Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations.
Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts.
We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa βGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response.
Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer.
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Kroemer, G., Galluzzi, L., Kepp, L. O. & Zitvogel, L. Immunogenic cell death in cancer therapy. Ann. Rev. Immunol. 31, 51–72 (2013).
Pol, J., Vacchelli, E., Aranda, F., Castoldi, F., Eggermont, A., Cremer, I. et al. Trial watch: immunogenic cell death inducers for anticancer chemotherapy. OncoImmunology 4, 1008866 (2015).
Dudek, M. A., Garg, A. D., Krysko, D. V., De Ruysscher, D. & Agostinis, P. Inducers of immunogenic cell death. Cytokine and Growth Factor Rev. 24, 319–333 (2013).
Fridman, W. H., Zitvogel, L., Sautes-Fridman, C. & Kroemer, G. The immune contexture in cancer prognosis and treatment. Nat. Rev. Clin. Oncol. 14, 717–734 (2017).
Mallucci, L. & Wells, V. The end of KRAS, and other, cancers? A new way forward. Drug Discov. Today 19, 383–387 (2014).
Blaser, C., Kaufmann, M., Muller, C., Zimmerman, C., Wells, V., Mallucci, L. et al. β-galactoside-binding β-GBP protein secreted by activated T cells inhibits antigen-induced proliferation of T cells. Eur. J. Immunol. 28, 2311–2319 (1998).
Wells, V. & Mallucci, L. Identification of an autocrine negative growth factor: mouse β-galactoside binding protein is a cytostatic factor and cell growth regulator. Cell 64, 91–97 (1991).
Wells, V., Davies, D. & Mallucci, L. Cell cycle arrest and induction of apoptosis by β galactoside binding protein in human mammary cancer cells. A potential new approach to cancer control. Eur. J. Cancer 35, 978–983 (1999).
Ravatn, R., Wells, V., Nelson, L., Vettori, D., Mallucci, L. & Chin, K.-V. Circumventing multi-drug resistance in cancer by βGBP, an antiproliferative cytokine. Cancer Res. 65, 1631–1634 (2005).
Wells, V. & Mallucci, L. Phosphoinositide 3-kinase targeting by the β-galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death. Breast Cancer Res. 11, 1–10 (2009).
Mallucci, L., Shi, D.-Y., Davies, D., Jordan, P., Nicol, A., Lotti, L. et al. Killing of Kras-mutant colon cancer cells via Rac-independent actin remodelling by the βGBP cytokine, a physiological PI3K inhibitor therapeutically effective in vivo. Mol. Cancer Ther. 11, 1184–1193 (2012).
Wells, V., Downward, J. & Mallucci, L. Functional inhibition of PI3K by the βGBP molecule suppresses Ras-MAPK signalling to block cell proliferation. Oncogene 26, 7709–7714 (2007).
Marino, G., Niso-Santano, M., Baehrecke, E. H. & Kroemer, G. Self-consumption: the interpay of autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 5, 81–94 (2014).
Kroemer, G., Marino, G. & Levine, B. Autophagy and integrated stress response. Mol. Cell 40, 280–293 (2010).
Michaud, M., Martins, I., Sukhurwata, A. Q., Adjemian, S., Ma, Y., Pellegatti, P. et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science 334, 1573–1577 (2011).
Obeid, M., Tesniere, A., Ghiringhelli, F., Fimia, G. M., Apetoh, L., Perfettini, J. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61 (2007).
Krysko, D. V., