Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo

Purpose: 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 (MBD) with that from single-targeted MBs (MBS) in a murine model of tumor angiogenesis. Materials and 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 (MBC) and nontargeted nonlabeled MBs (MBN) 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 (MBV), anti-alpha(v)beta(3) integrin-targeted MBs (MBI), MBD, and MBC 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 MBD 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 MBD than for MBV (mean, 0.58 +/- 0.09), MBI (mean, 0.42 +/- 0.21), MBC (mean, 0.11 +/- 0.13), and MBN (mean, 0.01 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MBD (mean, 16.7 +/- 7.2) than for MBV (mean, 11.3 +/- 5.7), MBI (mean, 7.8 +/- 5.3), MBC (mean, 2.8 +/- 0.9), and MBN (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature. Conclusion: 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. (C) RSNA, 2008.