Calreticulin exposure is a critical mediator of high-dose vitamin C-induced immune surveillance in the tumor microenvironment

Abstract High-dose vitamin C (vitC) has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immunodependent mechanism of action. Because high-dose vitC generates substantial reactive oxygen species (ROS), we 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. 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). 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. Collectively, our study indicates that high-dose vitC promotes ICD by mobilizing calreticulin and HMGB1, and that calreticulin is a determinant of its immune-mediated antitumor activity. Implications: vitC induces ICD, supporting its use as an adjuvant to anticancer immunotherapy.