laduree, S. 

Schmidt, G. Laumond and A.-M. Aubertin, J. Enzyme 

Inhib. Med. Chem., 2007, 22, 591. C. Fossey, N.-T. Huynh, 

A.-H. Vu, A. Vidu, I. Zarafu, D. Laduree, S. Schmidt, G. 

Laumond and A.-M. Aubertin, J. Enzyme Inhib. Med. 

Chem., 2007, 22, 608. 

3. Y. Meyer, J. A. Richard, M. Massonneau, P. Y. Renard and 

A. Romieu, Org. Lett., 2008, 10, 1517-1520. 

4. Y. Meyer, J. A. Richard, B. Delest, P. Noack, P. Y. Renard 

and A. Romieu, Org. & Biomol. Chem., 2010, In Press 

DOI: 10.1039/b926316k.


Olefin Metathesis for the functionalization of superparamagnetic nanoparticles 

Salinas B. (1) , Ruiz-Cabello J. (1) , Desco M. (2) , Herranz F. (1) . 

(1) Universidad Complutense Madrid- CIBERES, Madrid, Spain 

(2) Hospital Gregorio Marañón, Spain 

besalina@pdi.ucm.es 

Introduction: One of the most important points in 

the synthesis of new nanoparticles (NPs) for molecular 

imaging is the functionalization of the surface 

with new molecules. There is a necessity for new approaches, 

allowing more versatile synthesis and the 

introduction of biomolecules for specific interactions. 

This modification of the surface should be done covalently 

in mild conditions. Olefin metathesis offers 

many of those features thanks to the new family of 

catalysts, especially Hoveyda-Grubbs 2 nd generation. 1 

Methods: Iron oxide nanoparticles were synthesized 

by the decomposition of organic precursors redendering 

hydrophobic Fe 3 O 4 NPs, with oleic acid as 

surfactant. We carried out the olefin metathesis between 

the double bond in oleic acid structure and 

methyl acrylate. After the hydrolysis of the ester, 

water stable dispersion of superparamagnetic nanoparticles 

were obtained. 

Results: Fe 3 O 4 NPs were synthesized (10 ± 2 nm) by 

the decomposition of organic precursors. 2 The NPs 

were reacted in dry conditions with methyl acrylate 

in the presence of catalytic amounts (4%mol) of 

Hoveyda-Grubbs 2 nd generation catalyst. After hydrolysis 

of the ester water stable NPs, with narrow 

size distribution (30 ± 5 nm) and a Zeta potential 

corresponding to the introduced carboxylic acid 

(-46 mV) were obtained. The presence of the new 

diacid on the surface was confirmed by 1 H-NMR 

and FTIR. 

Conclusions: Here we report, for the first time, 

the use of olefin metathesis for the synthesis of 

water stable Fe 3 O 4 NPs with excellent yield and 

selectivity. This synthesis opens up the possibility 

for the attachment of new molecules in one step 

from the hydrophobic nanoparticles in a mild 

and selective way. 


by MAT2008-01489 and SAF2008-0512-C02-01. 

References: 

1. Rybak, A.; Meier, M. A. Green Chem. 2008, 1099-1104. 

2. Herranz, F.; Morales, M. P.; Roca, A. G.; Desco, M.; Ruiz- 

Cabello, J., Chemistry A European Journal 2008, 14, 

(30), 9126-30. 


P-068 

poStEr 

PROBE DESIGN

182 


P-069 Pyridine-based lanthanide complexes : towards bimodal agents operating as near infrared 

luminescent and MRI reporters 

Toth E. (1) , Bonnet C. (1) , Villette S. (1) , Suzenet F. (2) , Buron F. (2) , Shade C. (3) , Petoud S. (1) . 

(1) Centre de Biophysique Moléculaire, CNRS, Orleans, France 

(2) Université d’Orléans, France 

(3) University of Pittsburgh, USA 

eva.jakabtoth@cnrs-orleans.fr 

Introduction: Among the state of the art bioimaging 

modalities, some are characterized by high 

resolution but low sensitivity (magnetic resonance 

imaging, MRI) and others by high sensitivity but 

low macroscopic resolution (optical imaging). Luminescent/MRI 

bimodal imaging offers the advantage 

of combining the high resolution of MRI 

with the high sensitivity of luminescence and the 

development of contrast agents active for both 

techniques is of prime importance. Lanthanides 

offer unique opportunity to develop such bimodal 

contrast agents given their magnetic and optical 

properties. Nevertheless, it was long thought that 

the conditions required for both applications were 

non-compatible. Here, we report on a versatile pyridine-based 

scaffold for Ln 3+ complexation where 

MRI and near infrared (NIR) luminescence requirements 

are both satisfied using the same ligand. 

Methods: The synthesis of the ligands and the complexes 

will be briefly described. Potentiometric 

titrations have been performed to assess to thermodynamic 

stability of the complexes, together 

with kinetic measurements to quantify their inertness. 

To characterize the MRI properties, the exchange 

rate of the water molecules directly bound 

the Gd 3+ was determined by measuring 17 O longitudinal 

relaxation times. Finally the NIR spectra 

of the corresponding Nd 3+ and Yb 3+ were recorded, 

as well as the lifetimes of the excited states and the 

quantum yield of the complexes to quantify their 

luminescent properties. 

Results: The bishydrated complexes are found to be 

thermodynamically stable and the ligands show a significant 

selectivity for Ln 3+ over endogenous cations 

such as Zn 2+ , Ca 2+ and Cu 2+ . The kinetic inertness is 

also remarkable for such bishydrated complexes. The 

chelates do not form ternary complexes with endogenous 

donors which does not limit relaxivity in biological 

media. All the complexes give rise to NIR emission 

and the quantum yields are remarkable. 1 They are in 

the same range as those of non-hydrated complexes optimized 

for fully protecting the NIR emitting Ln 3+ for 

aqueous applications. The modification of the pyridine 

into quinoline was successful in shifting the excitation 

wavelength of the system towards higher values. 

imaging life 

Conclusions: The pyridine synthon is a prime candidate 

for the development of bimodal NIR/MRI imaging 

probes, as the bishydrated Ln 3+ complexes are thermodynamically 

and kinetically stable and display a high 

NIR quantum yield. The successful modification of the 

pyridine into a quinoline did not modify the thermodynamic 

properties of the complexes, but it resulted 

in a shift of the excitation energy towards lower values 

preventing damages to biological samples and allowing 

deeper tissue penetration of the excitation photons. The 

pyridine platform offers also easy routes for coupling 

the probes to biological vectors and optimizing the 

MRI properties. 

Acknowledgement: This work was financially supported 

by the Institut National du Cancer, La Ligue contre 

le Cancer, France, and was carried out in the frame of 

the COST action D38. 

References: 

1. L. Pellegatti, J. Zhang, B. Drahos, S. Villette, F. Suzenet, 

G. Guillaumet, S. Petoud, E. Toth, Chem. Commun., 

2008, 6591-6593. 

2. S. Comby, D. Imbert, C. Vandevyver, J.-C. G. Bünzli, 

Chem. Eur. J., 2007, 13, 936.


Delivery of multiple F-18 tracers from a single automated platform (FASTlab TM synthesizer) 

Bhalla R. . 

GE Healthcare, Amersham, United Kingdom 

rajiv.bhalla@ge.com 

Introduction: The majority of F-18 radiotracers 

prepared in the clinic are synthesized on be-spoke 

automated rigs, which require modifications (and 

time) in order to switch to the production of different 

tracers. The increasing demand to facilitate 

the production of multiple F-18 tracers from a single 

clinical site (and preferably from a single Hot Cell) 

has necessitated the need to develop an easy to use 

automated platform which can readily accommodate 

the production of multiple F-18 tracers. 

O 

18 

TsO n F 

18 

F 

O 

R 

Fig1: Example of radiochemistry on FASTlabTM 

18 

R n F 

Methods: The FASTlab TM is an automated PET radiochemistry 

synthesis platform incorporating a 

disposable cassette. The disposable cassette contains 

a reaction vessel, pre-filled reagent vials and SPE 

cartridge(s). The FASTlab TM performs transfers (of 

F-18, reagents, solvents etc) in and out of the reaction 

vessel using a combinations of syringe drivers, pressure 

and vacuum – this combination of processes provides 

a high degree of control, allowing manipulations to be 

carried out with a high degree of precision and accuracy. 

The F-18 labelling is preformed in the reaction vessel 

and subsequent purification is performed either on the 

cassette using SPE cartridges or externally via a HPLC. 

Results: Figure 1 presents a snapshot of some the 

chemistry which has been successfully transitioned 

onto the FASTlab TM , demonstrating that this platform 

is capable of accommodating a diverse range 

of chemistry. Examples of F-18 tracers transferred 

to the FASTlab TM and currently in use in the clinic 

will be presented. 

N 

R 

18 

E n F 

R 

18 

F 

18 

F 

Synthon chemistry 

Fluoroalkylkation 

Direct labelling 

S N 2 

Direct labelling 

S N Ar 

Synthon chemistry 

Oxime synthesis 

Conclusions: FASTlab TM is an automated synthesizer 

which is capable of producing GMP quality F-18 

tracers. We have demonstrated the versatility of 

FASTlab TM by transitioning a wide variety of chemistry 

onto the platform to produce a large number of 

F-18 tracers, many of which are now being assessed 

in the clinic. We are now focussing on expanding 

the range of chemistry and the number of tracers 

available on FASTlab TM (both GE Healthcare proprietary 

and non proprietary). 


P-070 

poStEr 

TECHNOLOGY

184 


P-071 Monte-carlo modelling of a silicon detector insert combined with a PET scanner 

Brzezinski K. , Oliver J.F. , Llosá G. , Solevi P. , Linhart V. , Cabello J. , Lacasta C. , Rafecas M. . 

Instituto de Física corpuscular, CSIC/Universidad de Valencia, Spain 

brzezinski@ific.uv.es 

Introduction: Recent works have explored the capabilities 

of insert devices to improve the performance 

of clinical [1,2] as well as small animal [3,4] PET scanners. 

The development of a high resolution probe to 

be mounted inside a conventional PET scanner to 

increase its spatial resolution and sensitivity is one of 

the goals of the EU project MADEIRA. This paper describes 

the modelling of the MADEIRA set-up using 

Monte-Carlo (MC) techniques. These techniques are 

vital for optimizing the configuration of the proposed 

system by maximizing efficiency and spatial resolution. 

Furthermore, simulations allow characterization 

of the system and are useful for modelling its physical 

response, which can be included in image reconstruction. 

In the probe-and-ring system, a pair of annihilation 

photons has several ways of being detected. This 

work focuses on the classification and quantification 

of the basic detection modes. 

Methods: A preliminary study was conducted to validate 

the physics models of GEANT4 for the purpose of 

modelling the silicon (Si) detector probe. One layer of 

a Si detector was irradiated by a Ba-133 source and energy 

spectra measured. The full probe-and-ring system 

was modelled using the MC toolkit GATE (based on 

GEANT4). The model includes a probe consisting of 

ten layers of 26x40 pixel Si detectors, with 1x1x1 mm 3 

pixels, in coincidence with an existing partial-ring PET 

scanner with two opposing groups of 12 block detectors. 

The scanner diameter is 1 m and each partial ring 

spans 67.5 o out of 180 o so that it must be rotated to three 

different positions to cover the full FOV. Each detector 

block consists of an array of 32 BGO crystals of 6x2x30 

mm 3 . Initial simulations were run with points sources 

of 0.1 and 1.5 mCi in the centre of the FOV and the 

probe at a 50 mm. A temporal resolution of 5 ns was 

applied. The coincidences were classified as ring-ring 

and ring-probe; for each type, the contribution of accidental 

coincidences (randoms) was estimated. 

Results: The spectra measured with the Si detector 

were successfully reproduced by the GEANT4 code. 

For the full probe-and-ring system, coincidences 

where constructed using a 10 ns time window. The 

simulations permitted to quantify the kinds of coincidences 

as well as to evaluate the sensitivity and random 

fractions for each type (see table I). 

imaging life 

random fraction (%) 

Sensitivity (%) 

ring-ring ring-probe 

0.1 mCi 0.013 1.3 

1.5 mCi 0.2 15.5 

0.1 mCi 9.7 0.42 

1.5 mCi 9.0 0.39 

Table1: Random Fractions and Sensitivities 

Simulations are now being carried out with more 

complex activity distributions. An iterative reconstruction 

algorithm, based on ML-EM, developed 

for previous two-dimensional simulations of the 

MADEIRA system is now being modified to reconstruct 

the new three-dimensional data. 

Conclusions: The goal of a high resolution Si detector 

probe in coincidence with a conventional PET 

scanner is improving its spatial resolution and sensitivity. 

MC simulations of the proposed system 

have been performed and basic processes quantified. 

Other types of events such as those with various 

detections in the probe are now being studied and 

could potentially be exploited to increase the system 

sensitivity. The GATE toolkit will be shown to be 

useful in simulating such a non conventional scanner 

geometry. The coincidence data is now being applied 

to reconstruction. 

Acknowledgement: Supported by TEC2007-61047 

References: 

1. Janecek M et al; IEEE Trans Nucl Sci. 53 No.3: 1143-1149 

(2006) 

2. Zhou J et al; Phys Med Biol. 54:5193-5208 (2008) 

3. Wu H et al; J Nucl Med. 49:1668-1676 (2008) 

4. Park SJ et al; Phys Med Biol. 52 4653-4677 (2007)


Autofluorescence corrected multispectral red-shifted fluorescent protein tomography 

Deliolanis N. (1) , Wurdinger T. (2) , Tannous B. (2) , Ntziachristos V. (1) . 

(1) Helmholtz Zentrum Muenchen, Neuherberg, Germany 

(2) Massachusetts General Hospital, United States 

ndeliolanis@yahoo.com 

Introduction: The development of fluorescent proteins 

(FPs) that operate in the far-red and nearinfrared 

part of the spectrum, enable the macroscopic 

visualization of FP activity deep in tissues. 

We demonstrate herein a multispectral fluorescence 

tomography method that allowed the visualization 

of labeled glioma tumors in animal brains operating 

with two-orders of magnitude better sensitivity 

compared to imaging GFP. We discuss how the 

detection sensitivity can be improved by at least an 

order of magnitude using further shifted FP’s and 

the potential of the method for accelerating discovery 

associated with functional genomics, stem cell 

research and systems biology. 

Methods: The method makes use of a novel spectral 

inversion scheme that integrates three-dimensional 

image reconstruction and auto-fluorescence 

correction that works seamlessly in the steep absorption 

transition from visible to near-infrared. 

The method is based on non-contact full angular 

projection Fluorescence Molecular Tomography. 

Results: We have successfully imaged mCherry labeled 

glioma tumors located deep in tissue in animal 

brains. The results show almost perfect anatomical 

localization of the tumors that is verified 

by MRI and histology. 

Conclusions: The approach offers therefore the ability 

for tomographically visualizing the emerging 

new class of red-shifted fluorescent proteins though 

entire animals. We discuss how the detection sensitivity 

can be improved by at least an order of magnitude 

using further shifted FP’s and the potential 

of the method for accelerating discovery associated 

with functional genomics, stem cell research and 

systems biology. 

Acknowledgement: This research is supported by a 

Marie Curie Intra-European Fellowship within the 

7th European Community Framework Programme. 

References: 

1. Shaner, N. C. et al. “Improved monomeric red, orange 

and yellow fluorescent proteins derived from 

Discosoma sp red fluorescent protein”. Nat. Biotechnol. 

22, 1567-1572, 2004. 

2. N. Deliolanis et al. “Free-space fluorescence molecular 

tomography utilizing 360° geometry projections”, Opt. 

Lett. 32 382-384 (2007) 


P-072 

poStEr 

TECHNOLOGY

P-073 

186 


Acquiring the sample surface from co-registered FMT/MR measurements of a murine tumor 

model 

Dikaiou K. (1) , Stuker F. (1) , Vats D. (1) , Ratering D. (1) , Keist R. (1) , Klohs J. (1) , Ripoll J. (2) , Rudin M. (1) . 

(1) Institute for Biomedical Engineering, Zurich, Switzerland 

(2) Institute of Electronic Structure and Laser, Heraclion, Greece 

dikaiou@biomed.ee.ethz.ch 

Introduction: The combination of MRI and FMT 

is promising in preclinical imaging, combining the 

high spatial resolution and soft tissue contrast of 

MRI for deriving structural and physiological information 

with high sensitivity of fluorescence imaging 

for studying molecular targets/interactions. Moreover, 

MR information can be used as a prior in the 

FMT reconstruction. The necessary steps to this end 

are the co-registration of the two datasets, the acquisition 

and use of the sample surface for reconstructing 

fluorescence images, and the characterization of 

different tissue types within the sample. The first two 

steps are presented here for an in vivo measurement. 

Methods: We measured nude mice bearing 

subcutaneous tumors on the thigh flank. 106 

colon cancer-derived C51 cells had been injected 

9 days prior to measurement. The protease-activatable 

probe ProSense 680 (VisEn 

Medical, Bedford, USA) was administered 

via the tail vein in two doses of 13nmol each, 

48h and 24h prior to measurement. 

We used a custom-made FMT/MR compatible 

animal support equipped with an 

MR transceiver surface coil. Each animal 

was fixed under anesthesia on the stage and 

measured on FMT. The optical signal from a 

1.5x1.2cm2 ROI around the tumor was collected 

upon excitation with a 671nm cw laser 

at 680nm and 720 nm. Thereafter, the stage 

was inserted into a Biospec 94/30 MR scanner 

(Bruker BioSpin MR, Ettlingen, Germany) operating 

at 9.4 T. 14 transversal slices with a FOV of 3.8x1.5cm2 

and 1.0mm thickness covering the tumor were acquired 

with a FLASH sequence (TE/TR = 5/250ms) 

for high sample/background contrast. The elapsed 

time between FMT/MR measurements was 30min. 

The MR data was automatically segmented according 

to [1]. Iso-surfaces were determined in 

order to compute the top surface height map, 

which was interpolated to match the optical image 

pixel dimensions. As both the height map and 

the optical white light image are oriented along 

yz on the coordinate system, they were used for 

co-registration. Reference points on the tumor 

imaging life 

outline were interactively selected to compute 

an affine transformation between the two images. 

The co-registered surface was subsequently used 

in FMT reconstruction. 

Results: The co-registered FMT/MR dataset for one 

mouse is shown, zoomed on the ROI for clarity. The 

reconstructed fluorescence is plotted on the whitelight 

image. The top surface is shown in green. 

Conclusions: A framework for co-registering FMT/ 

MR data and recording the sample surface was presented. 

It constitutes an essential step towards full 

FMT/MR data integration and future use of MR a 

priori information in the FMT reconstruction. 

Acknowledgement: This work was supported financially 

by the EU FP7 FMT/XCT project. 

References: 

1. N.Otsu, A Threshold Selection Method from Gray-Level 

Histograms, Automatica, 1975


fDOT/ PET/CT imaging of biological processes in tumors 

Garofalakis A. , Dubois A. , Kuhnast B. , Dupont D. , Jassens I. , Mackiewicz N. , Dollé F. , Tavitian B. , Ducongé F. . 

CEA, Institut d’Imagerie BioMédicale, Service Hospitalier Frédéric Joliot, Laboratoire d’Imagerie Moléculaire Expérimentale, Orsay, France, Metropolitan 

anikitos.garofalakis@cea.fr 

Introduction: Small animal Fluorescence Diffuse 

Optical Tomography (fDOT) is a relatively new 

technology that can provide quantitative imaging 

of molecular processes. It allows using smart activatable 

probes which can measure biological processes 

that are inaccessible using nuclear probes. For 

instance, fluorescent sensors have been designed 

to monitor specific proteolytic activities using 

quenched NIRF probes that become fluorescent 

when they are cleaved by a protease of interest. In 

this work, we performed experiments aiming in integrating 

fDOT imaging of fluorescent probes with 

PET-FDG and a X-ray micro-CT imaging. 

