1 anikitos garofalakis cea, i 2 bm, shfj, lime, inserm u803 wp6: cancer imaging with focus on breast...
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1Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Wp6: Cancer imaging with focus on breast cancer
Anikitos GAROFALAKIS
CEA, DSV, I2BM, SHFJ, LIME, INSERM U 803Laboratoire d’imagerie de l’expression des gènes
FMT – XCT meeting, Munich, April 24th
2Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Overview
• Background
• Animals models of breast cancer
• Fluorescent probes
• Instrumentation and measuremements
• Conclusions
3Anikitos GAROFALAKIS CEA, I2BM, 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
4Anikitos GAROFALAKIS CEA, I2BM, 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
5Anikitos GAROFALAKIS CEA, I2BM, 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)
CT
SPECT TcO4
FDG PET
Late CarcinomaEarly CarcinomaAdenomaHyperplasiaHistologicalstaging
CT
SPECT TcO4
FDG PET
Late CarcinomaEarly CarcinomaAdenomaHyperplasiaHistologicalstaging
weeks5 15
Optical Imaging
6Anikitos GAROFALAKIS CEA, I2BM, 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 namedSELEX ("Systematic Evolution of Ligands byExponential Enrichment") .
• We have validated a whole cell-SELEX protocolspecific for high lung metastatic cells(versus low lungmetastatic cells).
7Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
SELEX
7
Target
Selection
Removal of low affinity sequences
Specific selection of ligands of interest
mutation
Amplification
Evolution
1st round
8Anikitos GAROFALAKIS CEA, I2BM, 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)
9Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
D4-36
18015012090604030201053 500Time (min)
D4-36scramble
photo
D4-36
18015012090604030201053 500Time (min)
D4-36scramble
photo
D4-36
18015012090604030201053 500Time (min)
D4-36scramble
photo
Planar Optical Imaging
The in vivo biodistribution of aptamers directed against RET2A
10Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Resolution (~ 1mm in the x-y planeAnd 3mm in the z plane)
Depth resolution
Whole body imaging
Quantitative imaging at any depth
x
yz
Tomographic(3D) Imaging
11Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Mammary Tumor Xenografts MDAMB-231
Planar Imaging using AngioSense™ 680
Intravenous administration
12Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
z = 9 mm z = 10 mm z = 11 mm z = 12 mm z = 13 mm 5000
13100
10400
7700
5000
13100
10400
7700
C4
M10
Tomographic(3D) Imaging
Mammary Tumor Xenografts MDAMB-231
13Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Mean reconstructed signal
mmp/control =1.14
M8, M9, M10 vs C4, C5 M6 vs C3
mmp/control =1.44
14Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Distribution of voxels over Intensity
Interval of 2500 a.u. Interval of 1250 a.u.
15Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Distribution of voxels over Intensity
Distribution over a threshold/crossing pointof 5000 counts
16Anikitos GAROFALAKIS CEA, I2BM, 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
17Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Mammary tumor transgenic mice models
Evaluation of the vascular permeabilityEvaluation of the vascular permeability
intra venous injection of Superhance™680 (Visen Medical) Superhance binds to albumin and has a half-life in plasma of approximately 2 h
increased vascular permeability of the tumours during tumoral development.
8 weeks 10 weeks 12 weeks
25 min post i.v.
60 min post i.v.
120 min post i.v.
0
200000
400000
600000
800000
1000000
0 100 200 300 400 500
PyMT 2001 8 weeksPyMT 2001 10 weeksPyMT 2001 12 weeks
Surf
.Act
ivit
y(c
pm
/mm
2)
Time post i.v. injection (min)
18Anikitos GAROFALAKIS CEA, I2BM, 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.
19Anikitos GAROFALAKIS CEA, I2BM, 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 achievement6.1-6.3 : achieved and continued.
6.4: to start year 2
6.5: to start when FMT becomes available
6.6-6.7: 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 achievement6.1-6.3 : achieved and continued.
6.4: to start year 2
6.5: to start when FMT becomes available
6.6-6.7: 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 achievement6.1-6.3 : achieved and continued.
6.4: to start year 2
6.5: to start when FMT becomes available
6.6-6.7: 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 achievement6.1-6.3 : achieved and continued.
6.4: to start year 2
6.5: to start when FMT becomes available
6.6-6.7: to begin after 6.5
20Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
Aknowledgements
Experimental molecular imaging laboratory (LIME) Bertrand Tavitian
Frédéric Ducongé
Raphael Boisgard
Stéphanie Ricaud
Carine Pestourie
21Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
6.3 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
22Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
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Target
Selection
Removal of low affinity sequences
Specific selection of ligands of interest
mutation
Amplification
Evolution
23Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
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Target
Selection
Removal of low affinity sequences
Specific selection of ligands of interest
mutation
Amplification
Evolution
1st round
24Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
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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
Contreseection 2
PC12 MEN2A
PC12 MEN2B
PC12 wtContraselection 1
a.
25Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U803
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Target
Amplification
Evolution
Selection
Specific selection of ligands of interest
Removal of low affinity sequences
Cloning and sequencing
N rounds
Aptamers