VEGFR1-Targeted Contrast-Enhanced Ultrasound Imaging Quantification of Vasculogenic Mimicry Microcirculation in a Mouse Model of Choroidal Melanoma

Purpose: Investigate the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in vasculogenic mimicry (VM) formation in ocular melanoma, as well as whether or not VEGFR1-targeted contrast-enhanced ultrasound (CEUS) can evaluate and quantify VM perfusion and function in the ocular melanoma model. Methods: The expression of VEGFR1 was examined using immunofluorescence, western blot, and quantitative polymerase chain reaction. VM networks were analyzed with tube formation and periodic acid Schiff staining. Targeted microbubbles (MBs) were constructed and used for targeted CEUS imaging in vivo. Comparisons were made in perfusion parameters of tumors between targeted and non-targeted CEUS imaging. Results: VEGFR1 was highly expressed, and knockdown of VEGFR1 significantly decreased VM protein expression and disrupted VM formation in MUM-2B melanoma. VEGFR1-targeted MBs specifically bind to MUM-2B cell surfaces. CEUS with VEGFR1-targeted MBs showed significant imaging enhancement throughout the entire perfusion phase compared with CEUS with IgG MBs. VEGFR1-targeted imaging was able to detect a decrease in maximum intensity and mean transit time in VEGFR1 knockdown melanoma compared with control melanoma. The pathological VM patterns were consistent with VEGFR1-targeted CEUS findings. Conclusions: VEGFR1 was responsible for VM network formation and was required for efficient choroidal melanoma tumor growth. This study shows that VEGFR1-targeted CEUS can track VM levels in animal models of ocular melanoma at morphological levels in vivo. This experiment is noninvasive and reproducible and indicates the possibility of real-time in vivo imaging technology for VM evaluation. Translational Relevance: Based on our study results, VEGFR1 could prove to be a promising treatment that targets VM formation in choroidal melanoma. Our findings also suggest the potential use of VEGFR1-targeted CEUS for quantitative monitoring of VM processes at the molecular level in the future.