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
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