High-dose vitamin C induces ROS-dependent calreticulin exposure to drive immunogenic cell death and cancer immune surveillance

Background: High-dose vitamin C (VitC) has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immune-dependent mechanism of action. Because high-dose VitC generates substantial reactive oxygen species (ROS), we hypothesized that ROS might 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. Methods: Spatial transcriptomic and immunofluorescence analyses were performed on breast and colon tumors from mice treated with high-dose VitC to quantify oxidative stress, DAMP mobilization, and immune cell infiltration. To dissect the functional role of ROS and ICD, mice were treated with high-dose VitC in combination with the antioxidant N-acetylcysteine or an antibody blocking calreticulin (an early DAMP essential for ICD induction). Results: VitC treatment caused marked oxidative stress in 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. Conclusions: High-dose VitC promotes ICD through ROS-dependent mobilization of calreticulin and HMGB1, and calreticulin is required for its immune-mediated antitumor activity. These results provide a mechanistic rationale for ongoing translational analyses within the ALFEO clinical trial (ECTR2022-502101-15-00), which is evaluating high-dose VitC in combination with nivolumab and ipilimumab in mismatch-repair-proficient colorectal cancer. Funded by the Italian Ministry of Health Next Generation EU - PNRR M6C2 - PNRR-MAD-2022-12376593.