1 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Wp6: Cancer imaging with focus on breast cancer Anikitos GAROFALAKIS CEA, DSV, I 2 BM, SHFJ, LIME, INSERM U 803 Laboratoire d’imagerie de l’expression des gènes FMT – XCT meeting, Munich, April 24th

2 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Overview Background Animals models of breast cancer Fluorescent probes Instrumentation and measuremements Conclusions

3 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Background Objectives: 6.1 To provide key fluorescence probes and quantify the sensitivity and contrast achieved in the animal models developed and as a function of tumor growth. 6.2 To develop animal models of breast cancer for studying FMT-XCT performance. 6.3 To develop animal models of other cancers for studying FMT-XCT performance. 6.4 To perform in-vivo imaging of key animal models of cancer and correlate the findings with standard laboratory tests and growth measures 6.5 To predict clinical utility Phantoms the standard way of evaluating the performance of an optical tomographer For in-vivo imaging of the FMT-XCT there is need of using animal models to achieve realistic conditions Objectives: 6.1 To provide key fluorescence probes and quantify the sensitivity and contrast achieved in the animal models developed and as a function of tumor growth. 6.2 To develop animal models of breast cancer for studying FMT-XCT performance. 6.3 To develop animal models of other cancers for studying FMT-XCT performance. 6.4 To perform in-vivo imaging of key animal models of cancer and correlate the findings with standard laboratory tests and growth measures 6.5 To predict clinical utility

4 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 MDAMB-231 human breast adenocarcinoma cells Over-expression MT4-MMP RAG-1 immunodeficient mice Mammary Tumor Xenografts erb – B2 expressing cell cultures Target for trastuzumab (Herceptin®), used in breast cancer chemotherapy

5 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Mammary tumor transgenic mice models polyoma middle T oncoprotein(PyMT), under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) Optical Imaging

6 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Fluorescent Probes i)commercial probes(Angiosense 680, Superhance 680) ii)Home-made probes (Trastuzumab, Aptamers) Aptamers Aptamers are nucleic acid ligands selected by an iterative selection procedure named SELEX ("Systematic Evolution of Ligands by Exponential Enrichment"). We have validated a whole cell-SELEX protocol specific for high lung metastatic cells(versus low lung metastatic cells).

7 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 SELEX 7 Target Selection Removal of low affinity sequences Specific selection of ligands of interest mutation Amplification Evolution 1st round

8 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 General Scheme Animal models xenografts: MDAMB – 231 (human breast adenocarcinoma over expressing MT4 - MMP) human erb – B2 + / - transgenic mice models: PyMT transgenic Fluorescent Probes commercial probes (Angiosense 680, Superhance 680) aptamers home made Probes (Trastuzumab – AFluor680)

9 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Planar Optical Imaging The in vivo biodistribution of aptamers directed against RET2A

10 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Resolution (~ 1mm in the x-y plane And 3mm in the z plane) Depth resolution Whole body imaging Quantitative imaging at any depth x yz Tomographic(3D) Imaging

11 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Mammary Tumor Xenografts MDAMB-231 Planar Imaging using AngioSense™ 680 Intravenous administration

12 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 z = 9 mmz = 10 mmz = 11 mmz = 12 mmz = 13 mm C4 M10 Tomographic(3D) Imaging Mammary Tumor Xenografts MDAMB-231

13 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Mean reconstructed signal mmp/control =1.14 M8, M9, M10 vs C4, C5M6 vs C3 mmp/control =1.44

14 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Distribution of voxels over Intensity Interval of 2500 a.u.Interval of 1250 a.u.

15 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Distribution of voxels over Intensity Distribution over a threshold/crossing point of 5000 counts

16 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Mammary Tumor Xenografts erb – B2 expressing cells In collaboration with: Marie France Poupon, Institut Curie, Paris Laboratory of antibody imaging for health, Saclay (Didier Boquet) Planar Imaging using trastuzumub (Herceptin ® ) Xenografts of primary human mammary carcinoma over-expressing Her-2

17 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Mammary tumor transgenic mice models

18 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Applicable problems Difficulties: One breeding accident with the PyMT mice has delayed partly our programme with these animals No changes in planned activities.

