The environmental and metabolic pressures in the tumor microenvironment (TME) play a key role in molding tumor development by impacting the stromal and immune cell fractions, TME composition and activation. Hypoxia triggers a cascade of events that promote tumor growth, enhance resistance to the anti-tumor immune response and instigate tumor angiogenesis. During growth, the developing angiogenesis is pathological and gives rise to a haphazardly shaped and leaky tumor vasculature with abnormal properties. Accordingly, aberrantly vascularized TME induces immunosuppression and maintains a continuous hypoxic state. Normalizing the tumor vasculature to restore its vascular integrity, should hence enhance tumor perfusion, relieving hypoxia, and reshaping anti-tumor immunity. Emerging vascular normalization strategies have a great potential in achieving a stable normalization, resulting in mature and functional blood vessels that alleviate tumor hypoxia. Biomarkers enabling the detection and monitoring of tumor hypoxia could be highly advantageous in aiding the translation of novel normalization strategies to clinical application, alone, or in combination with other treatment modalities, such as immunotherapy.

2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.