3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

Clear cell renal cell carcinoma (ccRCC) is largely driven by Von Hippel Lindau (VHL) protein deficiency, promoting epithelial-mesenchymal transition, invasion, and hypervascularization, mediated by vascular endothelial growth factor (VEGF) signaling resulting in a structurally abnormal capillary network, which remains insufficiently defined. Previous studies correlating patient outcome with microvascular density yielded diverse results, underscoring the limitations of conventional parameters to fully capture vascular complexity. While VEGF-targeted anti-angiogenic first-line therapies such as sunitinib prolong progression-free survival in metastatic ccRCC, their efficacy is hampered by resistance mechanisms. This study aims to elucidate the three-dimensional architecture of ccRCC-specific vasculature in the tumor microenvironment, and its response to targeted therapies. Analysis of human ccRCC samples identified two distinct vascular structures, markedly differing from tumor capillaries, termed ponds and sheets, which were further characterized in patient-derived xenografts using advanced 3D microscopy on optically cleared samples. Ponds are large, dilated, irregular structures with wide cavity, whereas sheets are thin, elongated, and collapsed structures. To further dissect endothelial network morphogenesis, we developed an innovative in vitro 3D vascularized microtumor model, faithfully recapitulating the aberrant pond architecture. Dynamic live imaging unraveled the temporal relationship between tumor invasion and pond morphogenesis. Additionally, drug sensitivity assays demonstrated that ponds exhibit lower responsiveness to sunitinib compared to tumor capillaries. Altogether, our 3D model not only captures a specific architecture of ccRCC vascular network but also provides mechanistic insight into its development and therapeutic sensitivity. This model offers a promising avenue for personalized treatment assessment and for identification of novel therapeutic strategies. Statement of significanceA co-culture-engineered model integrating tumor spheroid invasion and capillary morphogenesis recapitulates the ccRCC endothelial structures and their response to treatments.