The molecular bases of the anticancer efficacy of high-dose Vitamin C

Several preclinical studies have shown that high doses of vitamin C (vitC) can impair tumor growth by creating intense oxidative stress in tumor cells and causing metabolic collapse due to glutathione and NAPDH depletion. VitC can also increase replication stress due to its ability to activate TET / JmjC enzymes leading to chromatin remodeling and methylation shifts. In addition to its cancer cell–intrinsic effects, vitC has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immune-dependent mechanism of action. Despite intense preclinical investigations, clinical trials in unselected cancer patients receiving VitC have yielded controversial results, indicating that its mechanisms of action remain incompletely understood. I hypothesized that gaining further mechanistic insights will support the development of biomarker-driven therapeutic strategies combining high-dose vitamin C with chemotherapy or immunotherapy. In the first chapter of this thesis, I investigated the molecular basis underlying the immune mediated anticancer efficacy of vitC. Because high-dose vitC generates substantial reactive oxygen species (ROS), I hypothesized that ROS could induce immunogenic cell death (ICD)—a regulated form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that enhance antigen presentation and promote antitumor immunity. VitC treatment caused marked oxidative stress in breast and colon tumors from both immunocompetent and immunocompromised mice; however, tumor growth inhibition occurred exclusively in immunocompetent animals. High-dose vitC triggered ROS-dependent exposure and release of the DAMPs calreticulin and HMGB1, respectively. This was accompanied by substantial remodeling of the tumor microenvironment, including increased infiltration of cytotoxic CD8⁺ T cells and natural killer cells, and a reduction in immunosuppressive regulatory T cells. Blocking calreticulin effectively disrupted the ICD cascade, prevented immune microenvironment remodeling, and completely abrogated the antitumor efficacy of vitC. In the second chapter of this thesis, I investigated the molecular determinants of cancer cell intrinsic sensitivity to high dose vitC. I hypothesized that ROS could generate oxidative DNA damage to which tumors with defective DNA repair processes may be especially vulnerable. In vitro, homologous recombination–deficient (HRD) cancer cell lines showed increased sensitivity to vitC than HR proficient (HRP) lines. VitC treatment induced accumulation of 8- 4 oxoguanine and single-strand DNA breaks that persisted in HRD lines. During DNA replication, persistent lesions were converted into double strand DNA breaks that could not be repaired in HRD cells, as shown by impaired formation of RAD51 foci in VitC treated tumors. HRD tumors are known to be sensitive to DNA-damaging agents, including PARP inhibitors and chemotherapies. In vivo, vitC synergized with oxaliplatin in a HRD pancreatic ductal adenocarcinoma (PDAC) model, significantly extending mouse survival. Suppression of ROS production by the co-administration of the antioxidant N-acetylcysteine markedly reduced the anticancer effect of VitC, both alone and in combination with oxaliplatin. In conclusion, this thesis found that the induction of ICD contributes to the immuno mediated anticancer surveillance promoted by high-dose vitC. This work also identified HRD status as a predictive biomarker for vitC efficacy, suggesting a potential therapeutic strategy combining vitC with oxaliplatin in HRD PDAC tumors. These novel mechanistic insights pave the way for further preclinical and clinical investigation of vitC containing regimens