Abstract 6169: Immunogenic cell death mediates the anticancer efficacy of high dose vitamin C

Abstract Background: We previously reported that high-dose Vitamin C (VitC) can synergize with immune checkpoint blockade in immunocompetent mice bearing colorectal, pancreatic and breast tumors. Here, we aim to unveil the molecular mechanisms underlying the above preclinical observations. We hypothesized that VitC could trigger immunogenic cell death (ICD), thereby stimulating an immune-mediated anticancer response. Methods: Induction of ICD was assessed by the expression or emission of specific markers, including calreticulin, as well as by ex-vivo phagocytosis assays by co-culturing dendritic cells with breast or colon cancer cells treated with VitC. A calreticulin blocking antibody was administered in vivo in combination with VitC to functionally characterize the role of ICD induction in the anticancer effects of vitC. Ex-vivo spatial transcriptomic and immunofluorescence analyses were performed to characterize the impact of the treatment on tumor and immune cell populations. Results: Pharmacological doses of VitC were able to induce oxidative stress as indicated by spatial transcriptomic data and confirmed by ex vivo 8-hydroxyguanine staining in tumor sections. VitC treatment induced ICD markers and among these calreticulin and HMGB1. Although the induction of these danger markers was seen in tumors grown both in immunocompromised and immunocompetent VitC treated mice, anticancer activity was evident only in the presence of an intact immune system. In vivo, a calreticulin blocking antibody effectively prevented its translocation and inhibited HMGB1 release in murine tumors. Notably, preventing calreticulin exposure abrogated the immune mediated anticancer effect of VitC. VitC enhanced the infiltration of natural killers, activated CD8+ T lymphocytes, while it decreased the number of Tregs. Calreticulin blockade impaired the modulation of the immune tumor microenvironment induced by VitC. Conclusions: VitC treatment can mobilize calreticulin and HMGB1, which are among the established markers of immunogenic cell death. These results, alongside the observation that VitC exerts its maximal antitumor activity only in immunocompetent mice and its anticancer activity is abolished by anti-calreticulin blocking antibody demonstrated that VitC is an ICD inducer when given at pharmacological doses. These findings lend the preclinical rationale and inform the translational analyses planned within the ALFEO clinical trial (Clinical trial information: ECTR2022-502101-15-00), that is investigating pharmacological doses of vitC in combination with nivolumab and ipilimumab in mismatch repair proficient colon cancer patients. Funded by- Next Generation EU - PNRR M6C2 - PNRR-MAD-2022-12376593. Citation Format: Alessandro Cavaliere, Marco Macagno, Federica Maione, Vito Amodio, Marc Escobosa Olmo, Rosaria Chilà, Simona Lamba, Pietro Paolo Vitiello, Valeria Pessei, Daniela Grases, Elena Perez, Alice Bartolini, Noemi Congiusta, Giovanni Germano, Andrea Sartore-Bianchi, Teresa Troiani, Manel Esteller, Eduard Porta, Alberto Bardelli, Federica Di Nicolantonio. Immunogenic cell death mediates the anticancer efficacy of high dose vitamin C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6169.