Methods: For performing combined fDOT/PET/CT 

measurements we have developed a simple approach 

that incorporates a multimodal mouse supporting 

system that can fit in all three modalities. We performed 

multimodal imaging of two processes in a 

tumour using the fluorine-18- FluoroDeoxyGlycose 

([ 18 F]-FDG) and a fluorescent activatable probe. For 

optical monitoring we used probes for monitoring 

cathepsin activity and the localization of integrin 

alpha-v-beta-3 in tumor models by using different 

commercially available probes (Prosense680, Integrisense680, 

Visen Medical, USA and AngioStamp, 

Fluoptics, France). We used as models nude mice 

bearing a subcutaneous xenograft of two different 

cell lines, the MDA-MB-231 and the NIH-MEN2A. 

For the multimodal measurements we followed a 

protocol that allows for the optimal correlation of 

the fDOT and the PET data. Each mouse has been 

firstly measured by PET and was sequentially placed 

on the fDOT system for optical imaging. Once the 

mouse was placed in the PET, an intravenous injection 

of FDG with an activity of 200 μCi was performed 

and the dynamic PET scan was initiated. 

Optical measurements were performed 24h after 

the probe injection. 

Results: We reconstructed the 3D cathepsin activity 

and the localization of Integrin with respect to the 

tumor volume as given by PET. In the all xenograft 

models, we observed that the glucose consumption 

and the fluorescent probes were just partially 

co-localized in the tumour. Indeed, the [ 18 F]-FDG 

labelled all the tumour xenograft whereas the 

fluorescence signal was predominantly located at 

the base and only partially overlapped with the tumor 

volume as imaged by μPET. 

Conclusions: In this study we explored the potential 

of integrating information collected by nuclear and 

optical imaging taking advantage of the unique possibilities 

that each modality can provide. We found 

that the activatable probes used are concentrated 

underneath the tumor predominantly connected to 

the stroma while the highest glucose consumption 

appears to be uniformly distributed in the tumour 

tissue. Cathepsin activity is regulated by complex 

interactions between extracellular matrix components 

and cells populating the tumor like stromal 

cells and therefore it is expected to be reconstructed 

underneath the FDG signal. We proved that cancer 

imaging can benefit from the development of 

dual PET/Optical methods can act synergistically 

and that can provide complementary information 

enhancing thus the information collected from a 

single subject. 


grants from the European Molecular Imaging Laboratory 

(EMIL) network (EU contract LSH-2004- 

503569 and the european program FMT-XCT “Hybrid 

Fluorescence Molecular Tomography (FMT) 

– X-ray Computed Tomography (XCT) method and 

system” contract No 201792. 

References: 

1. Ntziachristos, V., Ripoll, J., Wang, L.V. & Weissleder, R. 

Looking and listening to light: the evolution of wholebody 

photonic imaging. Nat. Biotechnol 23, 313-320 (2005). 

2. Weissleder, R., Tung, C.H., Mahmood, U. & Bogdanov, A. 

In vivo imaging of tumors with protease-activated nearinfrared 

fluorescent probes. Nat. Biotechnol 17, 375-378 

(1999). 


P-074 

poStEr 

TECHNOLOGY

P-075 

188 


Deep tissue molecular imaging with fluorescent biomarkers using multispectral optoacoustic 

tomography. A simulation study 

Glatz J. , Deliolanis N. , Schulz R. , Razansky D. , Ntziachristos V. . 

Helmholtz Zentrum Muenchen, Neuherberg, Germany 

ndeliolanis@yahoo.com 

Introduction: Fluorescent protein 

markers have established themselves 

as a valuable tool in biomedical 

research. Their applicability in 

multispectral optoacoustic tomography 

(MSOT) has recently been 

demonstrated with fluorochromes 

and fluorescent proteins [1,2]. 

Methods: A simulation study was 

conducted for the proteins GFP, mRaspberry, IFP 

and AF750. Theoretical calculations, based on light 

and sound propagation models suggests that IFP and 

AF750 yield an acoustic signal that is acoustic signal 

that is three orders of magnitude higher than that of 

GFP. Two protein inclusions of a concentration of 

1 μM were simulated in a cervical mouse structure 

and reconstructed by the backprojection algorithm. 

Results: It was shown that the protein concentration 

can be unmixed using only three multispectral 

measurements. Using blind source separation 

techniques this could even be achieved without 

prior spectral information. The unmixed components 

of the different proteins are shown in Figure 

1. The distinctively weaker signal strength from 

GFP and mRaspberry is a consequence of the 

strong tissue absorption in their spectral region. 

In order to obtain a stronger signal from the center 

the reconstructions were normalized for the light 

fluence inside the sample. From the simulation study 

the optimal measurement wavelengths could be determined 

for each protein. Their usage, as well as the 

blind source separation, was verified in a practical 

experiment using a tissue mimicking phantom with 

fluorochrome inclusions. 

Conclusions: The new red-shifted proteins can boost 

the acoustic signal intensity by over three order of 

magnitude. Protein concentrations can be unmixed 

in deep tissue and correcting for the light attenuation 

is an important step towards quantitative unmixing. 

Acknowledgements: This research is supported by a 

Marie Curie Intra-European Fellowship within the 

7th European Community Framework Programme. 

imaging life 

Fig. 1: Unmixing of tissue and protein distribution for simulated data 

References: 

1. D. Razansky et al. Multispectral photoacoustic imaging 

of fluorochromes in small animals Opt. Letters, 32, 

2891-2893, 2007 

2. D. Razansky et al. Multispectral opto-acoustic 

tomography of deep-seated fluorescent proteins in 

vivo Nat. Photonics, 3, 412-417, 2009


Microfluidic [ 11 C]-carbonylation reactions for the rapid synthesis of radiolabelled compounds 

for PET 

Miller P. (1) , Audrain H. (2) , Bender D. (2) , Demello A. (1) , Gee A. (3) , Long N. (1) , Vilar R. (1) . 

(1) Imperial College London, London, United Kingdom 

(2) Arhus University Hospital PET Centre, Denmark 

(3) GSK, United Kingdom 

philip.miller@imperial.ac.uk 

Introduction: The synthesis of 11C compounds for 

PET requires fast and specialised chemical techniques 

owing to the short half-life of the 11C radioisotope 

(t1/2 = 20.4 min) and sub-micromolar reaction scales. 

[1] Microfluidic reactors are emerging as a valuable 

technology for the rapid and small scale synthesis of 

short-lived radiopharmaceuticals for PET imaging. 

[2] The palladium mediated 11C-carbonylation reaction 

is a highly versatile route for the preparation 

of a wide range of 11C-carbonyl compounds[3], 

however, the low molecular concentrations of 

11CO coupled with its poor solubility in organic 

solvents make this a particularly challenging 

transformation. Here we report the application of 

a microfluidic reactor for improving the synthesis 

of 11C labelled amide and ester molecules via the 

palladium mediated 11C-carbonylation reaction. 

Methods: The microfluidic reactor (figure 1) was 

fabricated from glass using chemical wet etching 

techniques and contains two inlets, one outlet and a 

5 metre long reaction channel. A simple mixing tee 

motif is used bring the gas and liquid reagents into 

contact with each other. The palladium mediated 

11C-carbonylation reaction of a range of aryl halides 

was investigated (scheme 1). In a typical 11CO 

labelling experiment the coupling reagents (aryl halide, 

palladium catalyst and amine) were premixed 

and loaded into a 50 uL loop on an injector port. 

11CO, produced via the high temperature reduction 

of 11CO2 over Mo, was preconcentrated and trapped 

onto molecular sieves. The coupling reagents were 

injected into the microfluidic device while at the 

same time 11CO was released from the molecular 

sieves and passed into the device for reaction. 

Results: The total reaction and processing times for 

a typical reaction, including trapping and release of 

X 

+ 

H 2 N 

R 

X = I or Br; 

R = CN, CF3 or OCH3 Scheme 1 

11 CO, Pd catalyst 

microfluidic chip 

R 

O 

* N 

H 

11CO, was 15 min from end of bombardment. A series 

of amide and ester molecules were labelled with 

11C using our microfluidic reaction system (scheme 

1). Radiochemical yields (RCY) of labelled products 

were found to be dependent on the type of aryl halide 

and nucleophile used for the reaction. Generally, 

iodoaryl substrates with activating groups (CF3 

or CN) gave consistently higher RCYs (>80%) and 

radiochemical purities (>95%) than aryl halide substrates 

with deactivating groups (OCH3). 

Conclusions: A glass fabricated microfluidic device 

has been used to effectively perform high speed 11CO 

radiolabelling reactions. The larger surface area-tovolume 

ratio within the microfluidic reactor improves 

the gas liquid contact area and is thought to enhance 

the problematic CO insertion step of this reaction. 

Acknowledgement: PWM is grateful to the EPSRC for the 

award of a Life Sciences Interface fellowship (EP/E039278/1). 

References: 

Figure 1 

1. P. W. Miller et al., Angew. Chem. Int. Ed., 2008, 47, 

8998. 

2. P. W. Miller, J. Chem. Technol. Biotechnol. 2009, 84, 309. 

3. B. Langstrom et al., J. Labelled Compd. Radiopharm., 

2007, 50, 794. 


P-076 

poStEr 

TECHNOLOGY


P-077 Boosting image quality in low-dose RC-gated 5D cone-beam micro-CT 

190 

Sawall S. (1) , Bergner F. (1) , Lapp R. (2) , Mronz M. (2) , Karolczak M. (1) , Kachelrieß M. (1) . 

(1) Institute of Medical Physics (IMP), Erlangen, Germany 

(2) CT Imaging GmbH, Erlangen, Germany 

stefan.sawall@imp.uni-erlangen.de 

Introduction: Micro-CT imaging of the animal 

heart typically requires respiratory and cardiac 

(RC) gating. This can either be done prospectively 

or retrospectively. For functional imaging, and 

for multi-modality imaging, it is often desired to 

obtain the full 5D information (volumetric + respiratory 

+ cardiac) and retrospective gating is 

the method of choice. The amount of information 

available to reconstruct one volume for a given respiratory 

and cardiac phase is significantly lower 

than the total amount of information acquired. 

For example the reconstruction of a volume from a 

10% wide respiratory and a 20% wide cardiac window 

uses only 2% of the data acquired. Achieving 

a similar image quality as a non-gated scan would 

typically require to increase the dose by a factor of 

up to 50. Our aim is to provide similarly high image 

quality at low dose levels (100 to 500 mGy). 

Methods: We implemented a two-step iterative image 

reconstruction algorithm based on the McKinnon-Bates 

approach. The first step, aiming at R 

gating only, uses a prior image consisting of the 

reconstruction of all data. The RC gating step uses 

the image of the first step as a prior. An edge-preserving 

anisotropic diffusion filter is applied to the 

volume data after each iteration to perform spatial 

and temporal resolution-preserving noise reduction 

in up to five dimensions. We demonstrate our 

new reconstruction approach using mouse data 

scanned with a dedicated in-vivo micro-CT scanner 

(TomoScope Synergy, CT Imaging GmbH, Erlangen, 

Germany). We derived an intrinsic gating 

signal (kymogram) from the rawdata to synchronize 

our reconstruction. The scan itself consisted 

of ten rotations with 7200 projections in total. 

imaging life 

The scan time was five minutes, the tube voltage 65 

kV. The gating window widths were set to 10% in the 

respiratory and to 20% in the cardiac cycle. 

Results: Although the mouse data available to us 

shows only an untypically low enhancement of about 

110 HU (blood vs. myocardium) our approach yields 

an image noise of as low as 35 HU while preserving the 

spatial resolution of about 100 µm. We also performed 

a standard RC-gated reconstruction. Its high noise 

levels of about 170 HU are prohibitive and significant 

artifacts are observed due to sparse view sampling. 

The dose of our protocol is about 500 mGy. Reconstructions 

using less than the available 10 rotations 

show that one can achieve good image quality in 

RC-gated micro-CT with about 100 mGy, provided 

that the contrast agent delivers sufficient contrast. 

Conclusions: Using advanced image reconstruction 

techniques enables us to perform high fidelity 

low-dose double-gated imaging of free breathing 

small animals. 


the Deutsche Forschungsgemeinschaft (DFG) under 

grant FOR 661.


Methodological approach using microscopy for quantitative evaluation of neo-angiogenesis 

and tumour progression in pre-clinical cancer models 

Thézé B. , Beynel A. , Chapotot L. , Boisgard R. , Tavitian B. . 

CEA /Inserm 1023, Orsay, France 

benoit.theze@cea.fr 

Introduction: In vivo molecular imaging methods can 

localize sites of angiogenesis and obtain functional 

data, whereas microscopic methods provide their 

highest resolution on preserved tissue specimens. To 

bridge the gap between molecular imaging methods 

and microscopy, we developed a method to evaluate 

neo-angiogenesis and tumour volume on formalinfixed 

tissues samples. 

Methods: In order to visualize the mammary tumour 

vessels density in the MMTV-PyMT transgenic model 

of mammary carcinoma, mice were injected i.v. 

with Horse Radish Peroxidase (HRP)– tomato lectin 

(at 6.66 g/kg), which binds surface glycoproteins of 

red blood cells and endothelial cells inside the vascular 

lumen. Tumours were immediately sampled, fixed 

in formalin and frozen. For this pilot study, four tumours 

from each of two mice were sectioned every 

300 µm . For every section, HRP-lectin was revealed 

with 3,3´-diaminodbenzidine (DAB) and the tissues 

were counterstained with haematoxylin. Panoramic 

views of each tissue section were acquired in brightfield 

with a motorized AxioObserver Z1 Zeiss microscope. 

Tissue staining and image acquisition parameters 

were standardized for quality control. Using 

imageJ image analysis software, a macro was developed: 

(i) to separate blue and brown colours from the 

RGB original image using the colour deconvolution 

plugin in batches of images, and (ii) to evaluate the 

area occupied by the tumour in the whole sample. A 

treatment pipeline was designed with the Cellprofiler 

software for threshold and segmentation of vessels 

(brown) versus tumour masses (blue). Object-based 

filtering was applied to vessels according to their size 

and to their localization inside or outside of the tumour 

masses in order to separate neo-vessels from 

pre-existing vessels and areas of necrosis. 

Results: Using this approach, we were able to measure 

areas occupied by vessels and tumours and, (i) 

to obtain an estimate of neo-angiogenesis defined as 

the ratio between tumour blood vessels’ volume and 

tumour volume (1.27% ± 0.61%), (ii) to determinate 

the real volume of tumour in the sample (75% 

± 7%), which also contain muscular, adipose and 

conjunctive tissue, (iii) to obtain the ratio between 

total blood volume and total sample volume (5.99% 

± 0.42%), and (iiii) to discriminate between small 

(55% ± 9.6%), medium (32% ± 2.8%), and large 

(12.9% ± 7.8%) blood vessels. 

Conclusions: The data from this feasibility study 

provides morphological information about the vascular 

network in the PyMT model. Further validation 

steps should now allow correlating the results 

with functional information obtained by PET imaging, 

and provide a method for the evaluation of antiangiogenic 

drugs in animal tumour models. 

Fig1 from left to right: original image, blue and brown colour deconvolved images and final segmented image 

Acknowledgement: This work was supported by the 

6th FW EU grants EMIL (LSHC-CT-2004-503569) 

and DiMI (LSHB-CT-2005-512146) 

References: 

1. Rasband, W.S., ImageJ, U. S. National Institutes of 

Health, 1997-2009 

2. Jones TR et al. (2008) CellProfiler Analyst: data 

exploration and analysis software for complex imagebased 

screens. BMC Bioinformatics 


P-078 

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TECHNOLOGY

192 


P-079 Changes of heart stroke volume index established by 99m Tc MIBI GSPECT scintigraphy after 

radioiodine treatment of patients with subclinical hyperthyroidism 

Kaminski G. , Podgajny Z. , Szalus N. , Bilski M. , Dziuk M. . 

Military Institute of Health Services, Warsaw, Poland 

gkam@wim.mil.pl 

Introduction: Stroke volume index (SVI) is an indicator 

of heart load. Increased heart load is one of 

causes of cardiac death. Subclinical hyperthyroidism 

(SH yper ) increases mortality mostly due to cardiovascular 

events. SH yper affects about 1% of population including 

patients with cardiovascular diseases. The 

aim of the investigation was to estimate an influence 

of cure with radioiodine of SH yper on heart stroke volume 

index (SVI) measured by heart scintigraphy - 

99m Tc MIBI GSPECT. 

Methods: 44 patients (37 women, 7 men) aged 

45.9±11, with 12.8±9.8 month history of only autonomous 

SH yper (TSH=0.16±0.1 IU/l), were examined 

with 99m Tc MIBI GSPECT twice: before 

and 5.7±4.2 months after TSH normalization 

(TSH=1.32±0.1 IU/l) after radioiodine treatment 

(at dose 12.1±5.7 mCi) . The radioiodine ( 131 I) 

and 99m Tc MIBI were carried by POLATOM/Poland. 

The average time between examinations was 

12.5 ± 6 months. The Local Ethical Committee approval 

for this investigation has been obtained. 

Results: The cure of SH yper caused decrease of SVI 

from 26.36 to 24.21 ml/m 2 (p=0.042) 

Conclusions: Cure of autonomous subclinical hyperthyroidism 

with radioiodine decreases heart 

load. This finding indicates to treat subclinical hyperthyroidism, 

especially in patients with cardiovascular 

diseases. 

imaging life 

[ml/m 2 ] 

27 

26 

26 

25 

25 

24 

24 

23 

26,36 

przed po 

24,21 

Fig.1. Mean values of SVI before (left) and after (right) radioiodine 

treatment p = 0,042) 

References: 

1. Parle J.V., Maissoneuve P., Sheppard M.C., Boyle P., 

Franklyn J.A.: Prediction of all- cause and cardiovascular 

mortality in elderly people from one low serum 

thyrotropin result: a 10 -year cohort study. 

2. Lancet, 2001, 358: 861 - 865.


PET/CT investigations with 68Ga-DOATATE in neuroendocrine tumors - first clinical 

experience 

Kunikowska J. (1) , Krolicki L. (2) , Pawlak D. (3) , Kobylecka M. (2) . 

(1) Medical University of Warsaw, Poland 

(2) Medical University of Warsaw, Poland 

(3) IEA POLATOM, Świerk, Poland 

jolanta.kunikowska@wum.edu.pl 

Introduction: Neuroendocrine tumors (NETs) have 

distinct biological and clinical characteristics, in 

particular a high density of somatostatin receptors 

at the cell membrane. It is this property that allows 

the use of radiolabeled somatostatin analogs for imaging 

of these tumors. Novel techniques PET/CT 

with 68Ga-DOTATATE open new possibilities in the 

diagnosis of patients with NET. Sensitivity of that 

techniques is depending on SSTR expression, but average 

range is 60-94%. 

The aim of this study was to evaluate the diagnostic 

usefulness of a new somatostatin analog, 68Ga- 

DOTATATE, for PET/CT in patients with diagnosis 

of neuroendocrine tumors. 

Methods: 55 patients with NET were examined (24 

men, 31 women; age range, 18-86 y; mean age +/- SD, 

51.4 +/- 12.5 y). For analysis, patients were divided into 

3 groups: detection of unknown primary tumor (n = 

13 patients), follow-up after surgery (n = 23 patients), 

staging of disease (n= 19). PET imaging was performed 

on PET/CT scanner Biograph 64, 60 minutes 

post injection of 120-185 MBq 68Ga-DOTATATE. 

Results: In the group patients with unknown primary 

tumors, 68Ga-DOTATATE revealed 9 primary 

foci. 7 were not visible in CT (4 in intestinum, 3 in 

pancreas) and 2 foci observed in CT and PET/CT 

- small nodule in lung. In 4 cases primary tumors 

were not found. 

In the cases of patients after surgery, examination 

shown new foci in 5 patients (liver-3, lymph nodes 

in abdominal cavity-2, peritoneum-1, pancreas-1). 

