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Calreticulin exposure dictates the immunogenicity of cancer cell death

Abstract

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

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Figure 1: Immunogenic cell death induced by anthracyclins.
Figure 2: Surface exposure of CRT in immunogenic cell death.
Figure 3: Requirement of surface CRT for phagocytosis of tumor cells by DC.
Figure 4: CRT is required for the immune response against dying tumor cells.
Figure 5: Induction of CRT exposure and immunogenic cell death by inhibition of the PP1/GADD34 complex.
Figure 6: Therapeutic effect of CRT or PP1/GADD34 inhibitors injected into tumors.

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Acknowledgements

We thank P. Aucouturier (Hôpital St. Antoine) for providing the transgenic mice and F. Coutant for help with the phagocytosis experiments. G.K. is supported by the Ligue Contre le Cancer (Equipe Labellisée), the European Commission (RIGHT), Cancéropôle Ile-de-France and Institut National Contre le Cancer (INCa). L.Z. is supported by the Ligue Contre le Cancer and INCa. M.P. is supported by a grant from the Associazione Italiana per la Ricerca sul Cancro and by a Ricerca Corrrente and Finalizzata grant from the Italian Ministry of Health. M.O. received a fellowship from the Lebanese Government (Centre National pour la Recherche Scientifique-L) and Centre National pour la Recherche Scientifique, A.T. from the Fondation pour la Recherche Médicale, L.A. from the Ligue Contre le Cancer, F.G. from INSERM and G.M. from the Association pour la Recherche Clinique et Translationnelle.

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Authors and Affiliations

Authors

Contributions

M.O., A.T., F.G., G.M.F., L.A., J.-L. P., M.C., T.P., D.M., N.L. and F.C. performed the in vivo and in vitro experiments. G.M.F. performed mass spectroscopy. N.C. and P.v.E. provided essential reagents. M.P. conducted data analysis. L.Z. and G.K. conceived the study and wrote the manuscript.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Incidence of tumors after inoculation of dying cells. The data show the actual frequency of tumor-free mice, for the experiment summarized in Fig. 1b. (PDF 475 kb)

Supplementary Fig. 2

Mass spectroscopic identification and subcellular localization of CRT. (PDF 880 kb)

Supplementary Fig. 3

Kinetics of CRT exposure. CT26 cells were treated with mitoxantrone for the indicated period, followed by immunofluorescence staining with a CRT-specific antibody and cytofluorometric analysis. (PDF 246 kb)

Supplementary Fig. 4

CT26 cells were cultured for different periods with mitoxantrone or doxorubicin and then confronted with DC to measure their phagocytosis (a), as in Fig. 3a or injected into mice, one week before challenge with live cells (b). (PDF 195 kb)

Supplementary Fig. 5

Requirements for CRT-mediated tumor cell immunogenicity. (PDF 316 kb)

Supplementary Fig. 6

Cells were treated with mitoxantrone or inhibitors of PP1/GADD34, after pre-incubation for 1 h with the indicated inhibitors of protein synthesis (cycloheximide), RNA synthesis (actinomycin D), microtubuli (nocodazol), or the actin cytoskeleton (latrinculin A). (PDF 271 kb)

Supplementary Methods

Generation of bone marrow-derived dendritic cells (DC) (PDF 83 kb)

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Obeid, M., Tesniere, A., Ghiringhelli, F. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 13, 54–61 (2007). https://doi.org/10.1038/nm1523

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