34Hypoxia and immune suppression shapes therapeutic outcomes and metastases in clear cell renal cell carcinoma

Abstract Background Vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitors (VEGFR-TKIs) and aPD1 combinations are effective in multiple solid tumors, particularly in clear cell renal cell carcinoma (ccRCC), due to its characteristic pseudo-hypoxic, hyper-angiogenic state driven by biallelic VHL-loss. However, long-term durability is inferior to dual aPD1/aCTLA4 regimens, yet the mechanisms underlying these differences remain unclear. Methods Since tumor-associated macrophages (TAMs) are implicated in therapeutic resistance, we used immunofluorescence staining and scRNAseq (n = 37) to investigate TAM evolution following VEGFR-TKI, aPD1 and combined VEGFR-TKI/aPD1 treatment in a genetically engineered (Ksp1.3cre-ERT2Vhlf/fp53f/f Rb1f/f) ccRCC mouse model. We further corroborate our findings in a novel human scRNAseq cohort (n = 15) of on-treatment tumor samples from patients who received VEGFR-TKI/aPD1 or aPD1/aCTLA4 therapies and experienced a range of clinical responses before undergoing cytoreductive nephrectomy. Results By quantifying hypoxia using pimonidazole in mice, we reveal that VEGFR-TKIs induced severe intratumor hypoxia. This enabled the identification of hypoxia-responsive SPP1+ TAMs that are absent in baseline pseudo-hypoxic ccRCC tumors. This proxy of true hypoxia tracks with successful response to VEGFR-TKI/aPD1 in both mouse and human on-treatment samples, reflecting treatment-induced hypoxic necrosis. Paradoxically, high levels of pretreatment hypoxic TAM signatures predicted worse outcomes across multiple VEGFR-TKI/aPD1 trials including JAVELIN101 and IMMOTION150/151, as well as an MSKCC real-world data cohort (n = 44). Furthermore, extended exposure to hypoxia-inducing VEGFR-TKIs and aPD1 exacerbated metastasis in two syngeneic models of ccRCC. Conclusions In conclusion, our study suggests that VEGFR-TKIs induce tumor hypoxia in responsive ccRCC tumors, but preexisting hypoxia renders tumors refractory to VEGFR-TKI/aPD1 therapies. Our data also suggests that chronic therapy-induced hypoxia and inflammation may promote metastatic evolution, thus offering potential mechanistic insight into the poor durability of VEGFR-TKI/aPD1 regimens across multiple cancer types.