In the group of staging of disease in 12/19 cases 

PET and CT shown the same foci. In 7/19 (37 %) 

patients in 68Ga-DOTATATE examination was revealed 

new lesions (intestinum-1, liver-3, bone-1, 

pancreas-4, lymph nodes in abdominal cavity-2) 

Conclusion: Results of our study shown that 68Ga- 

DOTATATE PET/CT is very useful non-invasive 

techniques in diagnosis of patients with NET. 

68Ga-DOTATATE PET/CT examination should 

be performed for staging and restaging of disease 

and it gives more clinically useful information 

than CT. 


P-080 

poStEr 

ENDOCRINE DISEASES

194 


P-081 Influence of number of subsets and iterations of Ordered Subsets Expectation Maximization 

(OSEM 3D Flash) reconstruction on quantitative assessment of small and medium detected 

lesions in SPECT study with used in 99m Tc[EDDA/HYNIC]Octreotate for patients with GEP-NET 

Lenda-Tracz W. . 

Nuclear Medicin Unit, Krakow, Poland 

wtracz@su.krakow.pl 

Introduction: The algorithm OSEM 3D Flash generally 

is the most appropriate available method of 

reconstruction. The choice of appropriate sets of 

OSEM 3D Flash reconstruction is crucial in interpretation 

of scintigraphy images. The number 

of subsets and iterations significantly influences 

the lesion to noise ratio. Therefore the aim of the 

study was to find the optimum number of subsets 

and iterations which present the most effective 

lesion to noise ratio in quantitative assessment 

of small and medium lesions detected with 

the use of 99m Tc[EDDA/HYNIC]Octreotate. 

Methods: The results of 20 patients with confirmed 

neuroendocrine tumors (GEP-NET) were 

analyzed. SPECT studies, acquisition 3-4 h after 

injection of 740 MBq 99m Tc[EDDA/HYNIC] 

Octreotate, were performed. The E.CAM 180 

(Siemens), double-head gamma-camera, was 

equipped with parallel, low-energy, high-resolution 

collimators. Data were acquired with 180 

rotation with 128 non-circular projections (30s 

per view) using a 128x128 matrix with 1.23 zoom. 

OSEM 3D Flash reconstruction (subsets number 

8, 16, 32 for 2-30 iterations) was performed. 

Results: Lesion to noise ratio in voxels was analyzed 

for different setting combinations (n,m) 

of number of subsets (n) and iterations (m) of 

OSEM 3D Flash reconstruction. An increasing 

number of subsets and iterations influences the 

increasing values of lesion to noise ratio. No difference 

for settings (16,m) and (32,m) was observed 

for quantitative assessment of small and 

medium lesions detected. Differences between lesion 

to noise ratio were observed in a group with 

8 subsets (8,m). However, the quality of images is 

variable for all setting combinations (n,m). 

Conclusions: The high number of subsets improves 

the image quality and the images are 

smoother. The increasing number of iterations 

on the one hand gives a little better contrast but 

on the other hand the shape of the lesions and organs 

is sharper. In spite of the fact that the image 

quality is changed with the increasing number of 

subsets and iterations, for quantitative assessment 

imaging life 

only (8,m) setting is changed significantly. Therefore, 

for quantitative assessment the best choice 

is setting with 8 subsets and 2-30 iterations but at 

least 6 and no more than 22 iterations with step 

equal at least 4 iterations. The appropriate number 

of iterations depends on the image quality 

assessment.


The precise localization of metastatic lesion with used SPECT/CT with CoDe system after 

131I – MIBG therapy in patients with disseminated pheochromocytoma 

Szalus N. (1) , Podgajny Z. (2) , Kaminski G. (3) , Mazurek A. (1) , Giżewska A. (1) , Dziuk E. (1) . 

(1) Department of Nuclear Medicine,Military Institute of Medicine, Warsaw, Poland 

(2) Department of Endocrinology and Radioisotope Therapy2 - Military Institute of Medicine, Warsaw, Poland 

(3) Department of Endocrinology and Radioisotope Therapy - Military Institute of Medicine, Warsaw, Poland 

nszalus@wp.pl 

Introduction: Pheochromocytoma is a rare tumor 

that originates from chromaffin cells such as the adrenal 

medulla and sympathetic ganglia. Malignant 

pheochromocytoma is uncommon, and metastases 

typically affect the bones, liver, lungs, and lymph 

nodes. Meta-iodo-benzyl guanidine (MIBG) is a 

norepinephrine analog, and 131I- and 123I-MIBG 

have been widely used for the diagnosis of pheochromocytoma. 

This technique has high specificity and 

detectability not only for primary tumors but also 

metastatic lesions when compared with morphologic 

imaging such as computed tomography (CT) and 

magnetic resonance imaging (MRI). Co-registered 

data have been shown to be useful in the evaluation 

of patients with cancer at diagnosis and staging, in 

monitoring the response to treatment, and during 

follow up, for early detection of recurrence. Gamma 

camera with CoDe system is a new modality to the 

PET/CT and SPECT/CT imaging to the precise localization 

metastatic lesions. 

Aim of study: The aim of this study is to investigate 

the precise localization 131-MIBG with used SPECT/ 

CT CoDe system for metastatic diseases in patients 

with malignant pheochromocytoma. 

Material and methods: Two patients with disseminated 

pheochromocytoma (the first patient with metastases 

to the liver, bones and lung; the second with 

metastases to the bone) were referred for study. Routine 

whole body scan with I-131 was performed with 

a dual head gamma camera (Infinia Hawkeye General 

Electric Milwaukee with CoDe system, USA) using 

a large field of view with high energy collimator 

a 20 % energy window centered at 364 keV and, 1024 

x 512 matrix. The data acquisition was performed 72 

hours after 131-MIBG therapy. Whole body anterior 

and posterior views were obtained. SPECT images of 

thorax and abdomen were obtained with 45 sec/frame, 

60 projections, 20 % window centered at 364 keV, 

matrix size of 128 x 128 and zoom factor of 1.0. This 

was followed by CT acquisition using single slice (1 

cm thickness). Using volumetrix software in Xeleris, 

fused images in coronal, sagittal and transaxial views 

were then obtained. Whole body planar images were 

first interpreted alone. Then, they were reassessed 

with the addition of SPECT/CT coregistered images. 

Results: 1. In these patients SPECT/CT revealed 

30 % more pathological lesions than planar studies 

alone. 2. SPECT/CT provided precise anatomical 

localization not clearly evident in planar images 

alone. 3. It also enabled exclusion of disease in sites 

of physiologic tracer deposition found suspicious in 

planar studies alone 

Conclusion: SPECT/CT allows more precise localization 

and interpretation of 131I-MIBG whole 

body scan thereby improving its diagnostic accuracy. 

It also has impact on correct restaging after therapy 

with 131I-MIBG preparation. 

References: 

1. Bas Havekes at al. Clinical Endocrinology (2010) 72, 

137–145 

2. Akie Takano at al. Ann Nucl Med (2008) 22:395–401 


P-082 

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ENDOCRINE DISEASES

196 


P-083 Multimodal in vivo imaging of pancreatic beta-cells via antibody mediated targeting of 

beta-cell tumors 

Vats D. (1) , Dikaiou K. (1) , Stuker F. (1) , Honer M. (2) , Wang H. (3) , Schibli R. (2) , Rudin M. (1) . 

(1) Institute for Biomedical Engineering, ETH Zurich, Zurich, Switzerland 

(2) Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland 

(3) F.Hoffman La-Roche, Basel, Switzerland 

vats@biomed.ee.ethz.ch 

Introduction: Pancreatic beta-cells regulate glucose 

metabolism by producing and secreting insulin in 

sufficient amounts. Progressive loss of beta-cell mass 

and function surmounts to acute diabetes. Diagnosis 

of diabetes and the evaluation of potential therapeutics 

suffer from lack of established methods for in 

vivo imaging of beta-cells. This study demonstrated 

the in vivo targeting of a novel antibody to a beta-cell 

surface protein in beta-cell tumor models. The pilot 

study evaluated the binding capacity of 89 Zr-lableled 

and alexa680 labeled monoclonal antibody against 

hTMEM27, a human beta-cell surface glycoprotein 

(1), for targeted imaging of beta-cell tumors in nude 

mice using positron emission tomography (PET) and 

fluorescence imaging. 

Methods: Insulinoma cell-line (beta-cells), Ins1E, 

was taken as the parental cell-line for generating 

stable cell lines (2) over-expressing hTMEM27 (Ins- 

TM) while blank Ins1E cells (Ins-only) were taken 

as controls. Ins-TM and Ins-only cells were used to 

generate subcutaneous tumors in nude mice. 1.5MBq 

of 89 Zr-anti-hTMEM27 monoclonal antibody ( 89 Zr- 

in vivo fluorescence imaging 

Ins-TM Ins-only Ins-TM-HA 

ROI ROI ROI 

alexa680-anti-hTMEM-mAb alexa680-anti-HA-mAb 

TM-mAb) and 2mg/kg alexa680-anti-hTMEM27 

monoclonal antibody (alexa680-TM-mAb) and 

alexa680-anti-HA control monoclonal antibody (alexa680-HA-mAb) 

were injected i.v. in tumor bearing 

nude mice. The animals were in vivo imaged three 

days after antibody administration: PET for 89 Z-TMmAb 

and fluorescence imaging (both near infrared 

fluorescence reflectance imaging and near infrared 

fluorescence molecular tomography (NIR-FMT)) for 

alexa680-TM-mAb and alexa680-HA-mAb. The antibody 

retention was quantified using gamma counting 

of excised tumors for the 89 Zr labeled antibody and 

the fluorescence intensity was derived from region 

of interest (ROI) quantification for reflectance mode 

and NIR-FMT data (using MATLAB tools). The 

data was compared between the hTMEM27 over expressing 

insulinomas (Ins-TM), control insulinomas 

imaging life 

(Ins-only) and control antibody in hTMEM27 over 

expressing tumors (TM-HA), for antibody specificity 

over a period of 6 days. Individual tumors from 

optical study were further subjected to histological 

analysis for accurate localization of the dye distribution 

within tumor tissue. 

Results: We found 6-7 times higher retention of the 

antibody in TMEM27 over expressing beta-cells, 

when compared to control beta-cells or with control 

antibody by both 89 Zr-PET and NIR-imaging (Figure 

1). The fluorescence microscopy results confirmed 

antibody retention to TMEM27 in target insulinomas 

(Ins-hTMEM-tumors). 

Conclusions: The pilot study could demonstrate 

the dual modal imaging of beta-cell mass, in a betacell 

tumor model, and identified a novel antibody 

for image-guided targeting of a beta-cell surface 

glycoprotein. 

Acknowledgement: This work is funded by 

F.Hoffmann La-Roche Ltd. 

Figure 1. 

Av. Fl. intensitiy 

References: 

1.40E+05 

1.20E+05 

1.00E+05 

8.00E+04 

6.00E+04 

4.00E+04 

2.00E+04 

0.00E+00 

Fluorescence quantification 

pre 24h 3d 6d 

Time after injection 

Ins-only 

Ins-TM 

Ins-TM-HA 

1. Akpinar P et al.; Cell Metab. 2(6):385-97 (2005) 

2. Wang H and Iyenedjian P B.; PNAS. 94: 4372-4377 

(1997)


6-[ 18 F]Fluoro-PBR28, a novel TSPO 18 kDa radioligand for imaging neuroinflammation with 

PET 

Boisgard R. (1) , Damont A. (1) , Blossier A. (1) , Jego B. (1) , Siquier K. (1) , Kassiou M. (2) , Dolle F. (1) , Tavitian B. (1) . 

(1) CEA, Orsay, France 

(2) Sydney University, Australia 

raphael.boisgard@cea.fr 

Introduction: The peripheral benzodiazepine receptor 

(PBR or TSPO 18 kDa) is expressed by microglial 

cells in many neuropathologies involving 

neuroinflammation. [ 11 C]PK11195 is today the 

most widely used radioligand for the in vivo imaging 

of PBR using PET, and this in spite of its low 

brain uptake and its high level of non-specific binding. 

Numerous PK11195 challengers are currently 

under investigation [1,2], and of particular interest 

are the N-benzyl-N-(2-phenoxyaryl)-acetamides, a 

series which includes [ 11 C]PBR28 [3]. A fluorinecontaining 

analogue, namely 6-fluoro-PBR28 (N-(2methoxybenzyl)-N-(6-fluoro-4-phenoxypyridinyl- 

3-yl)acetamide), has been labeled with the longer 

half-life positron-emitter fluorine-18 and pharmacologically 

evaluated in a rat model of neuroinflammation 

(unilaterally, AMPA-induced, striatum-lesioned 

rats) with PET. 

A 

18 F 

N 

N 

O 

O 

O 

B 

Fig1: Structure of 6-[18F]fluoro-PBR28 (A) and microPET images (B) obtained in AMPA lesioned rat (60 min p.i.) 

Methods: 6-Fluoro-PBR28, as well as the corresponding 

precursors for labeling, were synthesized 

from 4-chloro-3-nitropyridine. 6-Fluoro-PBR28 was 

labeled with fluorine-18 by nucleophilic heteroaromatic 

substitution using K[ 18 F]F-Kryptofix ® 222, 

purified by HPLC (Waters Symmetry ® C-18) and 

formulated for i.v. injection. The AMPA rat model 

was used to study in vitro and in vivo specific and 

non-specific binding using autoradiography and 

µPET imaging on a Concorde Focus P220 PET 

camera, including displacement with PK11195 

and non-labeled 6-fluoro-PBR28 (1 mg/kg). 

Results: Starting from a 37 GBq cyclotron-produced 

[ 18 F]fluoride batch, 3.3-3.7 GBq of 6-[ 18 F] 

fluoro-PBR28, > 99% radiochemically pure and 

ready-to-inject, were obtained within 90 minutes 

(Figure 1A). In PET experiments, 6-[ 18 F]fluoro- 

PBR28 showed a higher contrast between the lesioned 

area and the corresponding area in the intact 

contralateral hemisphere (ratio ipsi/contra at 

60 min post-injection: 6-[ 18 F]fluoro-PBR28 : 2.2) 

(Figure 1B). Furthermore, 6-[ 18 F]fluoro-PBR28 

was displaced by PK11195 or 6-fluoro-PBR28. Finally, 

modelisation of the PET data using the “simplified-reference-tissue-model” 

showed increased 

binding potential (BP) in comparison to the BP 

of [ 11 C]PK11195 measured in the same model 

(2.0±0.6 versus 1.1±0.2). Immunohistochemical 

analyses correlate with PET-imaging and showed 

strong activation of microglia in and around the 

lesion. 

Conclusions: Dynamic µPET studies in rats demonstrate 

the potential of 6-[ 18 F]fluoro-PBR28 to 

image neuroinflammation. 

Acknowledgements: Supported in part by the EC - 

FP6-project DiMI (LSHB-CT-2005-512146) and 

EMIL (LSH-2004-503569). 

References: 

1. Chauveau F et al; Eur J Nucl Med Mol Imag 35: 2304- 

2319 (2008) 

2. Dollé F et al; Curr Med Chem 16: 2899-2923 (2009) 

3. Briard E et al. J Med Chem 51: 17-30 (2008) 


P-084 

poStEr 

INFECTION and INFLAMMATION


P-085 Neuroinflammation is increased in the brain of ageing corpulent (JCR:LA-cp) rats: a positron 

emission tomography study 

198 

Boutin H. (1) , Drake C. (1) , Denes A. (1) , Mccoll B. (1) , Prenant C. (1) , Brown G. (1) , Kassiou M. (2) , Herholz K. (2) , Rothwell N. (2) . 

(1) University of Manchester, United Kingdom 

(2) University of Sydney, Australia 

herve.boutin@manchester.ac.uk 

Introduction: Despite intense research and development 

of several animal models, drugs efficient in 

preclinical model of cerebral ischaemia so far have 

failed when reaching clinical trial [1] . One striking 

feature of the animal models is the lack of co-morbidities 

and risk factors when compared to clinical 

set-up, in which patients have atherosclerosis, high 

blood pressure, chronic and/or acute inflammation 

due to chronic inflammatory diseases and infections. 

Inflammation and neuroinflammation in particular 

are known aggravating factors of stroke outcome [2] . 

Here we investigate the impact of known risk factors 

of stroke such as obesity and atherosclerosis in 

JCR:LA-cp (corpulent) rats on neuroinflammation as 

measured by TSPO expression in activated microglia. 

Methods: Neuroinflammation was assessed with 

[ 18 F]DPA-714 by PET in Lean (control: Cp/?)) and 

JCR:LA-cp (corpulent: Cp/Cp) rats PET imaging. 2 

groups of rats were scanned at 9 months or 12 and 

15 months of age (n=4 per group). PET images were 

co-registered with a MRI template [3] for analysis 

and automatic segmentation performed for userindependent 

ROI determination [4,5] . Euthanasia was 

performed at 9 months and 15 months of age 3 to 7 

days after PET imaging to assess various neuroinflammation 

biomarkers (GFAP, IBA1, VCAM) by 

immunohistochemistry (IHC). 

Results: Our results show an increase (+38%) in 

neuroinflammation in the brain of corpulent rats 

when compared to control. In older rats (12 and 

15 months old), neuroinflammation was even further 

increased but in both lean and corpulent rats. 

imaging life 

Neuroinflammation, as quantified by PET, was 

mainly localised in peri-ventricular and thalamic 

areas of the brains. IHC for microglial activation 

confirmed the PET data in 15 months old animals 

(Figure below). 

Conclusions: Our data show here the importance 

of including risk and co-morbidity factors in preclinical 

models of stroke as they have a significant 

impact on neuroinflammation. This study shows 

the crucial role of molecular imaging with the 

high translational value of PET imaging to investigate 

such paradigms. A more detailed study of 

the localisation of neuroinflammation as localised 

by PET and IHC is now ongoing. Further investigation 

either in animals 

with spontaneous 

stroke or with induced 

cerebral ischaemia will 

be required to compare 

the impact of co-morbidity 

and risk factors 

in this animal model. 

Acknowledgement: Prof. N. Rothwell and Dr H. Boutin 

are funded by MRC. This study was carried out 

within the EC-FP6 project DiMI (LSHB-CT-2005- 

512146) framework. 

References: 

1. Dirnagl U. (2006) J Cereb Blood Flow Metab. 26:1465- 

1478; 

2. McColl B.W. et al. (2009) Neuroscience. 158:1049- 

1061; 

3. Schwarz A.J. et al. (2006) Neuroimage. 32:538-550; 

4. Maroy R. et al. (2008) IEEE Trans.Med.Imaging. 27:342- 

354; 

5. Maroy R. et al. (2010) J Nucl Med (submitted).


Detection of inflammatory diseases by NIRF imaging with specific probes targeting 

leukotriene receptor CysLT 1 R 

Busch C. (1) , Passon M. (1) , Lehmann F. (2) , Socher I. (1) , Kaiser W.A. (1) , Hilger I. (1) . 

(1) University Hospital Jena, IDIR, Jena, Germany 

(2) DYOMICS GmbH, Jena, Germany 

Corinna.Busch@med.uni-jena.de 

Introduction: Leukotriene synthesis occurs early 

in inflammatory processes and plays a major role 

for the recruitment of leukocytes to the inflamed 

region (1). A key representative of these eicosanoids, 

leukotriene D4 (LTD4), shows high affinity 

to G-protein coupled cysteinyl leukotriene 

receptor 1 (CysLT 1 R). Detection of CysLT 1 R by 

molecular imaging (2) with near-infrared (NIR) 

fluorophores could be a suitable diagnostic tool 

for early identification of inflammatory processes. 

Aim of this study was the design and characterization 

of NIRF-based contrast agents specifically 

targeting CysLT 1 R in order to develop future diagnostic 

tools for inflammatory diseases, particularly 

in early stages. 

Methods: Polyclonal rabbit CysLT 1 R antibody 

(CysLT 1 R*DY-734) or polyclonal rabbit-IgG 

(IgG*DY-734) as well as the corresponding Fab 

fragments (Fab-CysLT 1 R*DY-734, Fab-IgG*DY- 

734) were bound to activated NHS ester of NIRfluorophore 

DY-734 (Dyomics, Jena, Germany). 

