Dual-targeted Contrast Agent for US Assessment of Tumor Angiogenesis in Vivo

Purpose: To develop and validate a dual-targeted ultrasound imaging agent that attaches to both vascular endothelial growth factor receptor-2 (VEGFR2) and αvβ3-integrin, and to compare the ultrasonic imaging signal obtained from this dual-targeted agent with singly-targeted microbubbles in a murine model of tumor angiogenesis. Materials and Methods: Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Singly-targeted and dual-targeted ultrasound imaging agents were prepared by attaching either anti-VEGFR2 (MBVEGFR2), anti-αv (MBαvβ3), or both antibodies (MBDual) onto the shell of perfluorocarbon-filled microbubbles. Binding specificities of targeted microbubbles compared to control microbubbles (isotype-matched IgG-labeled [MBControl] and non-labeled microbubbles [MBNon-targeted]) were tested on VEGFR2- and αvβ3-integrin-positive cells (mouse SVR-cells) and control cells (mouse 4T1-cells). In vivo imaging signal of contrast-enhanced ultrasound using MBVEGFR2, MBαvβ3, MBDual, and control microbubbles were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, αvβ3 integrin, and CD31. Results: Attachment of MBDual was significantly higher to SVR-cells (0.74±0.05 microbubbles per cell) than to 4T1-cells (0.04±0.03) and attachment to SVR-cells was higher for MBDual than for MBVEGFR2 (0.58±0.09), MBαvβ3 (0.42±0.21), MBControl (0.11±0.13), and MBNon-targeted (0.01±0.01) (P<.05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P<.0001) for MBDual (16.7±7.2) than for MBVEGFR2 (11.3±5.7), MBαvβ3 (7.8±5.3), as well as for both MBControl (2.8±0.9) and MBNon-targeted (1.1±0.4). Immunofluorescence confirmed expression of VEGFR2 and αvβ3-integrin on tumor vasculature. Conclusion: Dual-targeted contrast-enhanced ultrasound imaging directed at both VEGFR2 and αvβ3-integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice.