Evaluating <scp>VEGFR2</scp> as a Target for Anti‐Tumour Therapy in Canine Melanoma

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for anti-angiogenic oncotherapy, as inhibiting this receptor on tumour vasculature slows tumour development and enhances drug- and immune infiltration, improving therapy outcome. Although VEGFR2 is primarily expressed on endothelial cells (ECs), it is also found on neoplastic cells in both humans and dogs, including canine malignant melanoma (CMM). In this study, we compared current methods for assessing VEGFR2 expression in CMM and healthy tissues to elucidate the targets of anti-VEGFR2 therapy in canines. VEGFR2 protein expression was analysed using various anti-VEGFR2 antibodies for immunohistochemistry, and mRNA expression was analysed using RT-PCR. Surprisingly, a marked difference in the detection of VEGFR2 was observed between the anti-VEGFR2 antibody clones used, despite recognition of the same sequences. Supported by additional Western blot and confocal fluorescence microscopy analysis, we observed two distinct staining patterns: a specific pattern predominantly labelling ECs and a more nonspecific pattern predominantly labelling non-ECs, including neoplastic melanocytes. Notably, the more specific pattern demonstrated significantly more VEGFR2 expression in ECs within CMM. These findings indicate that the interpretation of VEGFR2 expression on neoplastic cells is highly dependent on antibody specificity, leading to potential overestimation in some studies. We therefore suggest that anti-VEGFR2 therapy primarily targets the tumour vasculature rather than the tumour cells. This highlights the need to reconsider the aims of anti-VEGFR2 therapies in companion animals.