Probes were characterized by determining dye/protein 

ratios. After verification of CysLT 1 R expression 

(PCR, flow cytometry) in HL-60 cells, binding 

of the synthesized probes in vitro was assessed by 

flow cytometry. In vivo, an ear edema was induced 

in mice (3), and NIR fluorescence was measured 

with whole body imaging system Maestro2.2 after 

i.v.-probe administration. Ex vivo biodistribution 

studies were performed. 

Results: Flow cytometry proved binding of CysLT 1 R*DY- 

734 and IgG*DY-734 to CysLT 1 R-expressing HL-60. In 

vivo, all probes revealed stronger binding to the edematous 

than to the corresponding healthy region. 6 

h post injection, specific CysLT 1 R*DY-734 and Fab- 

CysLT 1 R*DY-734 demonstrated 1.9 and 1.2 fold higher 

binding than IgG*DY-734 and Fab-IgG*DY-734, 

respectively. Investigation of isolated organs revealed 

that full length IgG´s are eliminated via liver, while 

Fab fragments additionally accumulated in kidney. 

Conclusions: A novel NIRF-based probe specifically targeting 

Leukotriene D 4 receptor CysLT 1 R allows detection 

of inflammatory processes in ear edema-induced 

mice in early stages. 

Acknowledgement: The present investigations were 

supported by the “Deutsche Forschungsgemeinschaft” 

within the DFG program Hi 689/6-1. 

References: 

1. Hui Y, Funk C. Cysteinyl leukotriene receptors. Biochem. 

Pharmacol. 2002;64:1549-57. 

2. Weissleder R, Mahmood U. Molecular Imaging. 

Radiology 2001;219:316-33. 

3. Kurnatowska I, Pawlikowski M. Anti-inflammatory 

effects of somatostatin analogs on zymosaninduced 

earlobe inflammation in mice: 

comparison with dexamethasone and ketoprofen. 

Neuroimmunomodulation 2001;9:119-24. 


P-086 

poStEr 


200 


P-087 PET imaging of Hypoxia by 18 F-Fluoromisonidazole ([ 18 F]FMISO) to detect early stages of 

experimental arthritis 

Fuchs K. (1) , Grießinger C. (1) , Fischer K. (1) , Mannheim J. (1) , Wiehr S. (1) , Judenhofer M. (1) , Reischl G. (2) , Röcken M. (3) , 

Pichler B. (1) , Kneilling M. (3) . 

(1) Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, Department of Radiology, Eberhard- Karls University, 

Tübingen, Germany 

(2) Radiopharmacy, Department of Radiology, Eberhard- Karls University, Tübingen, Germany 

(3) Department of Dermatology, Eberhard- Karls University, Tuebingen, Germany University Hospital Tübingen - Department of Radiology, Tübingen, Germany 

Kerstin.Fuchs@med.uni-tuebingen.de 

Introduction: Early detection of autoimmune diseases 

such as rheumatoid arthritis is essential for 

early interventional anti-inflammatory treatment 

to prevent cartilage- and bone deterioration. Hypoxia 

can induce angiogenesis via stabilization of 

the transcription factor hypoxia inducible factor 

(HIF)-1α/2α in resident and infiltrating cells by induction 

of pro-angiogenic mediators. Also, hypoxia 

is hypothesized to be one of the early indicators of 

inflammation. The aim of our study was to examine 

initial phases of hypoxia-induced angiogenesis and 

inflammation in rheumatoid arthritis, by in vivo 

small animal PET, even before clinical symptoms 

or histological joint inflammation are detectable. 

Therefore, we used the hypoxia tracer 18 F-Fluoromisonidazole 

([ 18 F]FMISO), which selectively accumulates 

in hypoxic tissue, and [ 18 F]Fluordesoxyglucose 

([ 18 F]FDG). 

Methods: We induced arthritis in BALB/c mice via 

intraperitoneal injection of serum containing autoantibodies 

against glucose-6 phosphate-isomerase 

(GPI). Mice underwent [ 18 F]FMISO-, or [ 18 F]FDG 

- PET investigations, 6 - 52 hours after induction 

of GPI-arthritis. Additionally, we performed H&Estaining, 

Western Blot (HIF-1α/2α) and real-time 

PCR analysis of gene expression patterns (bFGF, 

VEGF, IL-1β, TNF, IL-6, and COX-2) in joint tissue 

6 and 12 hours after initiation of arthritis. 

Results: Starting 6 hours after induction of GPI-arthritis 

we detected an enhanced [ 18 F]FMISO uptake 

in arthritic joints compared to healthy joints. Differences 

in [ 18 F]FMISO uptake between arthritic 

and healthy joints reached a level of significance 13 

hours after induction of GPI-arthritis. In contrast 

to [ 18 F]FMISO, no increase in [ 18 F]FDG-uptake was 

detectable at these early time points (6-13 hours). 

Comparable to the in vivo [ 18 F]FDG-PET data, no 

histological visible signs of arthritis were examined 

in H&E-stained slices of arthritic joint tissue. In line 

with the in vivo [ 18 F]FMISO-PET-data, RT-PCR analysis 

performed 6 hours after GPI-serum injection 

showed a 7.5-fold enhanced expression of HIF-2α 

mRNA. Interestingly, mRNA-levels of pro-angiogenic 

imaging life 

and pro-inflammatory mediators such as bFGF and 

VEGF, TNF, IL-6, and COX-2 were not elevated 6h 

after induction of GPI-arthritis. Starting 54h after 

induction of GPI-arthritis we detected significant 

differences in [ 18 F]FDG uptake in arthritic ankles, 

and a 6.5-1550 fold enhanced expression NF- k B induced 

genes such as COX-2, IL-6, IL-1β and, TNF. 

Conclusions: Non invasive in vivo examination of 

hypoxia-induced angiogenesis using [ 18 F]FMISO 

is a powerful tool to detect initial phases of angiogenesis 

in autoimmune diseases such as rheumatoid 

arthritis even before joint inflammation becomes 

detectable by other methods.


Intracellular [ 64 Cu]PTSM and extracellular [ 64 Cu]DOTA-antibody labelling of ovalbuminspecific 

Th1 cells for in vivo PET investigations of Th1 cell trafficking in OVA-specific lung 

inflammation 

Griessinger C.M. (1) , Wiehr S. (1) , Bukala D. (1) , Kesenheimer C. (1) , Röcken M. (1) , Ehrlichmann W. (3) , Reischl G. (3) , Pichler B. (1) , 

Kneilling M. (2) . 

(1) Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, Department for Radiology, Eberhard Karls University of 

Tübingen, Germany 

(2) Department for Dermatology, Eberhard Karls University of Tübingen, Germany 

(3) Radiopharmacy, Eberhard Karls University of Tübingen, Germany 

Christoph.Griessinger@med.uni-tuebingen.de 

Introduction: T helper cells play an important role 

in the development of autoimmune diseases. For detailed 

in vivo analysis of the migration properties of 

Th1 cells, high sensitive imaging modalities, such 

as small animal PET are powerful tools. So far basic 

migration properties like kinetics, homing, and 

sites of T cell proliferation in animal models for autoimmune 

diseases are still poorly understood. The 

aim of our study was to establish new T cell labelling 

strategies to gain new insights in Th1 cell trafficking 

in vivo using small animal PET. In our studies Th1 

cells were in vitro labelled intracellularly with the 

lipophilic tracer [ 64 Cu]PTSM or extracellularly with 

[ 64 Cu]DOTA-linked antibodies prior to injection 

into diseased mice and tracking by small animal PET. 

Methods: To investigate whether intracellular [ 64 Cu] 

PTSM labelling or extracellular [ 64 Cu]DOTA-antibody 

labelling impair ovalbumin (OVA)-specific 

Th1 cells, we analysed cell viability and functionality. 

OVA-T cell receptor (TCR) transgenic CD4+ T 

cells were isolated from spleen and lymph nodes of 

DO.11.10 mice and cultured together with irradiated 

antigen presenting cells (APC), Oligo 1668 peptide, 

anti-IL-4, and IL-2 for 12-14 days. 10 6 OVA-Th1 cells 

were labelled with 0.7 MBq [ 64 Cu]PTSM for 3 hours 

or with 0.7 MBq radiolabelled OVA-TCR-specific 

antibody (KJ1-26), which was linked to [ 64 Cu] via 

the chelator DOTA, for 0.5 hours. Th1 cell viability 

was assessed by trypan blue staining after incubation 

with increasing amounts of activity. Specific 

Th1 cell functioning was analyzed through interferon-gamma 

(ELISA) levels in supernatants of specific 

activated OVA-Th1 cells (T cells + irradiated APC + 

OVA peptide). In vivo T cell migration was investigated 

in an animal model for OVA-induced lung 

inflammation. Mice were sensitized with OVA (i.p.) 

and challenged intranasally twice after four weeks 

to induce OVA-specific lung inflammation. A total 

of 10 7 [ 64 Cu]PTSM or [ 64 Cu]DOTA-KJ1-26 antibody 

labelled OVA-Th1 cells were injected i.p. into diseased 

and healthy mice. Static PET-scans in combination 

with CT, biodistribution, and autoradiography 

were performed 24 and 48 hours after OVA-Th1 

cell transfer. 

Results: In vitro investigations revealed an activity 

dependent impairment of OVA-Th1 viability and 

functionality after [ 64 Cu]PTSM or [ 64 Cu]DOTA- 

KJ1-26 antibody labelling. After a time period of 24 

hours post labelling, the cell viability sunk to 80% 

and functionally was decreased by 20% compared 

to unlabelled control cells. Analyzing OVA-specific 

Th1 cell migration in the mouse model for lung inflammation, 

we detected an accumulation of [ 64 Cu] 

PTSM labelled OVA-Th1 cells in lung tissue and the 

thymus already 24h after the final challenge. Detection 

of [ 64 Cu]DOTA-KJ1-26 antibody labelled OVA- 

Th1 cells was even possible for up to 48 hours. In 

vivo PET data were further confirmed by ex vivo 

biodistribution and autoradiography. Compared to 

[ 64 Cu]PTSM labelled OVA-Th1 cells we gained a 

more defined distribution of extracellularly [ 64 Cu] 

DOTA-KJ1-26 labelled cells in the peritoneum (the 

site of injection), draining lymphatic tissue and the 

sites of OVA-specific lung inflammation. Furthermore 

we detected a significant accumulation of 

OVA-Th1 cells in the omentum majus. 

Conclusions: Both [ 64 Cu]-based labelling methods 

cause an impairment of Th1 cells viability and functionality. 

However, both labelling strategies are applicable 

for in vivo PET-imaging, revealing a detection 

limit of 500 OVA-Th1 cells in draining lymph 

nodes and sites of OVA-specific lung inflammation 

over a time period of 48 hours. 


P-088 

poStEr 


202 


P-089 [ 18 F]DPA-714, [ 18 F]PBR111 and [ 18 F]FEDAA1106: Radiosyntheses on a TRACERLAb FX-FN 

synthesizer 

Kuhnast B. , Damont A. , Demphel S. , Le Helleix S. , Boisgard R. , Tavitian B. , Dollé F. . 

CEA, Orsay, France 

bertrand.kuhnast@cea.fr 

Introduction: Neuroinflammation is involved in 

acute and chronic neurological disorders through 

the activation of microglial cells or the recruitment 

of peripheral macrophages. Both microglia and 

macrophage activation results in the notable overexpression 

on the outer mitochondrial membranes 

of the so-called translocator protein (TSPO 18 kDa), 

supporting today extensive efforts in the design of 

radioligands for the in vivo imaging of this pharmacological 

target by Positron Emission Tomography 

[1]. Of particular interest are DPA-714 [2], PBR111 

[3] and FEDAA1106 [4], three ligands belonging to 

different chemical classes (the pyrazolo[1,5-a]pyrimidineacetamides, 

the imidazo[1,2-a]pyridineacetamides 

and the N-benzyl-N-(2-phenoxyaryl)acetamides, 

respectively) but all designed for a 

labelling with the positron-emitter fluorine-18 via a 

tosyoxy-for-fluorine nucleophilic aliphatic substitution. 

Production of [ 18 F]DPA-714, [ 18 F]PBR111 and 

[ 18 F]FEDAA1106 on the advanced, commercially 

available, automated module TRACERLab TM FX-FN 

synthesizer is presented. 

Methods: The automated process implemented on 

the TRACERLab TM FX-FN synthesizer involves: (A) 

the preparation of the K[ 18 F]F-Kryptofix ® 222 complex 

in two heating steps, first at 60°C for 7 min 

under a stream of N 2 and then at 120°C under reduced 

pressure for 5 min, followed by (B) reaction 

of K[ 18 F]F-Kryptofix ® 222 with the appropriate tosylprecursors 

(4 to 5 mg) at 165°C for 5 min in DMSO 

(0.7 mL), then (C) SepPak ® Plus Alumina N cartridge 

pre-purification after dilution of the reaction mixture 

with HPLC solvent (4 mL) and finally (D) semipreparative 

HPLC purification. HPLC purifications 

were performed on a X-Terra® column for [ 18 F]DPA- 

714 (Solvent : NH 4 OAc 0.1M pH 10 / MeCN : 60/40 

(v/v), Flow rate : 5 mL/min, Rt = 11-12 min), on a 

Symmetry ® C-18 column for [ 18 F]PBR111 (Solvent : 

H 2 O / MeCN / PicB7 : 60/40/2 (v/v/v), Flow rate : 5 

mL/min, Rt = 14-15 min) and on a Symmetry ® C-18 

column for [ 18 F]FEDAA1106 (Solvent : H 2 O / MeCN 

/ TFA : 60/40/0.1 (v/v/v), Flow rate : 5 mL/min, Rt = 

21-22 min). The HPLC-collected fraction containing 

pure [ 18 F]DPA-714, [ 18 F]PBR111 or [ 18 F]FEDAA1106 

imaging life 

were automatically formulated using a SepPak ® Plus 

C-18 cartridge ((i) HPLC-collected fraction dilution 

and loading on the cartridge, (ii) cartridge washing 

with 10 mL of water, (iii) cartridge elution with 2 mL 

of EtOH, and (iv) final dilution with 8 mL of saline). 

The process was programmed on the TRACERLab TM 

FX-FN synthesizer in one single “method” divided 

in three “time-lists”. 

Results: Starting from a 37.0 GBq cyclotron-produced 

[ 18 F]fluoride batch, 6.7 to 8.5 GBq (18-23% 

non decay corrected yields) of [ 18 F]DPA-714 or [ 18 F] 

PBR111 or [ 18 F]FEDAA1106, > 99% radiochemically 

pure and ready-to-inject, were obtained within 50- 

60 min. The overall decay corrected radiochemical 

yield reached up to 30%. Specific radioactivities 

ranged from 74 to 222 GBq/µmol (2-6 Ci/µmol). 

Conclusions: Radiosynthesis of [ 18 F]DPA-714, [ 18 F] 

PBR111 and [ 18 F]FEDAA1106 has been successfully 

implemented on a TRACERLab TM FX-FN 

synthesizer. 

Acknowledgements: Supported in part by the EC - 

FP6-project DiMI (LSHB-CT-2005-512146) and 

EMIL (LSH-2004-503569). 

References: 

1. Dollé F et al; Curr Med Chem 16: 2899-2923 (2009) 

2. Damont A et al; J label Compds Radiopharm 51: 286- 

292 (2008) 

3. Dollé F et al; J label Compds Radiopharm 51: 435-439 

(2008) 

4. Zhang MR et al; J Med Chem 49: 2735-2742 (2006)


In vivo near-infrared fluorescence imaging of lung matrix metalloproteinases in an acute 

cigarette smoke-induced airway inflammation model in different mice strains 

Perez-Rial S., Del Puerto-Nevado L., Giron-Martinez A., Gonzalez-Mangado N., Peces-Barba G. . 

Instituto de Investigacion Sanitaria-Fundacion Jimenez Diaz-CIBERES, Madrid, Spain 

sperezr@fjd.es 

Introduction: Recent studies have suggested that 

pulmonary macrophage-derived metalloproteases 

(MMPs) are the critical mediators of cigarette smoke 

exposure (CSE)-induced emphysema, but it has never 

been seen via imaging techniques [1] . The purpose 

of this study was to visualize and quantify acute pulmonary 

inflammation by real-time near-infrared 

fluorescence (NIRF)-mediated molecular imaging 

[2] in the lung tissue evaluating MMPs activity in response 

to CSE in mice strains with different susceptibility 

to develop smoking-induced emphysema. 

Methods: To accomplish this task, susceptible 

(C57BL/6j) and resistant (129S2/SvHsd) mice [3] 

were exposed to acute CSE using a whole-body exposition. 

24h after CSE MMPs activity was assessed 

via optical imaging system by a MMPs-sensitive activatable 

fluorescence probe in order to characterize 

the distinctive profile of CSE-induced acute inflammation. 

Furthermore MMPs protein levels in the 

lung tissue were analyzed by Western blot analysis. 

Results: In vivo semiquan- in vivo lung imaging 

titative optical imaging 

analysis of MMPs activity 

in the lung revealed 

increased acute-CSE-associated 

MMPs activity in 

C57BL/6j but not in 129S2/ 

SvHsd. Only in susceptible 

mice most important 

MMPs protein levels were 

significantly increased in the lung tissue of smokers 

compared with the non-smokers group. 

Conclusions: Optical imaging via NIRF offers a simple, 

effective, and rapid technique for noninvasive 

monitoring and semiquantitative analysis of lung 

inflammation and MMPs expression. We are able to 

distinguish between susceptible and resistant mice 

strains in terms of the profile of MMPs activity in 

the early stages of pulmonary disease. The results of 

our study suggest that mechanisms underlying different 

susceptibilities to CSE can be detected at the 

beginning of the disorder. They produce different 

response to acute tobacco smoke. 

Acknowledgement: This work is supported in part by the 

Spanish “Ministerio de Ciencia e Innovación” (SAF2008- 

05412-C02- 02) and CIBERES (CB06/06/0009). 

References: 

1. Churg A et al; Am J Respir Crit Care Med. 167(8):1083-9 

(2003) 

2. Haller J et al; J Appl Physiol. 104(3):795-802 (2008) 

3. Morris A et al; J Pharmacol Exp Ther. 327(3):851-62 

(2008) 


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204 


P-091 Development and pre-clinical evaluation of a novel class of 18 F labelled PET ligands for 

evaluation of PBR/TSPO in the brain 

Trigg W. , Ahmad R. , Arstad E. , Avory M. , Hirani E. , Jones P. , Khan I. , Luthra S. , Morisson-Iveson V. , O’shea D. , 

Passmore J. . 

GE Healthcare, Amersham, United Kingdom 

william.trigg@ge.com 

Introduction: The peripheral benzodiazepine receptor 

(PBR; otherwise known as TSPO (18kDa)) is a 

well-established target for imaging activated microglial 

cells and macrophages in inflammatory diseases 

such as MS, Alzheimer’s disease and a wide range 

of both peripheral and brain diseases 1 . Starting from 

a core tetracyclic indole pharmacophore 2 , which displayed 

high affinity for the PBR, we have designed 

a series of molecules to assess the SAR around the 

pharmacophore and to determine the best site to introduce 

the radiolabel. 

Methods: Compounds have been assessed for PBR 

affinity in a radioligand binding assay and in a range 

Compound (Ki) 

[ 11C]PK11195 (1.24nM) 

[ 18F]AH114011 (0.37nM) 

[ 18F]AH114629 (0.40nM) 

imaging life 

Initial brain 

uptake (%ID/g) 

OB @ 2 min 

(%ID/g) 

* Whole brain value not calculated 

** [3H]PK11195 used for autoradiography studies 

OB @ 30 

min 

(%ID/g) 

2:30 Striatum 

ratio 

OB : Striatum 

ratio 

(30 min) 

Metabolism 

Profile - % Parent 

in brain at 60 

min p.i. 