19 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Advancement of programme Planned Deliverables (month of delivery planned at start of first year) 6.1 To develop and characterize molecular probes (mo.9, 18) 6.2 Prepare and characterize mammary cancer animal models (mo.18) 6.3 Breed and making available PyMT animal models (mo.24) 6.4 Develop U87 animal models (mo.27) 6.5 Study and report the quantitative accuracy of FMT-alone and FMT-XCT in resolving tumors (mo. 36) 6.6 Study and report cancer detection performance in various organs (mo. 40) 6.7 Report the overall imaging performance. (mo.42) Status of achievement : achieved and continued. 6.4: to start year 2 6.5: to start when FMT becomes available : to begin after 6.5 Planned Deliverables (month of delivery planned at start of first year) 6.1 To develop and characterize molecular probes (mo.9, 18) 6.2 Prepare and characterize mammary cancer animal models (mo.18) 6.3 Breed and making available PyMT animal models (mo.24) 6.4 Develop U87 animal models (mo.27) 6.5 Study and report the quantitative accuracy of FMT-alone and FMT-XCT in resolving tumors (mo. 36) 6.6 Study and report cancer detection performance in various organs (mo. 40) 6.7 Report the overall imaging performance. (mo.42) Status of achievement : achieved and continued. 6.4: to start year 2 6.5: to start when FMT becomes available : to begin after 6.5 Planned Deliverables (month of delivery planned at start of first year) 6.1 To develop and characterize molecular probes (mo.9, 18) 6.2 Prepare and characterize mammary cancer animal models (mo.18) 6.3 Breed and making available PyMT animal models (mo.24) 6.4 Develop U87 animal models (mo.27) 6.5 Study and report the quantitative accuracy of FMT-alone and FMT-XCT in resolving tumors (mo. 36) 6.6 Study and report cancer detection performance in various organs (mo. 40) 6.7 Report the overall imaging performance. (mo.42) Status of achievement : achieved and continued. 6.4: to start year 2 6.5: to start when FMT becomes available : to begin after 6.5 Planned Deliverables (month of delivery planned at start of first year) 6.1 To develop and characterize molecular probes (mo.9, 18) 6.2 Prepare and characterize mammary cancer animal models (mo.18) 6.3 Breed and making available PyMT animal models (mo.24) 6.4 Develop U87 animal models (mo.27) 6.5 Study and report the quantitative accuracy of FMT-alone and FMT-XCT in resolving tumors (mo. 36) 6.6 Study and report cancer detection performance in various organs (mo. 40) 6.7 Report the overall imaging performance. (mo.42) Status of achievement : achieved and continued. 6.4: to start year 2 6.5: to start when FMT becomes available : to begin after 6.5

20 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U803 Aknowledgements Experimental molecular imaging laboratory (LIME) Bertrand Tavitian Frédéric Ducongé Raphael Boisgard Stéphanie Ricaud Carine Pestourie

21 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U To develop animal models of other cancers for studying FMT-XCT performance. 1. Develop rodent models of brain tumors 6.4 To perform in-vivo imaging of key animal models of cancer and correlate the findings with standard laboratory tests and growth measures 6.5 To predict clinical utility Future tasks

22 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U Target Selection Removal of low affinity sequences Specific selection of ligands of interest mutation Amplification Evolution

23 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U Target Selection Removal of low affinity sequences Specific selection of ligands of interest mutation Amplification Evolution 1st round

24 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U Target Amplification Evolution Selection Specific selection of ligands of interest Removal of low affinity sequences Bank of RNA 2’F-Py Transcription DNA library RT-PCR Extraction Selection Contre seection 2 PC12 MEN2A PC12 MEN2B PC12 wt Contra selection 1 a.

25 Anikitos GAROFALAKIS CEA, I 2 BM, SHFJ, LIME, INSERM U Target Amplification Evolution Selection Specific selection of ligands of interest Removal of low affinity sequences Cloning and sequencing N rounds Aptamers