VEGF

Application of MRI for imaging
vascular remodeling
Michal Neeman
[email protected]
Athens July, 2014
1um
What can we image
10 um
• Angiogenesis is required but
not sufficient for exit from
100 um
dormancy
• VEGF as a regulator of
vascular permeability
1 mm
• Vascular maturation defines
the window for
1 cm
antiangiogenic therapy
• Perivascular myofibroblasts
affect permeability,
10 cm
vasoreactivity and induce exit
from tumor dormancy
1m
mutation
transformation
dormancy
Angiogenesis?
detection
metastasis
fPV
C0/Cplasma
60 min
post-contrast
C6-pTET-VEGF s.c. tumors in the
limb of cd-1 nude mice
[Biotin-BSA-GdDTPA] (µM)
6
APS
dC/dt
Delayed enhancement maps interstitial convection and lymphatic drain
Time2Max
55
0.2
0.035
Min
µM/min
5
4
3
2
1
0
0
20
1
3
2
40
5
7
Time (min)
4
6
60
9
8
Time intervals (min)
11
10
5
Min
0
0
High VEGF
-tetracycline
C6-pTET-VEGF tumor: response to VEGF withdrawal
Vascular leak provides a functional biomarker for VEGF activity
Low VEGF
+tetracycline
min
Time2Max
fPV
APS (µM/min) in the interstitium
VEGF provides60
the
driving force for movement
of macromolecules
post-contrast
60 min
post-contrast
Dafni et al, Cancer Res 2002
(Convection)
fPV
APS (µM/min)
Time2Max
(Convection)
Interstitial convection and Lymphatic drain
Late lymphatic drain (6 h post-contrast)
+ second contrast
Contrast media
Biotin-BSA-GdDTPA(FAM/ROX)
Tumor
C6-pTET-VEGF s.c. tumors in the limb of
cd-1 nude mice
3D-GE MIPs
TR 10 ms, TE 3.6 ms, Flip angle 30
Resolution 156 µm X 156 µm X 312 µm
Acquisition time 163 s (2 averages)
Dafni et al Cancer Res 2002; MRM 2003
Avidin Chase
VEGF
VEGF-R
b
BSA
b
BSA
Blood
Gd
Gd
b
b
Rapid elimination
from the blood
BSA
BSA
Gd
Gd
Albumin
b
Avidin
b
Lymph
b
BSA
Gd
Avidin Chase
VEGF
VEGF-R
Blood
b
b
BSA
BSA
Gd
Gd
Albumin
b
Avidin
b
Lymph
b
BSA
Gd
Separation of vascular leak and lymphatic drain:
in vivo avidin chase
Avidin
Contrast enhancement
(0-3 min postcontrast)
Avidin chase
(60-72 postcontrast)
0.02
Red: signal increase
Green: signal decrease
Dafni et al Cancer Res 2002; MRM 2003
-0.02
min -1
Overcoming antiangiogenic inhibition
Mature extracellular matrix
Anti-angiogenic
MRI of Heparanase
expressing tumors
HSPG
heparanase
Release of
bFGF, VEGF
High MW
hyaluronan
hyal
low MW
Hyaluronan
NIR imaging of thrombin
fibrinogen
Provisional extracellular matrix
Pro-angiogenic
MRI of
hyaluronidase
NIR & MRI of
transglutaminase
fibrin
tTG
angiostatin
plasminogen
uPA
endostatin
collagen
thrombin
Fibrin targeted
contrast media
Cross-linked fibrin
MRI of angio/endostatin
expressing tumors
Neeman et al JMRI 2007
MRI of
MMP activity
plasmin
Mapping vascular function and maturation by
BOLD contrast MRI
Vascular maturation is associated with gain of vasoreactivity and with resistance to VEGF
withdrawal.
Abramovitch et al Cancer Res. 59: 5012-6, 1999.
Vascular obliteration by VEGF withdrawal: BOLD contrast MRI
Functional vessels
Response to hyperoxia
Mature vessels
Response to hypercapnia
No tetracycline
High VEGF
T-test
p value
0.05
With tetracycline
Low VEGF
0
Abramovitch et al Cancer Res. 59: 5012-6, 1999.
