Wp6. Cancer Imaging with focus on
bbreast cancer
Anikitos Garofalakis
‘Frederic Joliot Hospital Service(SHFJ), Institute of Biomedical
imaging(I2BM), Atomic Energy Commission(CEA)’g g( ) gy ( )
1Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
background
The use of animal models for the evaluation of FMT under realistic conditions
Objectives of Wp6:
To provide cancer animal models
To provide key fluorescence probes
Quantitatively examine FMT performance to visualize disease processes in-vivo
To predict clinical utility (48 month deliverable)
2Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Animal modelsmodels and probes
MCF 7MDA 231 U87PC12 MEN2A
a. xenografted tumors
PyMT
b. spontaneous tumors
Animal models
MCF-7MDA-231 U87 human glioma
PC12-MEN2A rat pheochromocytoma
PyMT
Fluorescent probes
Prosense RGD-based Angiosense
a. commercial probes
Aptamers Nano-micelles
b. custom-made probes
Prosense RGD basedoptical probes(Integrisense680,Angiostamp680)
Angiosense
3Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Cathepsin activity Integrin avβ3 Blood volume
fluorescent probes for tumor-related processes
FDG
Integrin avβ3 Neo-angiogenesis
CathepsinECM d d tiECM degradation
Blood volume
4Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Quantitatively examine FMT performance to visualize disease processes in-vivo
targetedbiological molecule 1
FDG
μPET FMT
Type of information Molecular Molecular
Resolution ~ 1 mm3 ~ 1 mm3
Kinetics Fast (~seconds) 5-15 min/scan
Whole Body Imaging YES NO
Time range of follow up ~ hours ~days
Cost $$$ $Cost $$$ $
Activatable probes NO YES
Labelling Need radiochemistry Simple
Quantification Linear from pM ?
5Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Q p ?
calibration of FMT
Use of tubes filled with fluorophores
Use of the quantification capacity of PET imaging in combination with a dual PET/Optical probe
targetedbi l i l biological molecule
PET
vs [C]
FMT
6Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
FMT-PET-CT imaging
in vivo
7Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
fDOT-PET-CT imaging/protocol
PET PETFMT CT
0 60 min30 90
CT contrast agenttargetedbiological molecule
8Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
calibration/FMT-PET-CT imaging
PET FMT PET CT
FMT/CTul
ated
es
) 800
1000
quan
tity
calc
y PE
T (
pmol
e
600
PET/CT
Prob
e by
200
400 PET/CT
Time (min)
0 20 40 60 80 100 120 140
9Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b/./calibration/fDOT-PET-CT imaging/volume validation
PET/CT fDOT/CT
Coronal view
Axial viewAxial view
PET segmented volume
Optical signal segmented volume
CT contrast
10Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
CT contrast
b/./calibration/fDOT-PET-CT imaging/quantification
1000
(M
) 10
oles
by
PET
10
100
tion
by P
ET
0,1
1
pm
o
0 1
1
Con
cent
rat
0 001
0,01
fDOT (a.u.)101 102 103 104 105
0,1
fDOT concentration (a.u.)0,1 1 10 100
0,001
Sensitivity limit ~ 1 pmoles, 5nM(C)
11Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Garofalakis et al, Optics Letters (2010)
PET-FMT dual imaging
targetedbiological molecule
μPET FMT
Type of information Molecular Molecular
Resolution ~ 1 mm3 ~ 1 mm3
Kinetics Fast (~seconds) 5-15 min/scan
Whole Body Imaging YES NO
Time range of follow up ~ hours ~days
Cost $$$ $Cost $$$ $
Activatable probes NO YES
Labelling Need radiochemistry Simple
Quantification Linear from pM Linear from nM
12Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Linear from nM
cancer imaging in combination with PET
Angiostamp680ControlAngiostamp680Angiosense750
FMT PET CT FMT
day -1 day 0 day 1 day 2 ….
Prosense680
13Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b
MDA-231/FDG-Cathepsin
a b
3D Optical( )
0.92
0.75 mm(Prosense)
0.59μM
PET [F18]FDG
c
Volume Mean
Prosense_DAY_1 317.781 3.5
Prosense_DAY_2 412.45 3.5
Prosense_DAY_3 373.337 4.0
14Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
MDA-231/FDG – Integrin avβ3
RGD-based ligand Control ligand
0.24
0 12
0.04
0 02
0
0.12
uM0
0.02
uM
15Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
models and probes
MCF-7MDA-231 U87 human glioma
PC12-MEN2A rat pheochromocytoma
a. xenografted tumors
PyMT
b. spontaneous tumors
human gliomarat pheochromocytoma
Probe to tumor
MDA231/ Cathepsin
activity
MDA231/ Integrin
localization
MDA231/ Nano-micelle labelling
PC12-MEN2A/Cathepsin activity
PC12-MEN2A/Cathepsin activity/tumor
% signaloverlapping 6,6 ± 2,5 6,7 ± 5,0 34 ± 12 31,8±12,0
p yIntegrin localization
% volumeHMGU@MunichD l 7 4% volume
overlapping 37,6 ± 8,1 39,0 ± 18,6 40,03 ± 19,02 41,2±12,5 Del 7.4
16Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
MCFS7 xenografted tumor/aptamers ACE8
0.80.9
0 30.40.50.60.7
D in
tum
our
“Annexin II” as potential target ?
