In Vivo Targeted Contrast Enhanced Micro-Ultrasound to Measure Intratumor Perfusion and Vascular Endothelial Growth Factor Receptor 2 Expression in a Mouse Orthotopic Bladder Cancer Model

Abstract

We evaluated the feasibility of using targeted contrast enhanced micro-ultrasound imaging to assess intratumor perfusion and vascular endothelial growth factor receptor 2 expressions in a mouse orthotopic bladder cancer model. We created an orthotopic mouse model by implanting MBT-2 murine bladder cancer cell lines in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Successful tumor implantation was confirmed by transabdominal micro-ultrasound imaging on post-implantation day 11. Contrast enhanced micro-ultrasound imaging was done on days 14 and 21. Vascular endothelial growth factor receptor 2 targeted contrast agent was prepared by adding biotinylated anti-vascular endothelial growth factor receptor 2 monoclonal antibodies to streptavidin coated microbubbles. The targeted contrast agents were injected via the retro-orbital route. We quantified intratumor perfusion, vascular endothelial growth factor receptor 2 endothelial expression and blood volume in real time. In the initial study intratumor perfusion data and vascular endothelial growth factor receptor 2 expression could only be measured in 10 of 14 mice (71%) due to motion artifact. We modified our technique by applying an elastic band over the lower abdomen to minimize body wall movement. After the modification complete images were acquired in all mice at 2 consecutive imaging sessions. Measurements were made of intratumor perfusion and in vivo vascular endothelial growth factor receptor 2 expression. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. Targeted contrast enhanced micro-ultrasound imaging enables investigators to detect and monitor vascular changes in orthotopic bladder tumors. It may be useful for direct, noninvasive, in vivo evaluation of novel anti-angiogenesis therapeutic agents. With the modified technique target enhanced contrast ultrasound can be applied in an orthotopic bladder cancer model.