Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to α_vβ₃

Background - Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrast-enhanced ultrasound (CEU) with microbubbles targeted to α_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 α_vβ₃ by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular α_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 mm², 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, α_vβ₃-targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from α_vβ₃-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 α_vβ₃-targeted microbubbles was greatest at the periphery of tumors, where α_v-integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r = 0.86). Conclusions - CEU with microbubbles targeted to α_vβ₃ 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.