ACE8 Control0.00.10.20.3
%ID
*
17Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
summary
To provide cancer animal models
Del 6.3 To develop animal models of other cancer for studying FMT-XCT performance (U87 glioma cells)
To provide key fluorescence probes
2nd year deliverable (target of aptamer ACE8 has to be identified)2 year deliverable (target of aptamer ACE8 has to be identified)
Quantitatively examine FMT performance to visualize disease processes in-vivop
Del 6.4 To perform in-vivo imaging of key animal models of cancer and correlate the findings with standard laboratory tests and gro th meas resand growth measures
To predict clinical utility (48 month deliverable)
18Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
aknowledgements
Albertine Dubois Dr
Laboratory of experimental molecular medicine (LIME)
Albertine Dubois, DrXiao Tong, Phd studentVincent Brulon, TechnicianRaphael Boisgard, Dr
Chemistry Laboratory for life sciences(LCV)Bertrand Czarny, Techniciang
Bertrand Tavitian, ProfFrédéric Ducongé, Dr
y,
Group of radiochemistry et radio-pharmacy (GRR) Bertrand Kühnast, DrFrederic Dollé Drex-members:
Agnes Cibiel, DrCarine Pestourie, DrDaniel Dupont, Dr
Frederic Dollé, Dr
Nicolas Mackiewicz, DrIsabelle Jassens, Technician
19Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
20Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
21Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
a/fDOT-alone output
xyz
x
PC12-MEN2A with SentiDye® 700
22Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
coronal sagittal
a/fDOT with CT
Tumor model MEN2A/Integrisense(angiogenesis)
fDOT is often combined with a structural modality
23Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Unmixed Unmixed
b/./cancer imaging in combination with PET/FDG-Two color fDOT
a b
UnmixedAngiostamp680
UnmixedAngiosense750
5.0
11.0
0.9
7.7
c dCT/Optical CT/Optical
μM μM
11.0 4.4
[F18]FDG segmented volume
Angiostamp680 signal segmented volume
FDG/Optical FDG/Optical
5.0μM
0.9μM
Angiosense750 signal segmented volume
e f g
h i j
24Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b/./calibration/fDOT-PET-CT imaging/protocol
PET PETfDOT CT
0 60 min30 90
Kidney contrastagent
Oligo
+Oligo
+
Known biodistributionKnown biodistribution
Deep seated organ
25Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
No degradation
b/./calibration/fDOT-PET-CT imaging
PET/CT fDOT/CT
26Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b/./calibration/fDOT-PET-CT imaging/volume validation
a) b)
PET segmented volumePET segmented volume
Optical signal segmented volume
CT contrast
PET/CT fDOT/CTPET/CT fDOT/CT
27Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b/./calibration/fDOT-PET-CT imaging/quantification
1000 10
calc
ulat
ed
mol
es) 100
robe
cal
cula
ted
M)
1
robe
qua
ntity
cby
PE
T (p
m
1
10
entr
atio
n of
pr
by P
ET
(
0,01
0,1
Reconstructed fDOT fluorescence (a.u.)
101 102 103 104 105
P
0,1
Reconstructed fDOT fluorescence concentration (a.u.)
0,1 1 10 100
Con
c
0,001
Reconstructed fDOT fluorescence (a.u.) ( )
Limit ~ 1 pmoles, 5nM(C)
28Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
29Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
a/fDOT imaging
hCd2-GFP mouse
Garofalakis A et alThree-dimensional in vivo imaging of green fluorescent protein-expressing T cells in mice with noncontact fluorescence molecular tomography. Mol Imaging. 2007 Apr-Jun;6(2):96-107
30Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Diffuse light in a homogeneous medium
continuous wave point source
DSUU )()()( 022 rrr
D
p
D D
l i f h d i J
+
solution for the energy density Jn
+ SourceeUr
)(rr
)(
31Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Diffusive semi – infinite medium
Integrisense 0h Integrisense 3h
b/./MDA-231/Integrisense and AngioStamp comparison
gIntegrisense and AngioStamp comparison
g
Tumor volume ~ 730 mm3
60,2 % signal of optical in the surrounding
30,01 % volume of tumor in the common area
31,42 % signal of optical in the surrounding
32,55 % volume of tumor in the common area
RAFT-RGD 0h RAFT - RGD 2h
Tumor volume ~ 670 mm3
47,29 % signal of optical in the surrounding
29,98 % volume of tumor in the common area
36,2 % signal of optical in the surrounding
36,48 % volume of tumor in the common area
32Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Discretization and weight matrix
• Sample the fluorescent concentration in N points
r1 r2 r3N points(voxels)• Convert integral equations into a system of linear equationssystem of linear equations
rny
x
For M projections : P[M×1] = W[M×N] · N[N×1]
ART method
33Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
ART method
b/./cancer imaging
Commercial probes
P 680Cathepsin Prosense 680Cathepsinactivity
Integrinsense680 (VisenMedical, USA)AngioStamp (Fluoptics, France)
Integrin localization
Custom-made probesCustom made probes
MPEG nano-micelles
Passivebinding of tumors
34Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
b/./calibration
Use of fluorophores of controledconcentration
Use of the quantification capacity of PET imaging in compination with a dual PET/Optical probe
vs [C]
L t th d b t t li ti Hi h t th d b t li ti
35Anikitos GAROFALAKIS CEA, I2BM, SHFJ, LIME, INSERM U1023
Low cost method but not realistic High cost method but realistic