FNA Model lesion 

: non lesion ratio 

(in vitro) 

0.28-0.48* 0.42 0.21 3.20 2.10 100 2.8** 

0.32 0.39 0.31 1.73 2.07 96.0 2.3 

0.42 0.51 0.28 4.75 3.50 97.6 NT 

of in vitro ADME assays. Following on from the in 

vitro assessments, promising compounds were radiolabelled 

and assessed in vivo for biodistribution 

and metabolism profiles. The compounds with the 

most appropriate profiles were assessed using autoradiography 

in the Facial Nerve Axotomy (FNA) 

model described by Banati et al. 3 

The corresponding data for the archetypal PBR ligand 

PK11195 was generated for comparison. 

Results: The olfactory bulb (OB) has high expression 

of PBR and is used to measure specific uptake, 

with the striatum used as a low expression area for 

comparative purposes. Key data obtained are summarised 

in the table below. 

Conclusions: Preclinical evaluation of the 18F labelled 

tetracyclic indole class of ligands for PBR 

shows that this class of molecules represents a 

promising class of PET ligands that should be further 

evaluated. 

References: 

1. Cagnin et al., Neurotherapeutics, Vol. 4, No. 3, 443-452 

(2007) 

2. Okubo et al., Bioorg Med. Chem. 12(2):423-438 (2004) 

3. Banati et al., J. Neurocytol. 26:77-82. (1997)


Local administration of adeno-associated virus into the mammary gland ductules 

Hamm J. (1) , Cojoca R. (1) , Iezzi M. (2) , Mautino A. (1) , Turco E. (1) , Silengo L. (1) , Musiani P. (2) , Forni G. (1) . 

(1) University of Turin, Italy 

(2) University of Chieti Pescara, Italy 

jorg_hamm@yahoo.com 

Introduction: Adeno-associated virus (AAV) is a 

single-stranded DNA parvovirus with charateristics 

that render it attractive for gene therapy applications 

(1). More than 14 AAV serotypes have 

been characterized which differ in efficiency of 

infection and tissue tropisms. Cell entry seems to 

occur by AAV receptor mediated endocytosis, the 

receptors recognised by the capsid proteins vary in 

different serotypes and might be an determinant 

for tissue tropism. The DNA remains mainly extrachromosomal, 

does not replicate, and is unable to 

produce infectious particles, features desirable for 

gene therapy vectors. In the majority of the applications 

AAV2 vector is used for gene transfer experiments, 

but other serotypes have been explored 

recently because they can offer superior transduction 

efficiencies or different cell tropisms. Furthermore, 

neutralising antibodies against AAV2 

are frequently found in human sera and can compromise 

efficiency of gene transfer. AAV9 has been 

reported to allow a robust expression in heart and 

muscle, but other expression sites have also been 

indicated. 

Methods: AAV9 vectors expressing luciferase as a reporter 

gene driven by the cytomegalo virus promoter 

(AAV9-CMV-luc) or by the murine EF1alpha promoter 

(AAV9-EF1a-luc) were administered locally into 

the mammary gland network by intraductal injection 

(2) into BALB/c female mice. Expression profile and 

body distribution were determined by in vivo and 

ex vivo optical imaging, and by histological analysis. 

Results: AAV9-CMV-luc migrates from the injected 

gland to ipsilateral glands, but not to contralateral 

glands. Luciferase expression is observed predominantly 

in muscle cells and is stable for months. The 

observed expression of luciferase in the ipsilateral 

lymph nodes indicate that the virus might use the 

lymphatic system to reach all of the five ipsilateral 

mammary glands. 

Conclusions: AAV9 vectors appear to be well suited 

for targeted, long-lasting gene expression in muscle 

tissue. Mammary epithelia cells are apparently not 

infected efficiently, not even after site specific injection 

into the duct of mammary glands. 

References: 

1. Büning et al.; J Gene Med 2008; 10: 717–733. 

2. Murata et al.; Cancer Res. 2006 Jan 15;66(2):638-45. 


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P-093 Copper-64- and Gallium-68- NODAGA-conjugated bombesin antagonists as new PET tracers 

Mansi R. , Dumont R. , Jamous M. , Tamma M. , Nicolas G. , Fani M. , Cescato R. , Reubi J.C. , Weber W. , Maecke H. . 

Nuclear Medicine, Freiburg, Germany 

rosalba.mansi@uniklinik-freiburg.de 

Introduction: The gastrin-releasing peptide or 

bombesin receptor (GRPr) is frequently overexpressed 

in several human cancers including prostate, 

breast, small cell lung and pancreatic cancer 

[1,2]. Specific radiolabeled ligand for this receptor 

offer new opportunities for the diagnosis and 

treatment of these diseases. Many bombesin antagonists 

have shown high affinity for the GRPr 

[3]. In this study, we evaluated the potential of a 

statine-based bombesin antagonist, conjugated to 

NODAGA through a polyethyleneglycol spacer, to 

specifically target GRPr expressing cancer cells. 

We determine the effect of two different radioisotopes 

Ga-68 and Cu-64 on the tumor targeting efficacy 

and in vivo pharmacokinetics. Moreover we 

evaluated the PET imaging properties of the two 

radiotracers. 

Methods: The peptide assembling and NODAGAconjugation 

were accomplished on solid phase. The 

conjugate MJ5 was radiolabeled with Ga-68 and 

Cu-64 and the radioconjugates were evaluated in 

vitro and in vivo in tumor-bearing nude mice, using 

the GRP-receptor positive prostate carcinoma 

cell line PC-3. To study the bombesin antagonistic 

properties of MJ5 cells were treated either with 10 

nmol/L bombesin, or with 1 µmol/L MJ5 or, with 

10 nmol/L bombesin in the presence of a 100-fold 

excess MJ5 for 30 min at 37°C. GRPr internalization 

was studied by immunofluorescence. 

Results: The immunofluorescence assays confirmed 

the strong antagonist properties of the conjugates: MJ5 

failed to induce significant internalisation of GRPr and 

when given at a concentration of 1 µmol/L together with 

10 nmol/L bombesin, MJ5 was able to antagonize bombesin-induced 

receptor internalisation. Biodistribution 

studies revealed high and specific uptake of both conjugates 

in PC-3 tumors and in GRPr positive tissues such 

as pancreas and intestine. The Ga-68-MJ5 biodistribution 

data showed a good tumor uptake at 1h (4.54±0.52% 

I.A./g) and good tumor to kidney and tumor to blood 

ratios (4.1 and 10.9 respectively). These promising data 

prompted us to investigate the biodistribution of MJ5 

with a longer lived radionuclide. The tumor uptake of 

the Cu-64-MJ5 was 3.7 times higher than the Ga-68labeled 

analog at 1h (16.80±3.22% I.A./g) and remained 

imaging life 

high at 4h (13.97±3.68% I.A./g). At 4 h higher tumor to 

kidney (8.6), tumor to blood (36.2) and tumor to pancreas 

(18.2) ratios were achieved than for Ga-68-MJ5 at 

1 h. The PET/CT images demonstrated exellent visualization 

of the PC3 tumors by Ga-68- and Cu-64 MJ5. 

Conclusions: The labeling with the two radionuclides 

distinctly influences the pharmakokinetics of the radiopeptides 

in particular in regard to the uptake in 

the tumor and receptor positive organs. The high 

tumor uptake and the good tumor to background 

ratio of the Cu-64 labeled analog at 4h warrant clinical 

testing of this probe for PET imaging in humans. 

Acknowledgement: We acknowledge COSTD38 and 

the Swiss National Science Foundation for the financial 

support. 

References: 

1. R.Markwalder, J.C. Reubi, Cancer Res., 155 (1999) 1152. 

2. J.C. Reubi, M. Korner, et al., Eur.J.Nucl.Med.Mol.Imaging, 

31 (2004) 803 

3. R.T. Jensen, D.H. Coy, Trends Pharmacol. Sci., 12 (1991) 13


A novel indocyanine green nanoparticle probe for non invasive fluorescence imaging in vivo 

Mérian J. (1) , Navarro F. (1) , Josserand V. (2) , Texier I. (1) . 

(1) CEA LETI MINATEC, Grenoble, France 

(2) CRI INSERM U823, France 

juliette.merian@cea.fr 

Introduction: Fluorescence imaging, a non invasive, 

non ionizing and sensitive technology, is opening a 

new era in medical application such as image-guided 

surgery. Indocyanine Green (ICG), the only Near InfraRed 

optical contrast agent approved by the FDA 

[1], is encapsulated in lipidic nanoparticles (LNP). 

Using LNP as nanocargo presents advantages such as 

improvement of the dye chemical stability, biocompatibility 

and passive targeting, as well as suitable 

properties for fluorescence detection of tumors [2]. 

Methods: LNP are composed of a lipid core, stabilized 

by phospholipids and pegylated surfactants and are 

dispersed in aqueous buffer. ICG (Infracyanine) is 

incorporated into the lipid mixture as a concentrated 

solution in ethanol. Solvent is evaporated and this 

oily phase is mixed with aqueous phase before sonication 

is performed at 40°C for a whole 5min period 

(VCX750 Ultrasonic processor). Particle size, size 

distribution, and surface charges are evaluated by 

dynamic light scattering (DLS). Optical properties 

Nano ICG 

Free ICG 

characterizations are performed using visible absorbance 

and fluorescence spectrometries. MTT assay 

is performed using 3T3 fibroblasts to assess LNP cytotoxic 

index. The in vivo distribution of ICG-loaded 

LNP is investigated in Nude mice bearing xenografted 

sub-cutaneous TS/Apc tumors using a NIR 

whole animal imaging device (Fluobeam 800), and 

compared to that of free ICG in aqueous solution. 

Results: ICG loading does not modify the size (30nm 

diameter, one population) and the polydispersity index 

(from 0.11 to 0.13) of the LNP, but decreases 

the zeta potential, which becomes more negative 

(-5.8±2.5mV for nude LNP, versus -18.2±2.1mV for 

ICG loaded LNP). The dye encapsulation within 

LNP induces a 16nm red-shift of its absorption and 

emission (maximum at 820nm); as well as an improvement 

of its fluorescence quantum yield and a 

longer lifetime in comparison to free dye in water. 

LNP carrier is well tolerated in vitro (IC50~1 mg/mL 

of lipids). Furthermore, ICG loaded LNP injection 

in Nude mice implanted with subcutaneous Ts/Aps 

tumors, shows a better fluorescence signal in tumor 

site in comparison of free ICG at the same dose. 

Conclusions: LNP vectorization provides ICG a 

modification of its pharmacokinetics in addition 

to an improvement of its optical properties. These 

characteristics are suitable for long term and sensitive 

in vivo imaging required for efficient tumor 

detection. ICG loaded LNP, thanks to their low size, 

takes advantages of the Enhanced Permeability and 

Retention (EPR) effect and are passively accumulated 

with time into the tumor. 

Acknowledgement: J.Mérian1, F. Navarro 1 , V. Josserand 2 , 

I. Texier 1 : 1CEA LETI Minatec, 2 CRI-INSERM U823 

References: 

1. Frangioni, J.V. In vivo near infrared fluorescence 

imaging. Curr. Opinion Chem. Biol. 7, 2003:626-634 

2. Texier, I. Goutayer, M. and al Journal of Biomedical 

Optics, 14(5), 2009: 054005 


P-094 

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TARGETED THERAPY


P-095 Evaluation and optimization of the concept of an antibody directed enzyme-prodrug therapy 

using noninvasive imaging technologies 

208 

Napp J. (1) , Dullin C. (2) , Krewer B. (3) , Von Hof J.M. (3) , Schmuck K. (3) , Mathejczyk J. (4) , Hartung F. (4) , Stühmer W. (4) , Tietze L. F. (3) , 

Alves F. (4) , Pardo L. A. (4) . 

(1) MI for exp Medicine, Göttingen, Germany 

(2) University Medicine Göttingen, Germany 

(3) University Göttingen, Germany 

(4) MPI for exp Medicine, Göttingen, Germany 

jnowako1@gwdg.de 

Introduction: Relatively low selectivity to tumor versus 

normal cells challenges virtually all cancer chemotherapies. 

Site-specific activation of prodrugs in 

tumors is one strategy to achieve high efficacy and 

specificity of treatment, decreasing toxicity in normal 

tissues. Here, we present the design and validation 

of an Antibody Directed Enzyme-Prodrug Therapy 

(ADEPT) in which an antibody against Eag1 is used 

to carry the drug-activating enzyme, ß-galactosiase 

(ß-gal) to the tumor tissue. Eag1 (ether-à-go-go1) 

voltage-gated potassium channel has been chosen as 

a tumor-specific target since this plasma-membrane 

protein is easily accessible to extracellular interventions 

and Eag1 is aberrantly expressed (>75%) in tumors 

from diverse origin but basically not detected 

in healthy tissue outside the central nervous system. 

Methods: Two monoclonal anti-Eag1 antibodies, 

mAb62 and mAb56, a humanized 56 antibody, as 

well as a single chain fragment of the mAb62, scFv62, 

were tested for their feasibility to deliver the drugactivating 

enzyme ß-gal to the tumor. Near infrared 

fluorescence (NIRF) imaging was used to study 

the biodistribution and binding characteristics of 

the anti-Eag1 antibodies in vivo in a subcutaneous 

Eag1-expressing MDA-MB-435S tumor model in 

nude mice. Fluorescence intensity, lifetime and location 

of Cy5.5 labeled antibodies in vivo was measured 

with the time-domain NIRF imager, Optix 

MX2 (ART, Advanced Research Technologies; Canada), 

at certain time points. Fluorescence lifetime 

(the average time during the molecule stays in its excited 

state) was used to discriminate between nonspecific 

and probe-derived signals. Distribution of 

the fluorescent probe in tumor sections ex vivo was 

further investigated by the Odyssey infrared imaging 

system (LI-COR Biosciences, Germany) as well 

as by NIRF microscopy (Axiovert 200M, Carl Zeiss, 

Germany). For the tumor-specific activation of the 

prodrug, the mAb62 was conjugated to ß-gal, resulting 

in 62-gal. The ß-gal activity of the 62-gal conjugate 

and its ability to bind to the Eag1 epitope were 

tested in vitro on Eag1-expressing MDA-MB-435S 

cells and control AsPC-1 cells using a colorimetric 

CPRG (chlorophenolred-ß-D-galactopyranoside) 

imaging life 

assay. The ß-gal activity in mice was analyzed by 

NIRF imaging using the fluorescent activatable probe, 

DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one- 

7-yl) β-d-galactopyranoside) by NIRF imaging. 

Results: All tested antibodies targeting Eag1 bound 

specifically to MDA-MB-435S tumors with maximal 

intensity peaks at 24-48 h after application; fluorescence 

was still detectable for at least 1 week in vivo. 

We confirmed specific binding of the antibodies to 

the tumors by ex vivo NIRF imaging of tumors isolated 

from mice injected with fluorescently labeled 

antibodies 24 h prior to section, as well as by NIRF 

microscopy of tumor slices. Moreover, we show that 

monoclonal antibodies against Eag1, but not the 

scFv62 fragment, resulted in strong fluorescence 

signals in the area over liver, detectable for at least 4 

days in vivo. Similar fluorescence signals over liver 

were also observed in tumor-bearing mice injected 

with Cy5.5-labeled control IgGĸ2B, confirming that 

the liver signals did not result from Eag1-mediated 

binding of those antibodies to cells within liver. 

Furthermore, we show that the 62-gal conjugate 

specifically binds in vitro to Eag1-expressing MDA- 

MB-435S, but not to control Eag1-non expressing 

AsPC-1 cells and that 62-gal possess high ß-gal activity, 

when bound to MDA-MB-435S cells. Moreover, 

24 h after application of the 62-gal to the tumor 

bearing mice we detected ß-gal activity in vivo over 

the tumor area. 

Conclusions: Here, we successfully applied NIRF 

imaging to evaluate anti-Eag1 antibodies as tools for 

a novel concept of targeted cancer therapy. Since in 

vivo 62-gal 1) specifically binds to Eag1-expressing 

tumors and 2) shows measurable ß-gal activity at the 

tumor site, this conjugate can further be applied in 

the ADEPT for specific activation of cytotoxic prodrugs 

at the tumor site.


A novel [ 18 F] PET imaging agent for the epidermal growth factor receptor 

Pisaneschi F. (1) , Shamsaei E. (1) , Nguyen Q.D. (1) , Glaser M. (2) , Robins E. (2) , Kaliszczak M. (1) , Smith G. (1) , Spivey A.C. (1) , 

Aboagye E.O. (1) . 

(1) Imperial College London, London, United Kingdom 

(2) Medical Diagnostic Discovery (part of GE Healthcare), United Kingdom 

f.pisaneschi@imperial.ac.uk 

Introduction: The Epidermal Growth Factor Receptor 

(EGFR/c-ErbB1/HER1) is overexpressed in 

many cancers including breast, ovarian, endometrial 

and non-small cell lung cancer. 1, 2 An EGFR specific 

imaging agent could facilitate clinical evaluation of 

primary tumours and/or metastases. 

Methods: We designed and synthesized a small array 

of fluorine containing compounds based on 

a 3-cyanoquinoline core. 3 The compounds were 

screened in vitro for their affinity in cell-free EGFR 

autophosphorylation assay and for their activity in 

the inhibition of the EGFR tyrosine kinase in EGFR 

overexpressing A431 cell lines. 

Results: A lead compound, incorporating 2’-fluoroethyl-1,2,3-triazole 

was selected for evaluation as a 

radioligand based on its high affinity for EGFR kinase 

(IC 50 = 1.81 ± 0.18 nM), good cellular potency 

(IC 50 = 21.97 ± 9.06 nM), low lipophilicity and good 

metabolic stability. ‘Click’ labelling 4 afforded [ 18 F]- 

2’-fluoroethyl-1,2,3-triazole derivative in 7% end 

of synthesis (EOS) yield from aqueous fluoride in a 

total synthesis time of 3 h and >99% radiochemical 

purity. The compound showed good stability in vivo 

and a 4-fold higher uptake in A431 tumour xenografts 

relative to muscle. Furthermore, the radiotracer 

could be visualized in A431 tumour bearing mice 

by small animal PET imaging (NUV 60 = 0.13±0.02). 

Conclusions: The novel [ 18 F]-imaging agent constitutes 

a promising radiotracer for further evaluation 

for imaging of EGFR status. 

Acknowledgement: This work was funded by Cancer 

Research UK programme grant C2536/A7602 and 

UK Medical Research Council core funding grant 

U.1200.02.005.00001.01. 

References: 

Bazley, L. A.; Gullick, W. J. Endoncr. Relat. Cancer 2005, 12, 

S17. 

Marmor, M. D.; Skaria, K. B.; Yarden, Y. Int. J. Radiat. Oncol. 

Biol. Phys. 2004, 58, 903. 

Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, 

B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; 

Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; 

Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, 

H.-R. J. Med. Chem. 2003, 46, 49. 

Glaser, M.; Årstad, E. Bioconjugate Chem. 2007, 18, 989. 


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P-097 Structural methods in subcellular image analysis 

Dietlmeier J. , Whelan P.F. . 

Centre for Image Processing and Analysis, Dublin, Ireland 

julia.dietlmeier@ieee.org 

Introduction: From a computer vision perspective 

subcellular imaging is an extremely complex and dynamic 

environment as can be inferred from Fig.1(a). 

Mitochondria form an important category of membrane 

enclosed, on average 200nm large organelles 

which reside inside every living cell. Mitochondrial 

morphology is crucial to the understanding of 

apoptosis mechanisms and the subsequent development 

of therapies targeting age- and cancer-related 

diseases[1],[2]. There is a high demand in automated 

segmentation which can provide an objective quantitative 

information in a reasonable time frame[2]. 

However, the state-of-the-art is dominated by manual 

tools. Early attempts to automate the segmentation 

are based on the machine-learning framework[3]. 

