Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo.
OBJECTIVE
To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and alpha(v)beta(3) integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB(D)) with that from single-targeted MBs (MB(S)) in a murine model of tumor angiogenesis.


METHODS
Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti-alpha(v)beta(3) integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G-labeled control MBs (MB(C)) and nontargeted nonlabeled MBs (MB(N)) were tested with VEGFR2-positive and alpha(v)beta(3) integrin-positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material-enhanced US by using anti-VEGFR2-targeted MBs (MB(V)), anti-alpha(v)beta(3) integrin-targeted MBs (MB(I)), MB(D), and MB(C) were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, alpha(v)beta(3) integrin, and CD31.


RESULTS
Attachment of MB(D) to SVR cells (mean, 0.74 MBs per cell +/- 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 +/- 0.03), and attachment to SVR cells was higher for MB(D) than for MB(V) (mean, 0.58 +/- 0.09), MB(I) (mean, 0.42 +/- 0.21), MB(C) (mean, 0.11 +/- 0.13), and MB(N) (mean, 0.01 +/- 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB(D) (mean, 16.7 +/- 7.2) than for MB(V) (mean, 11.3 +/- 5.7), MB(I) (mean, 7.8 +/- 5.3), MB(C) (mean, 2.8 +/- 0.9), and MB(N) (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature.


CONCLUSIONS
Dual-targeted contrast-enhanced US directed at both VEGFR2 and alpha(v)beta(3) integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice.


BACKGROUND
http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1.
Willmann
Jürgen K
JK
Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Stanford University School of Medicine, the James H Clark Center, 318 Campus Dr, East Wing, 1st Floor, Stanford, CA 94305-5427, USA.
Lutz
Amelie M
AM
Paulmurugan
Ramasamy
R
Patel
Manishkumar R
MR
Chu
Pauline
P
Rosenberg
Jarrett
J
Gambhir
Sanjiv S
SS
D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D003287:Contrast Media / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods; D005260:Female; D006801:Humans; D007089:Image Enhancement; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D045423:Microbubbles; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging* / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000098:blood supply / Q000000981:diagnostic imaging* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014463:Ultrasonography; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D014408:Biomarkers, Tumor; D003287:Contrast Media; D039302:Integrin alphaVbeta3; D040301:Vascular Endothelial Growth Factor Receptor-2