Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to v 3

Background—Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrastenhanced ultrasound (CEU) with microbubbles targeted to v-integrins expressed on the neovascular endothelium could be used to image angiogenesis. Methods and Results—Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to v 3 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular v-integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2 1 versus 35 14 mm, P 0.001). Tumor blood volume increased by 35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, v 3-targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from v 3-targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7 0.4 versus 3.3 1.0 relative units, P 0.05). CEU signal from v 3-targeted microbubbles was greatest at the periphery of tumors, where v-integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r 0.86). Conclusions—CEU with microbubbles targeted to v 3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications. (Circulation. 2003;108:336-341.)