1 anikitos garofalakis cea, i 2 bm, shfj, lime, inserm u803 wp6: cancer imaging with focus on breast...

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

23

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