3D co-culture model of tumor spheroid and endothelial cells unveils ccRCC aberrant vasculature and distinct sensitivity to targeted treatments.

Clear cell renal cell carcinoma (ccRCC), the most common renal cancer, is largely driven by von Hippel Lindau (VHL) protein deficiency. VHL inactivation promotes epithelial-to-mesenchymal transition (EMT), invasion, and hypervascularization, through vascular endothelial growth factor (VEGF) signaling, resulting in an abnormally complex vasculature. Given the limited prognostic value of microvessel density and the frequent development of resistance to VEGF-targeted therapies, the architecture of the ccRCC vascular network is likely a critical, underexplored determinant of therapeutic response. This study investigates the three-dimensional (3D) architecture of ccRCC vasculature within the tumor microenvironment and its response to drugs. Examination of human ccRCC samples revealed two vascular structures, designated ponds and sheets, that are morphologically distinct from tumor capillaries. High-resolution 3D imaging of optically cleared patient-derived xenografts revealed that ponds formed large, irregular structures with wide luminal cavity, whereas sheets were thin, elongated, and collapsed. We engineered a 3D in vitro tumor spheroid-endothelial cell co-culture model, incorporating EMT-like tumor spheroids co-cultured with endothelial cells, that mimicked pond architecture. Time-lapse imaging revealed a temporal link between tumor invasion and pond morphogenesis. Drug testing demonstrated that temsirolimus, crizotinib, and sunitinib impaired capillary morphogenesis and tumor invasion to varying extents in monoculture, while co-culture model reduced overall drug efficacy. Importantly, ponds exhibited markedly reduced sensitivity to sunitinib compared with adjacent capillaries, suggesting that they may contribute to anti-angiogenic resistance. This study defines key features and heterogeneity of ccRCC vascular architecture and highlights ponds as candidate contributors to treatment failure and targets for future interventions.