PhD thesis defense. Multidisciplinary approach to study HIF2a/PD-1 axes in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is characterized by the inactivation of the Von Hippel-Lindau (VHL) tumour suppressor gene, leading to the stabilization and accumulation of hypoxia-inducible factor 2α (HIF2α) and constitutive activation of hypoxia-inducible pathways. This thesis aimed to investigate the role of HIF2α as a central driver of tumour progression, immune evasion and metabolic adaptation in ccRCC. A multidisciplinary approach was employed to assess the molecular and phenotypic consequences of HIF2α inhibition (using belzutifan) and genetic knockout (via CRISPR-Cas9) in both VHL-mutant and VHL-wild-type ccRCC cell lines. In parallel, the immunomodulatory effects of belzutifan and immune checkpoint blockade (pembrolizumab) were evaluated ex vivo using patient-derived tumour fragments (PDTFs). To explore metabolic adaptation, a custom-designed 3D-printed culture platform compatible with Raman spectroscopy was developed. This system enabled spatially and temporally resolved analysis of extracellular metabolites under controlled oxygen and nutrient gradients. The results showed that HIF2α inhibition modulates immune-related gene expression, reshapes the tumour microenvironment and may enhance the efficacy of pembrolizumab in combination therapy. Moreover, Raman-based metabolic profiling revealed that HIF2α knockout drives lactate accumulation and extracellular acidification in ischemic regions. Overall, this work demonstrates how integrating molecular, immunological and bioengineering strategies can advance our understanding of ccRCC and inform future therapeutic studies.