VHL restoration in clear cell renal cell carcinoma improves NK cell infiltration and function.

BACKGROUND: The von Hippel-Lindau (VHL) gene is frequently mutated in clear cell renal cell carcinoma (ccRCC) which results in stabilization of hypoxia-inducible factor (HIF). Despite the well-known immunosuppressive effect of HIF, ccRCC is considered an immunogenic tumor with high lymphocyte infiltration. Since NK cells have a prognostic value in ccRCC patients, it is important to understand how VHL mutations affect NK cell activity and anti-tumor immunity. METHODS: Tumor spheroids were generated from parental 786-O (VHL-mutated) and 786-O-pVHL (VHL-restored) ccRCC cell lines. Tumor phenotypes, proteome, and secretome were analyzed by flow cytometry, mass spectrometry, and Luminex assays, respectively. Quantitative proteomics analysis and quantitative gene ontology enrichment were used to correlate protein expression changes to ccRCC progression and immunosuppressive pathways. NK cell infiltration, activation, and cytotoxicity were assessed in co-cultures of ccRCC spheroids with NK cells from healthy donors using real-time imaging, immunostaining, and flow cytometry, respectively. RESULTS: VHL-mutated tumor spheroids were significantly less infiltrated by NK cells compared with VHL-restored tumor spheroids. pVHL-infiltrating NK cells showed an activated phenotype along with the ability to reduce tumor spheroid size. Proteomic analysis revealed that VHL-restored tumors express reduced levels of proteins associated with ccRCC progression and immunosuppression, including components of MHC class I processing and PD-1 signaling. Furthermore, VHL-restored tumors exhibited decreased levels of hypoxia-related and pro-tumoral cytokines, such as GROα, IL-8, IL-10, TRAIL, VEGF, and SCF. Within 768-O tumor spheroids, NK cells displayed a higher degree of hypoxia and expression of HIF1α, and inhibition of HIF1α resulted in higher NK cell infiltration into 786-O spheroids. Similarly, inhibition of the VHL-target gene, HIF2α, in 786-O spheroids resulted in increased NK cell infiltration. CONCLUSIONS: VHL mutant tumors are less infiltrated by NK cells due to immunosuppressive pathways driven by HIF stabilization. Restoration of VHL reprograms the tumor microenvironment, reducing ccRCC progression and immunosuppressive signaling while enhancing NK cell infiltration and activation. Inhibition of HIFα improves NK cell infiltration into VHL mutant tumors. Therefore, inhibition of HIFα should be explored as a therapeutic strategy in ccRCC to improve NK cell anti-tumor efficacy against VHL-mutated tumors.