Methods: Instead of pursuing the machine-learning 

pathway we approach the segmentation and localization 

problem from the structural point of view which 

also has an appealing theoretical aspect. Here, we 

apply the well developed and widely acknowledged 

framework of Mathematical Morphology. Our multistage 

segmentation targets (i) specific challenges 

of image acquisition such as low contrast, non-uniform 

illumination and speckle noise; (ii) structural 

challenges such as cluttered scene, clustered and deformed 

mitochondria and deformed cristae. 

Results: We outline our preliminary results by examining 

Fig.1. The first and the key step is localization 

which is based on the analysis of mitochondrial 

morphology. After extracting localiza tion markers 

we apply a seeded region grow algorithm which fills 

the inner-membrane space of each organelle, as can 

be seen in Fig.1(e). Gap-free control boundary is 

currently achieved by using morphological thickening 

and thinning operators on the binary image. In 

order to separate clustered mitochon-dria we apply a 

seeded watershed algorithm. The results of extracted 

sha-pes are provided in Fig.1(f)-(g). We verify that 

our segmentation works well on small-scale images 

containing a small number of mitochondria as can 

be seen in Fig.1(f)-(l). The other technique to obtain 

mitochondrial contour is recon-struction-by-dilation. 

Here, we correlate the localization markers with 

extracted contours in order to yield correct result. 

imaging life 

Fig.1 (a) TEM image of STS-treated DU-145 prostate cell. (b). Example 

of two clustered mitochondria. (c)-(l) Selected segmentation results. 

Conclusions: The major challenge is firstly to extract 

the mitochondrial shape within reasonable 

accuracy. Secondly, we attempt to expand our detection 

approach to a larger scale problem such as 

the whole cell image. We plan to validate this on a 

larger representative mitochondria image database 

and in doing so compare our approach to existing 

machine-learning algorithms. 

Acknowledgement: This research was supported by 

NBIP Ireland funded under the Higher Education 

Authority PRTLI Cycle 4, co-funded by the Irish 

Government and the European Union - Investing in 

your future. Special thanks to Royal College of Surgeons 

in Ireland (RCSI) for sample preparation and 

acquisition of TEM images. We would like to thank 

American Society for Cell Biology (ASCB) and Nature 

Publishing Group3 for the permission to use 

selected micrographs. 

References: 

1. Perkins GA et al; Methods in Enzymology. 456:29–52 

(2009) 

2. Sun MG et al; Nature Cell Biology. 9:1057–1065 (2007) 

3. Narasimha R et al; Pattern Recognition. 42:1067–1079 

(2009)


In vivo-post mortem multimodal image registration in a rat glioma model 

Dubois A. , Boisgard R. , Jego B. , Lebenberg J. , Hérard A. S. , Dollé F. , Lebon V. , Delzescaux T. , Tavitian B. . 

CEA-I2BM, Orsay, France 

albertine.dubois@cea.fr 

Introduction: Histological staining techniques can 

identify the regulation of specific biomarkers in 

small animal models of glioma and provide indicators 

of cellular dysfunction. The correlation of in 

vivo imaging signal changes and molecular indicators 

of tissue damage in histological brain sections 

represents an important means of understanding the 

cellular mechanisms responsible for these changes 

under pathological conditions. This requires an accurate 

image registration that can compensate the 

distortions that occur in the brain during the extraction, 

fixation, and staining process. Here, we present 

the preliminary results obtained by applying an 

overall registration strategy for the fusion of in vivo 

PET and MRI data with post mortem brain images in 

a rat 9L-glioma model. 

Methods: Experiments were conducted on male 

Wistar rats. Twelve days after intrastriatal injection 

of 9L rat glioma cells, we acquired T2-weighted MRI 

and 60 min dynamic PET using [18F]DPA-714 [1] 

for in vivo evaluation of peripheral benzodiazepine 

receptor (PBR) expression. Following in vivo imaging, 

animals were euthanized. The entire brains were 

cut into 20 µm-thick coronal sections and processed 

for H&E staining and PBR immunohistochemistry. 

A blockface photograph was also recorded prior to 

each section. The overall co-registration strategy relied 

on using the blockface photographs as an intermediate 

reference, onto which the in vivo MRI and 

PET images and the digitized histo- and immunohistochemical 

brain sections were registered separately 

and superimposed so as to obtain in vivo-post 

mortem registration [2;3]. 

Results: By way of feasibility study, the co-registration 

strategy was only performed on one rat’s right 

hemisphere. As illustrated in Figure 1, after each registration 

task, both the external contours, the outer 

edges of the cortex and inner structures such as the 

corpus callosum, the hippocampus, the striatum and 

even the tumor were correctly superimposed whatever 

the modality. 

Conclusions: We obtained promising registration 

results of T2-weighted MRI and [18F]DPA-714 PET 

with post mortem brain volumes. Once results from 

additional animals had been provided, our approach 

could be used to evaluate, quantify and compare 

tumor volume, pharmacokinetic and physiological 

parameters (e.g. ligand differential uptake areas 

Fig1: Fusion of (A) PET (rainbow) 

and MRI (black and 

white), (B) Blockface (black 

and white) and H&E (pink) 

volumes, (C) MRI (black 

and white) and blockface 

volume (rainbow), (D) MRI 

(black and white) and H&E 

volume (pink) and (E) PET 

(rainbow) and H&E volume 

(black and white) 

within the tumor) measured from in vivo MRI and 

PET data with those derived from corresponding 

post mortem histo- and immunohistochemistry. 

References: 

1. Damont A et al; J label Compds Radiopharm. 51 (7): 

286-292 (2008) 

2. Dauguet J et al; J Neurosci Methods. 164 (1): 191–204 

(2007) 

3. Lebenberg J et al; Neuroimage. In press (2010) 


P-098 

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MI DATA ANALYSIS METHODS

212 


P-099 Feasibility and success of Independent Component Analysis of resting state fmri data from 

the rat 

Jonckers E. , Van Auderkerke J. , Van Der Linden A. , Verhoye M. . 

Bio-Imaging Lab, Sint Lenaarts, Belgium 

elisabeth.jonckers@ua.ac.be 

Introduction: Resting state fMRI is used with growing 

interest with the purpose to spot age1 or pathology2 

induced abnormalities in functional connectivity 

(FC) in the human brain. Only recently resting state 

FC measurements were acquired in rats3, opening the 

potential of assessing FC in the rat brain in several 

imaging life 

Fig 1: 3 Components resulting from ICA of rat resting state data. Colors representing z-values, higher z-values 

meaning a higher convergence between time course of the voxel and mean time course for the component. 

neuropathologies (e.g. schizophrenia). The human 

resting state networks are identified using a seed-based 

approach or independent component analysis (ICA)4. 

Since ICA analysis of resting state fMRI of rodents is 

up to now not published, we performed a pilot study, 

implementing the technique in rodents and testing 

the reproducibility of the ICA outcome. 

Methods: A group of 5 male rats was imaged 4 times. 

On 2 time points with one week in between 2 consecutive 

resting state data were acquired using single 

shot gradient echo EPI. Rats were anesthetized with 

Medetomidine. Imaging was done on a 9.4T scanner 

using TR 2s and TE 16ms. 12 slices of 1 mm were 

acquired with a FOV of (3x3) cm and matrix size of 

128x128. ICA was implemented on the dataset using 

GIFT (Group ICA of fMRI toolbox), working in matlab2008. 

The number of components for the ICA of 

the individual subjects or group (5 rats x 4 repetitions) 

was set at 15. 

Results: The outcome of our ICA analysis, clearly 

revealed different brain regions within 10 of the 15 

components. Figure 1 demonstrates 3 components of 

clearly functional different cortical regions. 

Each ICA component was evaluated over the different 

time points. Mean components (for the 5 animals) 

were very reproducible both for measurements on the 

same day as measurements with one week in between. 

Also single subject components were similar over time. 

Although the differences are bigger between measurements 

with one week in between, indicating that there 

could be an influence of scanner variability and physiological 

state of the animal. 

Conclusions: This study implementing ICA on rat 

resting state data proves the usability of the technique 

for studying FC in the rat brain in a reproducible 

way. This paves the way to use this tool for assessing 

FC changes under different circumstances 

such as both physiological changes and pathologies. 

Implementing the technique in animal models 

can help to unravel the underlying processes. 

References: 

1. Damoiseaux JS et al (2008) Cereb Cortex. 18(8), 1856- 

1864. 

2. Greicius MD et al (2004) Proc Natl Acad Sci U S A. 101(13), 

4637-4642. 

3. Pawela CP et al (2008) Magn Reson Med. 59(5), 1021-1029. 

4. Beckmann CF et al (2005) Philos Trans R Soc Lond B 

Biol Sci. 360(1457), 1001-1013.


A fast and robust acquisition scheme for CEST experiments 

Longo D. , Cittadino E. , Terreno E. , Aime S. . 

University of Torino, Italy 

dario.longo@unito.it 

Introduction: Chemical Exchange Saturation Transfer 

(CEST) agents are a new class of MRI contrast 

agent based on the selective irradiation of a mobile 

proton pool in slow/intermediate exchange with 

water [1] . Long irradiation pulses at the resonant frequency 

(∆ω) are needed to obtain a complete saturation 

of the CEST pool and a correct saturation transfer 

(ST%) quantification requires the acquisition 

of many images at different frequency offsets (Zspectrum), 

thus making a CEST experiment a timeconsuming 

procedure depending on the number of 

sampled frequency offsets [2] . A trade-off between 

the number of frequency offsets and the accuracy of 

ST% determination has to be found in order to distinguish 

the characteristic ST% time course for individual 

voxels. The development of fast and robust 

methods are therefore mandatory to correctly detect 

CEST molecules in Molecular Imaging applications. 

In this work a computational procedure has been 

developed to evaluate the accuracy of ST% quantification 

as a function of the number of frequency offsets 

centered around the resonant frequency of the 

mobile proton pool with different levels of noise and 

of B 0 inhomogeneities. We also proposed a different 

acquisition procedure, by acquiring first a complete 

Z-spectrum followed by the sampling of a limited 

number of frequency offsets and then replacing 

these points in the first Z-spectrum, thus improving 

the temporal resolution of CEST contrast quantification, 

without sacrifying the ST% accuracy. 

Methods: Z-spectra were simulated by using a twopool 

model solving the Bloch equation (T 1A = 2 s, 

T 2A = 0.1 s, T 1B = 1 s, T 2B = 0.015 s, ∆ω = 4.2 ppm, k ex 

= 160 s -1 , 30 mM diacest agent, saturation pulse 3 

μT for 5 s). Gaussian noise was added both in the 

signal intensities and in the frequency axis and zero 

shifts were introduced in the range 0.1-1 ppm. Zspectra 

were interpolated by smoothing spline (with 

zero shift correction) by different number of points 

around the resonant frequency (with step of 0.1 ppm) 

both at positive (∆ω) and negative offsets (-∆ω): i) 7 

points, ii) 5 points, iii) 3 points, iv) 3 points with 0.2 

ppm step, v) 1-(∆ω/-∆ω) (ON/OFF scheme) with 

and vi) without zero-shift correction, compared to 

a full Z-spectrum in the range ±10 ppm with steps 

of 0.1 ppm. In in vivo CEST images were acquired 

on a 7 Tesla Bruker spectrometer with a complete 

Z-spectrum (single-shot RARE spin-echo, TR 6 s, 

centric encoding, MTX 64, saturation pulse: 3 μT 

for 5 s) followed, upon injection of a CEST agent by 

the dynamic acquisition of only 3 points around the 

resonant frequency (∆ω = 4.2 ppm) for 30 min (50 

scans, temporal resolution: 36 s). 

Results: The ON/OFF scheme with and without zero-shift 

correction showed the highest mean ST errors, 

up to 2 % and 5%, respectively and with high 

standard deviation values. The method iv) with 3 

points spaced of 0.2 ppm, in comparison with the 

more accurate but also time consuming i) and ii) 

methods, displayed mean ST errors lower than 1%, 

but with a drastic reduction in acquisition time. In 

in vivo the continous acquisition of only 3 frequency 

offsets around the resonant frequency and the correction 

for zero shifts by using the pre-contrast Zspectrum 

allowed to evaluate the ST% time course 

with a temporal resolution of few seconds and to 

discriminate between enhancing and not-enhancing 

pixels in kidneys. 

Conclusions: The acquisition of 3 points spaced of 0.2 

ppm around the resonant frequency of the mobile proton 

pool resulted to be sufficient enough to provide a robust 

and accurate ST% quantification, with a fast temporal 

resolution by using a modified Z-spectrum acquisition. 

Acknowledgement: Economic support from regional 

government ( NanoIGT project - CIPE 2007) and 

EC-FP7 project ( ENCITE: FP7-HEALTH-2007A). 

References: 

1. Ward KM, et al.; J Magn Reson. 2000; 143: 79 

2. Liu G, et al.; Magn Reson Med. 2009; 61: 399 


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P-101 Binding potential estimation in 11C-PE2I PET brain striatal images: impact of partial volume 

correction under segmentation errors 

Maroy R. , Dusch E. , Comtat C. , Leroy C. , Trebossen R. . 

CEA, Orsay, France, Metropolitan 

renaud.maroy@cea.fr 

Introduction: Numerous methods have been proposed 

for the correction of the Partial Volume Effect 

(PVE) that hampers striatal PET studies. While 

their quantification accuracy is known for most, 

their ability to recover correct pharmacokinetic parameters 

in case of segmentation errors is less clear. 

The work proposes to compare the binding potential 

estimations using 4 distinct Time Activity Curve 

(TAC) estimation methods. 

Methods: A phantom based on the Zubal phantom 

of a 11C-PE-2I PET exam with kinetics generated 

using the simplified tissue model of Lammertsmaa 1 

(R1=1.28, k2=.09, BP=17) was analytically simulated 

on a ECAT HRRT (Siemens, 2.4mm intrinsic 

resolution). PET images were reconstructed with 

1.2�1.2�1.2mm 3 voxels and 20 frames using both 

RM-OP-OSEM 2 , which compensate for PVE, and 

OP-OSEM. 

Twenty realizations of caudate segmentations containing 

errors ranging between 40% volume underestimation 

(-40%) and 40% volume overestimation 

imaging life 

(40%) were defined 3 . Four TAC estimation methods 

were compared for the caudate: the mean TAC measurement 

in the standard OP-OSEM image (ME) and 

in the RM-OP-OSEM image (MRO), the Geometric 

Transfer Matrix method (GTM) and an improved 

GTM 3 method using voxel selection (GTM20). The 

R1, k2 and binding potential (BP) estimated with 

the estimated TACs using 1 were compared based 

on the Apparent Recovery Coefficient (ARC). 

Results: Unlike k2 and R1, 

the BP is globally underestimated 

by the methods. 

The lowest performances 

are obtained without correction, 

using ME. The 

best ARC for the TACs (Fig 

1.A) and R1 (Fig 1.C) are 

achieved using MRO, but 

the use of MRO leads to 

suboptimal BP (Fig 1.D), 

which is the model parameter 

of major interest. The 

best ARC for BP and k2 

(Fig 1.B) are achieved using 

GTM20, which provides 

better TACs than GTM, 

leading also to better ARC 

than GTM. 

Conclusions: Our improved GTM method with voxel 

selection achieves the TAC estimation in terms of 

binding potential and k2 recovery. 

References: 

1. A A Lammertsma et al. Neuroimage, 1996 

2. F C Sureau et al. JNM, 2008 

3. R Maroy et al. SNM, 2009


Automated quantification scheme based on an adapted probabilistic atlas based 

segmentation of the brain basal nuclei using hierarchical structure-wise registration 

Maroy R. (1) , Leroy C. (1) , Douaud G. (2) , Trebossen R. (1) . 

(1) CEA, Orsay, France, Metropolitan 

(2) Radcliffe Hospital, Oxford, United Kingdom 

renaud.maroy@cea.fr 

Introduction: The work 

proposes: 1) a new basal 

ganglia segmentation 

method based on probabilistic 

atlas construction 

and compared to 

manual segmentation, 

2) a validation method 

for segmentation of structures 

using a co-acquired 

PET image. 

Methods: T1 MRI (T1) and 

18F-PEII PET images were 

acquired in 12 healthy 

adults and co-registered. 

The PET images, acquired 

on the HRRT PET system, 

were reconstructed with 

both the AWOSEM (NoPSF) and the 3D-OP- 

OSEM PSF (PSF) methods. The basal nuclei were 

segmented manually in a knowledge base (KB) of 

24 T1 and in the 12 acquired subjects T1 (IS). A 

probabilistic atlas adapted to IS was constructed 

using a structure-wise hierarchical registration of 

each KB image on IS. Segmentation was obtained 

simply through atlas probability maximization. 

A GTM correction was applied on the NoPSF 

using as spatial domain 1) the manually drawn 

structures or 2) the automatically drawn structures, 

with A) ROI defined as in [Frouin, 2002] 

or B) with adequate selection of 20% (GTM20) 

of the corresponding spatial domain voxels. 

The estimated values were compared for 1)+A) 

and 2)+B) to the mean TAC in PSF, considered 

as the reference, inside manually drawn 

eroded regions using the estimation error (E). 

Results: GTM20 performed significantly better 

than GTM for both the manual and automated 

delineation. Besides, the gain using together the 

automated segmentation and GTM20 (E1)+A)/ 

E2)+B)) was 6.1 for the caudate, 3.6 for the globus 

pallidus, 4.6 for the putamen and 1.4 for the 

thalamus for an increased reproducibility. 

Conclusions: We have proposed a quantification 

scheme using an adapted probabilistic atlas based 

segmentation of the basal nuclei and a partial volume 

correction method that enhances both the 

precision and the reproducibility of the PVE corrected 

measures. 


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216 


P-103 VHISTdiff - comparing workflow histories (VHIST/VINCI) 

Vollmar S. , Hüsgen A. , Sué M. , Nock J. , Krais R. . 

Max Planck Institut for Neurological Research, Köln, Germany 

vollmar@nf.mpg.de 

Introduction: VHIST [1] has been developed to 

document workflows in multi-modality imaging 

(however, it is not at all limited to this field). The 

VHIST file format is PDF compatible and it can be 

displayed with any PDF viewer (“human readable”). 

However, VHIST files also contain structured information 

on each workflow step (embedded XML) 

suitable for automated processing. In one current 

project, we are integrating VHIST into VINCI [2], 

our package for visualization and analysis of tomographical 

data. This enables us to natively create 

VHIST files which can store meta data of original 

file formats, filter operations, “image arithmetics” 

and registration parameters. 

Methods: An interesting application of VHIST files 

is the comparison of workflow histories belonging 

to similar but subtly different image data, e.g. image 

data of one subject which has been aquired by 

the same scanner but which has been processed in a 

different way (type and order of filter steps; thresholding; 

application of masks; different types of normalization, 

different reconstruction parameters; 

different approaches when converting raw data). For 

this kind of comparison we are currently developing 

a new tool, VHISTdiff, which allows to automatically 

find corresponding workflow steps and report 

the differences. 

The figure depicts a graphical representation of the 

“macroscopic” differences between two VHIST files 

(A.vhist, B.vhist) as they were found by the VHISTdiff 

algorithm: each tree represents the workflow 

history of one image. Dotted lines denote corresponding 

workflow steps, white text marks workflow 

steps that do not have a pendant. VHIST files 

can contain other VHIST files: in this example, the 

registration step of the A-tree contains file C.vhist, 

and likewise D.vhist is contained in the other registration 

step. The report of differences between 

corresponding workflow steps (e.g. different filter 

parameters) are not part of this particular graphical 

representation which has been rendered fully automatically 

using Graphviz [3]. 

VHISTdiff ’s algorithm uses some ideas of the traditional 

UNIX diff command for comparison of text 

imaging life 

files. However, the concept has been generalized to 

tree structures: VHISTdiff uses Dijkstra’s algorithm 

[4] to optimize a cost function which favours association 

strings of similar workflow steps. 

Outlook: First results of this new tool look promising. 

We will investigate strategies to define cost 

functions that try to be useful for a wide range of 

real-world use cases. 