Gilead Neoplasia, IJC 2004; Gilad IJC 2005
Vascular maturation
Ang-1
Ang-1
Ang-2
High
maturation
Rapid angiogenesis
Ang-2
0.05
Slow angiogenesis
VEGF
Ang-2
Vascular permeability:
Ang-1
APS (mM/min)
Vascular reactivity:
biomarker for expression of VEGF
biomarker for expression of Ang1/Ang2
VEGF
Vascular pruning
Vascular stability
Ang-2
VEGF
Low
0
0.05
VD (p-value)
0
Uncoupling between vascular maturation and permeability:
MLS human epithelial ovarian carcinoma in nude mice
Ang-1
Ang-2
VEGF-A
High
Low
Assaf Gilad et al Int J Cancer 2005
Ang-1
Ang-2
Tumor associated fibroblasts and tumor angiogenesis
Blue DAPI
Red aSMA
Green biotin-BSA
Gilead et al Int J Cancer 2004
25 µm
Fibroblasts recruitment into ovarian carcinoma tumors
5
Biotin-BSA-GdDTPA (labeling CM)
R1 (s-1)
Tumor
0
control
labeled
MRI, 4.7 T, 3D GE
Tumor
6
arbitrary units (*106)
1
Dorit Granot et al Cancer Res 2007
Two photon microscopy
Recruitment of myofibroblasts into ovarian carcinoma tumors
Addadi et al intravital microscopy
Fibroblast trans-differentiation to myofibroblasts
Green
Blue
Red
MLS EGFP ovarian tumor
CV-1 fibroblasts expressing
alphaSMA-DsRed
Blood vessels: DiD iv
Addadi 2012, unpublished
ES2-GFP CV1-Tom co-culture
A small preference for one's neighbors to be of the
same color could lead to total segregation
Schelling, Thomas C. 1971. "Dynamic Models of Segregation." Journal
of Mathematical Sociology 1:143-186.
ES2-GFP + CV1-Tom co-culture
3d
4d
Can fibroblast homing be used for tumor detection?
4x106 (GFP/ DiD)
CV1 fibroblast
1x106 (tomato) ES2
cells or spheroids
DiD/ FITC dextran
IP
IP
1 week
IV
Imaging at different
time points (2P/axio)
• CD1-NU female 8 weeks old mice.
• Even 2 weeks after tumor injection tumors were barely visible
by eye.
2 day after CV1 injection
Gut area
Red - tumor
Green – fibroblasts
axio microscope
Ferritin as a reporter gene for MRI
Fe3+
Fe2+
transferrin
transferrin
Transferrin-R
Transferrin-R
endosome
O2
O2
H2O2
OH*
Fe2+
Ferritin
Heme synthesis
and other iron enzymes
Recruitment of Ferritin-Expressing Fibroblasts to the Angiogenic Rim of Tumors
Vandsburger 2013 Radiology
Two compartment analysis of fibroblast
recruitment
Vandsburger 2013 Radiology
Application of MRI for imaging vascular remodeling
• BOLD contrast MRI of vasoreactivity reveals vascular
maturation as a critical regulator of VEGF dependence
(Abramovitch R)
• Vascular permeability and lymphatic drain (Dafni H)
• Hyaluronan control of angiogenesis (Shiftan L)
• Perivascular myofibroblasts, permeability, vasoreactivity
and exit from tumor dormancy (Gilad AA)
• Systemic recruitment of tumor associated fibroblasts
(Granot D)
• Tumor induced activation of fibroblasts (Addadi Y; Oren R)
• Ferritin as a reporter gene for detection of fibroblast
recruitment (Vandsburger M)
Acknowledgements
Rinat Abramovitch
Hagit Dafni
Assaf Gilad
Dorit Granot
Liora Shiftan
Yoseph Adaddi
Roni Oren
Michal Weiler-Sagi
Moriel Vandsburger
Marina Radoul
Gila Meir
Batya Cohen
Ofra Golani
Gera Neufeld, Technion
Eli Keshet, Hebrew University
Laura Benjamin, Harvard
Brian Hemmings, Basel
Leoni Kunz-Schughart, Dresden
Fabian Kiessling, Aachen
Moshe Oren, Weizmann
Raz Zarivach, Ben Gurion University