18P - Exposure of calreticulin is required for vitamin C immunomediated cancer surveillance

Background

Vitamin C (VitC) was shown to synergize with immune checkpoint blockade in tumor bearing immunocompetent mice, which provided the rationale for the ongoing ALFEO trial in colorectal cancer patients (Clinical Trial Registration Number 2022-502101-15-00). In this work, we aimed to elucidate the molecular mechanisms underlying these preclinical observations. We hypothesized that VitC could strain cancer cells, which react by emitting danger signal molecules, which in turn could stimulate an immune mediated anticancer response.

Methods

We assessed expression of the oxidative stress marker hydroxyguanosine (8-OHG) and danger signal molecules (calreticulin and HMGB1) in breast and colon murine tumors treated with VitC. A calreticulin blocking antibody was given in combination with VitC to functionally ascertain the role of this stress signal protein.

Results

Pharmacological doses of VitC were able to strongly induce oxidative stress as indicated by strong 8-OHG staining in tumor sections. VitC-stressed tumor cells mobilized the danger molecules calreticulin and HMGB1. Although the induction of these danger markers was seen in tumors grown both in immunocompromised and immunocompetent VitC treated mice, its 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 immunomediated 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

We unveil that VitC is able to 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 are clues that VitC could act as immunogenic cell death inducer when given at pharmacological doses. Our findings have implications for cancer patients enrolled in clinical trials based on VitC containing regimens

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

AIRC.

Disclosure

P.P. Vitiello: Financial Interests, Personal, Speaker, Consultant, Advisor: Biocartis, Merck. G. Germano: Other, Personal, Advisory Board: NeoPhore. A. Bardelli: Financial Interests, Personal, Advisory Role: Illumina, Inivata; Financial Interests, Personal, Advisory Board: NeoPhore. F. Di Nicolantonio: Financial Interests, Personal, Speaker, Consultant, Advisor: Illumina, Pierre Fabre. All other authors have declared no conflicts of interest.