References: 

1. VHIST, http://www.nf.mpg.de/vhist 

2. VINCI, http://www.nf.mpg.de/vinci 

3. The Graphviz software package, http://www.graphviz.org 

4. Dijkstra, E. W. (1959). “A note on two problems in 

connexion with graphs”. Numerische Mathematik 1: 

269–271


Fast matrix-free method for fluorescence imaging 

Zacharopoulos A. (1) , Garofalakis A. (2) , Ripoll J. (1) , Arridge S. (3) . 

(1) FORTH IESL , Heraklion, Greece 

(2) CEA, I2BM , France 

(3) UCL CS, United Kingdom 

azacharo@iesl.forth.gr 

Introduction: In non-contact Fluorescence Molecular 

Tomography, the large number of measurements 

poses a challenge since the reconstruction methods 

used rely on the inversion of a derivative operator 

and the explicit formulation and storage of this operator 

in a matrix is generally 

not feasible. We test a matrixfree 

method that addresses the 

problems of large data sets and 

reduces the computational cost 

and memory requirements 

for the reconstructions. More 

specifically we challenged 

the Matrix-Free method with 

in-vivo measurements from 

mice where fluorescence tubes 

of different but controlled 

concentrations are inserted, 

to assess the quantification 

performance of the method. 

Methods: Our approach initially presented in [1], is 

based on a formulation of the diffusion approximation 

for the fluorescence case and the TOAST Image 

Reconstruction in Optical Tomography FEM 

package, [2] and uses a matrix free formulation. 

The explicit calculation and storage of the Jacobian 

is avoided by replacing it by a vector times matrix 

operator and a vector times adjoint matrix operator. 

Since we wanted to assess the quantification properties 

of the matrix-free method we aquired in-vivo 

measurements using a balb/c mouse where capillary 

of Alexa Fluor 647 of controlled concentrations 

a)60.5uM, b)22uM, c)8.5uM, d)2.2uM and e)0.9 uM. 

was inserted hypodermically in the ventral side. For 

the reconstructions, a slab-like geometry of dimensions 

41mm x 41mm x 12mm was used with 5733 

nodes and 4800 voxel-elements. For the optical 

parameters we assumed μa =0.06 mm-1 and μs =1. 

mm-1 for the 615nm excitation and μa =0.05 mm-1 

and μs =1.5 mm-1 for the 700nm emission wavelengths. 

We set 1458 detectors positions by sampling 

the images, which for the 36 sources created 

two vectors of 48285 measurements each. 

Results: The results for the five different experiments 

are presented as iso-surfaces in 3D in the 

figure where the white light image of the mouse 

is rendered in the background as a reference. The 

average time for the reconstruction was 50sec, in a 

Pentium 2Ghz machine with 2GB of memory. 

Conclusions: Using the Matrix-free method we 

managed to reconstruct for the positions of the 

fluorescent tubes and a relative quantification accuracy 

and sensitivity. More specifically the algorithm 

demonstrated an almost linear response between 

the controlled concentrations and the reconstructed 

ones. 

Acknowledgement: This work was funded by the 

EC Seventh Framework Grant, FMT-XCT, grant 

201792. 

References: 

1. Zacharopoulos A, et al (2009) Opt. Express , 17, 3042- 

-3051. 

2. Arridge R S (1999) Inverse Problems, 15, 41--93 . 


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218 


TOPIC: Cancer Biology 

PET and MRI studies applied on characterization of Fisher/F98 rat glioma model 

Valtorta S. (1) , Ronchetti F. (2) , Lo Dico A. (3) , Politi L.S. (4) , Masiello V. (5) , Matarrese M. (5) , Zara G. (6) , Zenga F. (7) , Scotti G. (4) , 

Mauro A. (2) , Moresco R.M. (5) . 

(1) Università degli Studi di Milano; IBFM-CNR, Segrate, Italy 

(2) Istituto Auxologico Italiano, Italy 

(3) San Raffaele Scientific Institute; IBFM-CNR; L.A.T.O., Italy; 

(4) San Raffaele Scientific Institute, Italy; 

(5) San Raffaele Scientific Institute; IBFM-CNR; Università degli Studi di Milano-Bicocca, Italy; 

(6) Università di Torino, Italy; 

(7) Università di Brescia, Italy 

silvia.valtorta@unimi.it 

Introduction: Preclinical brain tumor models 

have provided a wealth of information on the 

biology, imaging and experimental therapeutics 

of brain tumors [1],[2] . The aim of our study is 

characterized Fisher/F98 rat glioma model using 

Positron Emission Tomography (PET) and 

Magnetic Resonance (MR) analysis to set up an 

experimental model useful to study the efficacy 

of new colloidal vectors for chemotherapy. 

Methods: Syngenic rat brain-glioma models 

(Fisher/F98) was obtained by stereotactic (x=2; 

y=5; z=3) implantation of different cell concentrations 

(10 2 , 10 3 , 10 4 and 10 5 ). To monitor tumor 

growth progression, rats underwent once a week 

Gadolinium enhanced T1-MRI studies followed 

by [ 18 F]FDG PET studies, starting from 7 days 

after surgery. A group of animals performed also 

[ 18 F]FAZA PET studies to evaluate regional tissue 

hypoxia. To improve quantification, PET and 

MRI images were fused using PMOD 2.7 software. 

Max radiotracers uptake was calculated for tumor, 

frontal cortex, cerebellum and background using 

region of interest (ROI) analysis. Radioactivity 

concentration values expressed in MBq/g were 

then transformed into percentage of injected dose 

per gram of tissue (%ID/g). Moreover, histological 

analysis of proliferation, apoptosis, differentiation, 

neoangiogenesis and hypoxia markers were 

performed. 

Results: Mean survival time of rats injected with 

10 4 and 10 5 cells was nine days. One week after 

surgery, MRI revealed a rapid growth that reached 

0.11 cm 3 mean tumour volume. Animals injected 

with 10 3 and 10 2 cells showed a mean survival time 

of 18 and 24 days respectively. In rats injected with 

10 3 cells, tumor was revealed 14 days after surgery 

at MRI and [ 18 F]FDG PET and successively tumors 

rapidly increased. Disease course in 10 2 cells 

injected rats was slower. Tumors were characterized 

by high [ 18 F]FDG uptake and hypoxic subareas 

which only partially overlapped. Hypoxic 

imaging life 

areas were mainly localized in correspondence 

to Gd-enhanced regions whereas hyper glucose 

metabolic areas were localized in the outer part 

of the tumors. At histological analysis tumoral 

masses showed an infiltrative pattern of growth 

and moderate neoangiogenesis. Tumors obtained 

at animals death showed diffused necrotic areas. 

HIF1 was clearly expressed by glial and neuronal 

cells in oedematous and hypoxic areas. 

Conclusions: Our study indicates that Fisher/ 

F98 rat glioma model reproduce s the characteristic 

of aggressiveness of human glioblastoma. 

Tumor was characterized by high glucose 

metabolism and by hypoxic sub-areas. The concentration 

of 10 2 cells permits to better monitor 

disease onset and progression and to plan 

experiments to test new anti-neoplastic therapies 

efficacy. 


by grants from: Regional AIRC 2008 n°6278 and 

FIRB-CNR n°RBIP06M8ZA. 

References: 

1. Spaeth N et al; Eur J Nucl Med Mol Imaging. 33:673- 

682 (2006) 

2. Brioschi AM et al; J Nanoneurosci. 1:65-74 (2009)

TOPIC: Cancer Biology 


Non-invasive “E2F sensing” system for monitoring DNA damage alteration induced by BCNU 

Monfared P. (1) , Rudan D. (1) , Viel T. (1) , Waerzeggers Y. (1) ,Hadamitzky M. (1) , Schneider G. (1) , Rapic S. (1) ,Neumaier B. (1) , Backes H. (1) , 

Winkeler A. (1) , Jacobs A.H.. (1,2). 

(1) Max Planck Institute for Neurological Research, Cologne, 

(2) European Institute for Molecular Imaging (EIMI), University of Muenster, Germany 

Parisa.Monfared@nf.mpg.de 

Introduction: Imaging transcriptional regulation of 

endogenous genes in living animals using noninvasive 

imaging techniques provide a clear perception 

of normal and cancer-related biological processes. 

Radiolabeled reporter probes and PET imaging can 

be translated into human studies in the near future. 

In our preliminary study, the endogenous expression 

of E2F, a gene that affects several important 

biological processes, has been imaged in vivo with 

bioluminescence imaging. A retroviral vector, Cis- 

E2F/LUC-IRES-TKEGFP, was generated by placing 

the reporter genes under control of an artificial 

cis-acting E2F-specific enhancer element. Following 

retroviral transduction of tumor cells in established 

xenografts, DNA damage induced alteration 

of E2F transcriptional activity, which correlated 

with the expression of E2F-dependent downstream 

genes as assessed by biolumencsnce imaging. 

Aim: To verify whether the cis-reporter system 

(Cis-E2F/LUC-IRES-TKEGFP) is sufficiently sensitive 

to image endogenous transcriptional gene 

regulation by [ 18 F] FHBG PET imaging. 

Methods: U87dEGFR-E2F-LITG cells were injected 

subcutaneously in nude mice and the 

development of the tumours was followed by 

Multimodal imaging. Two weeks after implantation, 

bioluminescence imaging, FLT and 

FHBG were performed before and 24h after 

treatment with low and high doses of BCNU. 

Results: Here, we validate the utility of [ 18 F]FLT- 

PET to image proliferation rate of the tumor in response 

to DNA damage and compare with [ 18 F] 

FHBG uptake which is associated with E2F transcriptional 

activity. In keeping with in vitro findings, 

after 24 h post-treatment, low dose of BCNU 

induced an increase in [ 18 F]FHBG uptake as compared 

to non-treated mice. However, with high 

dose of BCNU [ 18 F]FHBG uptake decreased in E2F 

xenografts. [ 18 F]FLT accumulation in E2F xenografts 

decreased with low and high doses of BCNU 

at 24 and 48 hours. Quantitative changes in tumor 

[ 18 F]FLT uptake were associated with decreased 

tumor proliferation and tumor [ 18 F]FHBG uptake 

correlated with transcriptional gene regulation of 

E2F in response to DNA damage 

Conclusions: We show the utility of [ 18 F]FHBG- 

PET to image DNA damage induced by BCNU and 

shown the tumor-specific activity of E2F. We propose 

that these types of reporter systems, will allow 

a detailed insight into the kinetics of cell cycle 

control and for the development of new cell cycle 

targeted molecular therapies. 


by the EC-FP6 European DiMI, (LSHC-CT-2004- 

503569) LSHB-CT-2005-512146 and Clinigene 

(LSHB-CT-06-018933). 


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220 


TOPIC: Neuroimaging 

Multi-tracer PET imaging of a mouse model of Alzheimer´s disease to assess microglial 

activation related to ageing and anti-inflammatory treatment 

Rapic S. (1) , Backes H. (1) , Viel T. (1) , Hadamitzky M. (1) , Monfared P. (1) , Rudan D. (1) , Vollmar S. (1) , Neumaier B. (1) , Hoehn M. (1) , Van der 

Linden A. (2) , Heneka M.T. (3) , Jacobs A.H. (1,4) 

(1) Max Plank Institute for Neurological Research, Cologne, Germany 

(2) Bio-Imaging Lab, University of Antwerp, Belgium 

(3) Department of Neurology, University of Bonn, Germany 

(4) European Institute for Molecular Imaging (EIMI) 

Sara.Rapic@nf.mpg.de 

Introduction: Alzheimer´s disease (AD), the leading 

cause of dementia in elderly people, is characterized 

by the deposition amyloid-β plaques in the 

brain surrounded by activated microglia. Activated 

microglia play an important role in amyloid clearance 

but also secrete pro-inflammatory molecules 

that lead to chronic neuroinflammation, and toxins 

that trigger cell death cascades in neurons, both 

eventually resulting in neurodegeneration. The 

nuclear peroxisome proliferator-activated receptor 

gamma (PPAR-γ) plays an important role in inflammatory 

processes. Agonists that bind and activate 

PPAR-γ, suppress inflammation by inhibiting the 

expression of pro-inflammatory molecules at transcriptional 

level. Determination of the contribution 

of neuroinflammation to the progression of AD can 

provide new insights in the development of therapy. 

Aim: To assess with in vivo positron emission tomography 

(PET) whether microglial activation is correlated 

with age in APP/PS1 transgenic mice and wildtype 

mice and if anti-inflammatory treatment with a 

PPAR-γ agonist can reduce neuroinflammation. 

Methods: Multi-tracer micro-PET imaging was performed 

in 13 months old APP Swe /PS1 dE9 transgenic 

mice (n = 5) and C57BL/6J wild type mice (n = 7). 

Dynamic imaging was performed after intravenous 

injection of [ 11 C]PK11195, a radioligand for the 

translocator protein-18 kDa which is upregulated in 

activated microglia and [ 18 F]FDG for the quantification 

of cerebral glucose metabolic rate. [ 18 F]Fluoride 

bone imaging was performed after every scan for coregistration 

purposes. The animals were imaged at 

baseline and treated for 4 weeks with a PPAR-γ agonist. 

Right after treatment and 4 weeks off treatment, 

all animals were investigated again using the same 

imaging protocol to assess the effect of the therapy. 

Results: Preliminary results show that uptake of 

[ 11 C]PK11195 is upregulated in transgenic mice 

compared to wild-type mice in the cortex (SUV 

of 75.15 and 57.20 respectively), thalamus (SUV 

of 78.90 and 63.63 respectively) and cerebellum 

(SUV of 82.12 and 65.47 respectively). Uptake of 

imaging life 

[ 18 F]FDG in transgenic mice is elevated as well, 

compared to wild-type mice, in the cortex (SUV 

of 162.10 and 106.78 respectively), thalamus (SUV 

of 172.07 and 115.33 respectively) and cerebellum 

(SUV of 166.50 and 114.46 respectively). Data were 

corrected for the total injected dose (ID). First image 

processing of the data obtained after treatment 

shows decreased brain retention of both tracers in 

transgenic mice. 

Conclusions: The higher uptake of [ 11 C]PK11195 in 

the brain of transgenic mice compared to wild-type 

mice indicates the presence of activated microglia, 

suggesting neuroinflammation of the Alzheimer 

brain. Reduction of [ 11 C]PK11195 brain retention 

after treatment with the PPAR-γ agonist reflects its 

anti-inflammatory effect. 

With the help of animal models for Alzheimer’s disease 

and in vivo molecular imaging, longitudinal detection of 

microglial cells is made possible and may be of vital importance 

in defining their role in the progression of the 

disease and in the development of therapeutic strategies. 

Acknowledgement: This work is supported in part by 

EC-FP6-project and DiMI LSHB-CT-2005-512146.

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INDEX

ANIMASCOPE 

High TECHNOLOGY for High TRANSPARENCY 

• High Technology for High Transparency 

o Non invasive imaging technologies 

o Small animals Preclinical imaging 

o Multimodality imaging services (Ultrason, MRI, Optical, PET, SPECT, CT Scan) 

• Reduce, Refine, Replace 

o Quantitative, Precise, Predictive 

o Reduces experimental Artifacts 

o Longitudinal Studies - Reduce Animal Usage (minus 70-80%) 

o Reduction and Refinement of animal works are Cost and Time-effective on R&D 

• Bridge the gap between preclinical and clinical research 

o Drug Delivery and Liberation (drug to target) 

o Efficacy, Safety, Pathology (desiderable and undesirable effects) 

o Ligand-Target interaction (receptors, enzymes, transporters) including Dose 

Occupancy relationships 

o Dynamic Biodistribution and Kinetics 

• One stop shop for : 

o biology, 

o cardiovascular, 

o cell therapy, gene therapy, immunization, 

o musculoskeletal, 

o neurology, nephrology, 

o oncology, 

o reproduction, development, 

o nutrition (nutraceuticals, dietary supplements and 

functional food). 

Contact : Daniel CHRISTIAEN 

ZI EUREKALP 38660 St Vincent de Mercuze (F) 

d.christiaen@animascope.eu 

Tel +33 (0)476 97 94 87 

www.animascope.eu 

Give to your study a new look

Index 

A 

Abasolo I. 126 

Abiraj K. 77 

Aboagye E.O. 209 

Adamczak J. 64 

Aelvoet S. A. 63 

Agarwal A. 41 

Agin V. 148 

Ahmad R. 204 

Aigner L. 62, 81, 169 

Aime S. 49, 79, 114, 

171,174, 176, 213 

Allard S. 90 

Alves F. 128, 208 

Anderson S. 155 

Arena F. 49 

Arridge S. 217 

Arstad E. 204 

Ashitate Y. 55 

Aswendt M. 171 

Audrain H. 189 

Autio A. 54, 130 

Avory M. 204 

Awde A. 160 

B 

Baatenburg De Jong R.J. 

