The influence of hypoxia on RIG-I-mediated melanoma immunotherapy

Hypoxia, being the inadequate tissue supply of oxygen, is a characteristic of many solid tumors that has negative implications for the patient’s prognosis. Cancer cells adapt to this hostile microenvironment by inducing transcriptomic and translational changes, which ultimately can lead to their epithelial-to-mesenchymal-transition (EMT) and resistance to anti-tumor treatments like radiation-, chemo- and immunotherapy. Thus, it is fundamental to investigate the impact of hypoxia on novel developing therapies, such as the intratumoral activation of the innate pattern recognition receptor retinoic acid-inducible gene I(RIG-I) using 5’-triphosphate RNA (3pRNA) ligands. Stimulation of RIG-I signaling elicits tumor cell death as well as anti-tumoral immunity and has shown promising results in preclinical studies against malignant melanoma. However, the influence of oxygen deprivation on the function of RIG-I has not been investigated. That is why this study aimed at uncovering the effects of hypoxia on the RIG-I signaling pathway and the immunostimulatory potential of 3pRNA in murine melanoma. As presented here, 3pRNA-induced RIG-I protein upregulation was attenuated upon hypoxic incubation (2% partial oxygen pressure) as compared to normoxia (20% partial oxygen pressure) in different melanoma cell lines, which occurred concomitant to the induction of EMT and downregulation of interferon alpha (IFNα) receptor expression. Interestingly, while IFNα lost its capacity to trigger an immune response under hypoxia, the RIG-I signaling outcome was largely unaffected in B16F10 melanoma cells, as indicated by unaltered induction of IFN-stimulated genes and equal expression of major histocompatibility complex (MHC) class-I and programmed cell death ligand 1(PDL-1). Besides, cytokine profiles were moderately modulated; exhibiting enhanced 3pRNA-stimulated type-I IFN production. Most importantly, the ability of 3pRNA-treated melanoma cells to activate melanocyte antigen specific CD8+ T-cells and NK cells in vitro was not inhibited by oxygen deprivation. Finally, treatment of melanoma cells with vitamin C as ROS scavenger or NFκB inhibitors, to protect cells from EMT, reinstated 3pRNA-induced RIG-I expression in hypoxic cells in vitro. Moreover, vitamin C also increased intratumoral proinflammatory cytokine production and anti-tumor efficacy in a subcutaneous melanoma model in vivo. In conclusion, this thesis provides evidence that the immunostimulatory potential of RIG-I activation is preserved under hypoxia and resists attenuation of RIG-I protein upregulation as well as tumor cell IFNα-desensitization. Thus, immunotherapy utilizing 3pRNA proved superior to IFNα by being effective also in hypoxic tumors. Furthermore, adjuvant vitamin C treatment has the potential to aggravate 3pRNA-mediated tumor eradication. These findings essentially contribute to the successful establishment of 3pRNA mono- or combinatorial therapies to the valuable benefit of melanoma patients