119 

Backes H. 134, 219, 220 

Baekelandt V. 63, 85 

Barnard P. 77 

Barré L. 78 

Barré W. 35 

Barrio J. 157 

Bauernfeind A. 155 

Baulieu J. L. 157 

Bauwens M. 116 

Beaufils E. 157 

Bednar B. 89 

Behe M. 117 

Béhé M. 52 

Bellard E. 127 

Bender D. 189 

Benoit D. 155 

Benyó Z. 37 

Berardi F. 151 

Bergner F. 190 

Bergström J. P. 78 

Bernhardt P. 52 

Bernsen M. 118, 163, 

166,167, 168 

Berthezene Y. 65 

Berti R.P. 172 

Beynel A. 191 

Bhalla R. 183 

Bijster-Marchand M. 118 

Bilski M. 192 

Biserni A. 90, 91 


Blanc A. 53 

Blankevoort V. 158 

Blockx I. 169 

Blossier A. 197 

Boehm-Sturm P. 64 

Bogaert-Buchmann A. 147 

Bogner W. 115 

Bohmer M. 103 

Boisgard R. 152, 160, 

191,197, 202, 211 

Bonetto F. 161 

Bonnet C. 182 

Bonzom S. 43 

Borelli M. 84 

Bormans G. 85, 170 

Boschi F. 42 

Bos P. 167 

Botnar R. 97 

Botta M. 48 

Boussel L. 68 

Boutin H. 198 

Brepoels L. 30 

Brown G. 198 

Brüggemann C. 171 

Brzezinski K. 184 

Bukala D. 201 

Burdinski D. 44 

Buron F. 182 

Busch C. 199 

Bzyl J. 72, 122 

C 

Cabello J. 184 

Calandrino R. 42 

Calderan L. 42 

Calotti F. 120 

Camus V. 157 

Cannet E. 68 

Carelli S. 56 

Carlsen H. 100 

Carpenter A. 36 

Carroll X. 155 

Casteels C. 33 

Catanzaro V. 79, 176 

Caveliers V. 29, 136 

Cemazar M. 127 

Cescato R. 206 

Céspedes M. V. 126 

Chapotot L. 191 

Chattopadhyay A. 151 

Chauveau F. 65 

Chazalviel L. 148 

Cheung P. 164 

Choe J. G. 153 

Choi H. S. 55 

Cho T. H. 65 

Christofori G. 52 

Ciana P. 90, 91 

Cibiel A. 173 

Ciobanu L. 160 

Cittadino E. 79, 114, 

174,213 

Clark C. 36 

Clerici M. 84, 165 

Clerici M. S. 56 

Cojoca R. 205 

Coleman P. 89 

Coll J. L. 139 

Comtat C. 214 

Contag C.H. 21 

Cordula D. 140 

Cottet M. 135 

Couillaud F. 83 

Crosby 61 

Cruz Ricondo L. J. 161 

Cuhlmann S. 100 

Curtis A. 41 

Custers E. 73 

D 

D´Alessandria C. 87 

D’Ambrosio D. 42 

Damont A. 197, 202 

Dastrù W. 114 

Debeissat C. 83 

Debyser Z. 63, 85 

Deckers N. 116 

Decristoforo C. 178 

Defraiteur C. 149 

De Geeter F. 101 

Degrassi A. 84 

Degroot T. 30 

De Jong M. 104, 107, 118 

Delest B. 180 

Deliolanis N. 185, 188 

Delli Castelli D. 114 

Delpassand E. S. 105 

Del Puerto-Nevado L. 203 

Del Vecchio S. 143 

Delzescaux T. 211 

Demello A. 189 

Demmer O. 87 

Demphel S. 202 

Denes A. 198 

De Poel M. 118 

De Rooij K. 142 

Deroose C. 85, 170 

De Saint-Hubert M. 30,116 

Desco M. 177, 181 

De Smet M. 103 

De Spirito M. 121 

De Swart J. 118 

De Vocht N. 57 

Devoogdt N. 29, 136 

Devos E. 30, 116 

Devos H. 101 

De Vries A. 73 

De Vries I. J. 161 

D’huyvetter M. 136 

Díaz-Lópeza R. 172 

Diependaele A. S. 148 

Dietlmeier J. 210 

Digilio G. 79, 176 

Dijkgraaf I. 87 

Dikaiou K. 186, 196 

Dilworth J. 77 

Doeswijk G. 166 

Dollé F. 160, 187, 197 

202,211 

Douaud G. 215 

Douek P. 68 

Drake C. 198 

Dresch C. 82 

Dubois A. 160, 187, 211 

Ducongé F. 173, 187 

Dullin C. 128, 208 

Dumont R. 117, 206 

Duong L. T. 89 

Dupont D. 147, 187 

Durroux T. 135 

Dusch E. 214 

Duyckaerts C. 179 

Dziuk E. 195 

Dziuk M. 192 


E 

Ehrlichmann W. 201 

Ell P. J. 52 

Emond P. 78 

Emonds K. 137 

Etard O. 148 

Euskirchen P. 123 

Evans P. 100 

Evens N. 85 

F 

Fani M. 77, 117, 178, 206 

Farde L. 75 

Farrell E. 167 

Farr T. D. 64 

Fattal E. 172 

Fedeli F. 79 

Fekete K. 37 

Fekete M. 48 

Fenzi A. 42 

Fernández Y. 126 

Figdor C. 161 

Figueiredo S. 174 

Finnema S. 75 

Fiorini C. 84, 165 

Fischer K. 200 

Flamen P. 138 

Fokong S. 122, 175 

Forni G. 205 

IndEx

226 


Fortin P. Y. 83 

Franci X. 149 

Frangioni J. 55 

Freret L. 172 

Fuchs K. 200 

G 

Gadella T.W.J. 60, 61 

Gaetjens J. 122 

Garcia C. 138 

Garcia E. 53 

Garnuszek P. 70 

Garofalakis A. 187, 217 

Gätjens J. 175 

Gauberti M. 66, 145, 148 

Gee A. 189 

Genevois C. 83 

Geraldes C. 179 

Geraldes C.F. 174 

Gerhard A. 32 

Gervais P. 152 

Geys R. 159 

Ghanem G. 138 

Gianolio E. 49, 79, 171 

Gillet B. 147 

Giron-Martinez A. 203 

Giżewska A. 195 

Glaser M. 209 

Glatz J. 188 

Gleason A. 89 

Glowa B. 70, 146 

Glowalla A. 144 

Goblet D. 129, 149 

Goedhart J. 61 

Goethals L. 101 

Golzio M. 127 

Gombert K. 173 

Gonzalez-Mangado N. 203 

Gorio A. 56 

Gourand F. 78 

Grabner G. 115 

Gremse F. 72 

Griessinger C. 178 

Grießinger C. 200 

Griessinger C.M. 201 

Gringeri C. 176 

Groetzinger C. 140 

Grote M. 131 

Grouls C. 122 

Gruber S. 115 

Gruell H. 44, 47, 73, 103 

Gruetter R. 46 

Gsell W. 100 

Guedin P. 148 

Guenoun J. 166 

Guglielmetti C. 162 

Guilloteau D. 76, 78, 157 

Gulyas B. 78 

imaging life 

H 

Haberkorn U. 102 

Hadamitzky M. 123, 134, 

219, 220 

Haeck J. 118 

Haiat G. 172 

Halldin C. 75, 78 

Hammers A. 156 

Hamm J. 205 

Hamplova A. 179 

Harlaar N. J. 99 

Hartung F. 208 

Haskard D. 100 

Hasselbalch S. G. 35 

Haubner R. 117, 178 

Heckemann R. A. 156 

Heerschap 31 

Heerschap A. 161 

Heijman E. 103 

Helbich T. 115 

Heneka M.T. 220 

Hennink W. 132 

Herance J. R. 126 

Hérard A. S. 211 

Herholz K. 198 

Hernandez L. 43 

Hernot S. 136 

Herranz F. 177, 181 

Herzog E. 99 

Higuchi T. 97 

Hijnen N. 103 

Hilger I. 199 

Hillebrands J. L. 99 

Himmelreich U. 171 

Hink M.A. 61 

Hirani E. 204 

Hoeben R. 120 

Hoehn M. 64, 171, 220 

Hogset A. 49 

Hommet C. 157 

Honer M. 196 

Horváth I. 37 

Houston G. 168 

Hubalewska-Dydejczyk A. 

70, 146 

Hüsgen A. 216 

Huskens J. 44 

Hutteman M. 55, 142 

Hwang Y. M. 153 

I 

Ibrahimi A. 30, 63, 85 

Iezzi M. 205 

Iommelli F. 143 

J 

Jacobs A.H. 123, 134, 

219, 220 

Jafurulla M. 151 

Jalkanen S. 54 

Jamous M. 206 

Janssens I. 173 

Jaron A. 154 

Jassens I. 187 

Jego B. 160, 197, 211 

Jikeli J. 64 

Jiskoot W. 132 

Jonckers E. 212 

Jones H. 100 

Jones P. 204 

Joshi A. 36 

Josserand V. 139, 207 

Jucker M. 34 

Judenhofer M. 200 

Justicia C. 150 

K 

Kachelrieß M. 190 

Kaijzel E. 106, 119, 120, 

124, 125, 132, 

142, 158 

Kaiser W. A. 199 

Kaisin G. 129 

Kaliszczak M. 209 

Kallur T. 64 

Kaminski G. 192, 195 

Kandasamy M. 169 

Karczmarczyk U. 70, 146 

Karhi T. 130 

Karolczak M. 190 

Kassiou M. 197, 198 

Kaufholz P. 131 

Keereweer S. 106, 119,125 

Keil B. 52 

Keist R. 53, 186 

Kepe V. 157 

Keramidas M. 139 

Kerrebijn J. 119 

Kesenheimer C. 201 

Kessler H. 87, 128 

Keupp J. 103 

Khan I. 204 

Kiessling F. 72, 122, 175 

Klaubert D. 90 

Klohs J. 186 

Kneilling M. 200, 201 

Knetsch P. 178 

Knödgen E. 123 

Knudsen G. M. 35, 156 

Knuuti J. 71 

Kobylecka M. 193 

Komm B. 91 

Kossodo S. 89 

Kotek G. 163, 166, 167 

Krais R. 216 

Krams R. 100 

Krautkramer M. 36 

Krenning E.P. 107 

Krestin G. 163, 166, 167, 

168 

Krewer B. 208 

Krolicki L. 193 

Krucker T. 41 

Kruttwig K. 171 

Krzanowski M. 70, 146 

Kubicek V. 179 

Kuhnast B. 187, 202 

Kumar-Singh S. 159 

Kunikowska J. 193 

Kuppen P. 55, 142 

Kuśnierz-Cabala B. 146 

Kyrgyzov I. 43 

L 

Labate V. 121 

Lacasta C. 184 

Lacivita E. 151 

Laget M. 135 

Lahoutte T. 29, 101, 136 

Laine J. 71 

Laitinen I. 71 

Lamberton F. 148 

Langereis S. 44, 103 

Lapp R. 190 

Lazar A. 179 

Lebenberg J. 211 

Lebon V. 211 

Lederle W. 72, 122 

Le Helleix S. 202 

Lehel S. 35 

Lehmann F. 199 

Lehmann S. 53 

Lemaire C. 129 

Le Masne Q. 43 

Lenda-Tracz W. 194 

Leopoldo M. 151 

Lepetit-Coiffé M. 83, 88 

Leppänen P. 71 

Leroy C. 214, 215 

Lettieri A. 143 

Levrey O. 43 

Liang J. 164 

Libani I. V. 56 

Libani I.V. 165 

Li G. 164 

Linhart V. 184 

Lin S. A. 89 

Li R. 155 

Liu Z. 175 

Llosá G. 184 

Lo Dico A. 218 

Long N. 189 

Longo D. 213 

López M. 126

López M.E. 126 

Löwik C.W.G.M. 55, 106, 

119, 120, 124, 125, 

132, 142, 158 

Lub J. 73 

Lucignani G. 56, 84, 165 

Lui R. 56, 165 

Luoto P. 130 

Luthra S. 204 

Luxen A. 129, 149 

M 

Mackeyev Y. 133 

Mackiewicz N. 187 

Macrez R. 145 

Madaschi L. 56 

Madsen K. 35 

Maecke H. 52, 77, 117, 206 

Maggi A. 90, 91 

Maier F. C. 34 

Maina T. 107 

Mainini F. 114 

Maitrejean S. 43 

Mali A. 130 

Mangues R. 126 

Mannheim J. 34, 200 

Mansfield J. 41 

Mansi R. 52, 206 

Marengo M. 42 

Marfia G. 56 

Markelc B. 127 

Marner L 35 

Maroy R. 214, 215 

Marra F. 56 

Marsouvanidis I.P. 107 

Martelli C. 84, 165 

Martin A. 152 

Martinelli J. 48 

Martins A. 179 

Masiello V. 218 

Massberg S. 26 

Matarrese M. 218 

Máthé D. 37 

Mathejczyk J. 208 

Mathejczyk J.E. 128 

Mathis G. 135 

Maubert E. 145 

Maulucci G. 121 

Maurin M. 70 

Mauro A. 218 

Mautino A. 205 

Mazurek A. 195 

Mccoll B. 198 

Meijering E. 166 

Mele M. 121 

Menchise V. 79, 176 

Mercier F. 129 

Mérian J. 207 


Merli D. 56 

Messeguer A. 150 

Meyer A. 131 

Meyer Y. 180 

Mezzanotte L. 120 

Michelini E. 120 

Mieog J. 142 

Mieog S. 55 

Mikołajczak R. 70, 146 

Miller P. 74, 189 

Miranda S. 126 

Mol I. 106, 119, 124, 125 

Monfared P. 123, 134, 219, 

220 

Montagne A. 66, 145 

Moonen C. 83, 88 

Moreira J.N. 174 

Moresco R. M. 218 

Morfin J.F. 179 

Morisson-Iveson V. 204 

Mortelmans L. 30, 116, 

137, 170 

Moser E. 115 

Mottaghy F. 30, 116, 137 

Mronz M. 190 

Musiani P. 205 

Muylle K. 138 

N 

Napolitano R. 79 

Napp J. 128, 208 

Narula J. 95 

Nataf S. 65 

Navarro F. 207 

Nekolla S. 97 

Neumaier B. 134, 219, 220 

Nguyen H. H. P. 169 

Nguyen Q. D. 209 

Nicolaij K. 47 

Nicolas G. 206 

Nicolay K. 73 

Niessen W. 166 

Nighoghossian N. 65 

Noack P. 180 

Nock B.A. 107 

Nock J. 216 

Ntziachristos V. 99, 185, 

188 

Nuyts J. 137 

O 

Odenthal J. 34 

Odoardi F. 51 

Oh S. Y. 153 

Oikonen V. 130 

Oliver J. F. 184 

Opalinska M. 70, 146 

Orset C. 66, 145, 148 

O’shea D. 204 

Ottobrini L. 56, 84, 165 

P 

Pach D. 146 

Palmowski M. 122 

Panieri E. 121 

Pani G. 121 

Pardo L. A. 208 

Paris J. 129 

Park E. 153 

Park K. W. 153 

Park S. 155 

Passmore J. 204 

Passon M. 199 

Pauli J. 128 

Pawlak D. 193 

Peces-Barba G. 203 

Peleman C. 29 

Pereson S. 159 

Perez-Rial S. 203 

Perkuhn M. 72 

Perrone R. 151 

Pesenti E. 84 

Pestourie C. 173 

Peterson J. 89 

Petoud S. 45, 182 

Petrik M. 178 

Pichler B. 34, 178, 200, 201 

Pickarski M. 89 

Pietzsch H.J. 178 

Pijarowska J. 154 

Pikkemaat J. 44 

Pinker K. 115 

Pirozzi G. 143 

Pisaneschi F. 209 

Pisani E. 172 

Planas A. 150 

Plenevaux A. 149 

Plenge E. 166 

Pluchino S. 80 

Podgajny Z. 192, 195 

Politi L. S. 218 

Ponsaerts P. 162 

Pontecorvo M. 36 

Praet J. 162 

Prenant C. 198 

Pucci B. 172 

Q 

Que I. 106, 120, 124, 132 

Quesson B. 83 

R 

Rafecas M. 184 

Rainone V. 84 

Rajopadhye M. 89 

Rakowski T. 70 

Rando G. 91 

Rangaraj N. 151 

Rangger C. 178 

Raphael B. 43 

Rapic S. 134, 219, 220 

Ratering D. 186 

Raynaud J.S. 145 

Razansky D. 99, 188 

Reder S. 97 

Reischl G. 34, 200, 201 

Renard P.Y. 180 

Resch-Genger U. 128 

Reubi J. C. 52, 77 

Reubi J.C. 206 

Reumers V. 63 

Reutelingsperger C. 116 

Rexin A. 140 

Riccardi P. 155 

Richard J.A. 180 

Righini C. 139 

Riou A. 65 

Ripoll J. 186, 217 

Rix A. 72, 122 

Robillard M. 47 

Robins E. 209 

Röcken M. 200, 201 

Roda A. 120 

Roivainen A. 54, 71, 130 

Rojas S. 126 

Romanowicz G. 36 

Romieu A. 180 

Ronchetti F. 218 

Rosell Y. 177 

Rossin R. 103 

Rothwell N. 198 

Rubio M. 145 

Rudan D. 123, 134, 219, 

220 

Rudin M. 39, 53, 186 

Ruiz-Cabello J. 177, 181 

Russo M. 84 

Rutten E. 138 


S 

Saanijoki T. 54, 130 

Saha K. 36 

Salerno M. 179 

Salinas B. 177, 181 

Salvatore M. 143 

Sanches P. 103 

Saraste A. 97 

Sawall S. 190 

Saxena R. 151 

Sbarbati A. 42 

Schäfers M. 98 

Schellenberger E. 69 

Schibli R. 77 

Schibli R. 53, 196 

IndEx

228 


Schmid A. 34 

Schmidt D. 155 

Schmuck K. 208 

Schneider G. 134, 219 

Schober A. 72 

Schulze A. 131 

Schulz P. 140 

Schulz R. 72, 188 

Schwaiger M. 97 

Schwartz Jr S. 126 

Scotti G. 218 

Sebrie C. 147 

Sée V. 24, 25 

Sersa G. 127 

Shade C. 182 

Shamsaei E. 209 

Sherif H. 97 

Sigovan M. 68 

Sihver W. 131 

Sijbers J. 159 

Silengo L. 205 

Silvola J. 71 

Sims-Mourtada J. 105, 133 

Sipilä H. 54, 71 

Siquier K. 197 

Siquier-Pernet K. 160 

Skovronsky D. 36 

Slütter B. 132 

Smith G. 209 

Snoeks T. 106, 119, 124, 

125, 158 

Socher I. 199 

Soema P. 132 

Solevi P. 184 

Soria G. 150 

Sowa-Staszczak A. 70, 141, 

146 

Spadaro M. 114 

Spinelli A. E. 42 

Spivey A. C. 209 

Srinivas M. 161 

Stadelbauer A. 115 

Stein J. 38 

Stiller D. 34 

Stompor T. 70, 146 

Strijkers G. 96 

Stühmer W. 208 

Stuker F. 186, 196 

Suárez L. 126 

Sué M. 216 

Suilamo S. 130 

Sułowicz W. 146 

Sur C. 89 

Suzenet F. 179, 182 

Svarer C. 156 

Swinnen J. 137 

Szalus N. 192, 195 

Szigeti K. 37 

imaging life 

T 

Takano A. 78 

Tamma M. 206 

Tamma M. L. 77 

Tannous B. 185 

Tapfer A. 97 

Taruttis A. 99 

Tatsi A. 107 

Tauber C. 157 

Taulier N. 172 

Tavitian B. 152, 160, 173, 

187, 191, 197, 

202, 211 

Tchouate Gainkam O 136 

Tei L. 48 

Teissie J. 127 

Teräs M. 130 

Terreno E. 114, 174, 213 

Texido G. 84 

Texier I. 207 

Thézé B. 152, 173, 191 

Thonon D. 129, 149 

Tiemann K. 103 

Tietze L. F. 128, 208 

Toelen J. 63, 85 

Torres E. 114 

Toth E. 179, 182 

Tousseyn T. 30 

Trabattoni D. 84 

Trattnig S. 115 

Trebossen R. 214, 215 

Trigg W. 204 

Trinquet E. 135 

Tsapis N. 172 

Tudela R. 150 

Turco E. 205 

Tworowska I. 105, 133 

U 

Uijtterdijk A. 167 

Urbach W. 172 

V 

Vahrmeijer A. 55, 142 

Vainio J. 130 

Vainio P. 130 

Valtorta S. 218 

Van Auderkerke J. 212 

Van Beek E. 158 

Van Beers B.E. 28 

Van Broeckhoven C. 159 

Van Buul G. 167 

Van Dam G. M. 99 

Van Den Haute C. 63 

Vandeputte C. 85 

Van Der Heiden K. 100 

Van Der Linden A. 57, 

138, 150, 159, 162, 

169,212, 220 

Van Der Vorst J. 55, 142 

Van De Velde C. 55, 142 

Vandormael S. 138 

Van Driel P. 106, 119, 125 

Van Duijnhoven S. 47 

Vaneycken I. 29, 136 

Vanhoutte G. 159 

Vanhove C. 101 

Van Laere K. 33, 85, 170 

Van Osch G. 167 

Van Santvoort A. 170 

Van Tiel S. 163, 168 

Van Weerden W. 137 

Van Weeren L. 61 

Varrone A. 75 

Vats D. 186, 196 

Venel Y. 157 

Veraart J. 159 

Verbruggen A. 30, 85, 116 

Vercouillie J. 157 

Verfaillie C. 170 

Verhaar J. 167 

Verhoye M. 169, 212 

Vermaelen P. 170 

Verwijnen S. 118 

Viel T. 123, 134, 219, 220 

Vilar R. 189 

Villette S. 182 

Vivien D. 66, 145, 148 

Vollmar S. 216, 220 

Von Hof J. M. 208 

Vreys R. 162, 169 

Vuillemard C. 152 

W 

Waerzeggers Y. 219 

Wang H. 196 

Watson J. 90 

Weber C. 72 

Weber W. 27, 117, 206 

Wehrl H. F. 34 

Weidl E. 97 

Weigelin B. 161 

Weinans H. 167 

Wester H.J. 87, 97, 178 

Whelan P.F. 210 

Wiart M. 65 

Wicki A. 52 

Wiedenmann B. 140 

Wiehr S. 34, 200, 201 

Wielopolski P. 163, 167, 168 

Wilczynski G. 67 

Wild D. 52 

Wilson L. J. 133 

Winkeler A. 160, 219 

Woff E. 138 

Wolfs E. 170 

Wouters L. 149 

Wurdinger T. 185 

X 

Xavier C. 29, 136 

Xie B. 106, 119, 125 

Y 

Yang E. 164 

Yang X. 164 

Yared W. 89 

Ylä-Herttuala S. 71 

Young A. 148 

Yudina A. 88 

Z 

Zacharopoulos A. 217 

Zannetti A. 143 

Zara G. 218 

Zeebregts C. J. 99 

Zenga F. 218 

Zheng J. 160 

Zhong P. 164 

Ziegler S. 40 

Zwier J. 135

230 


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imaging life

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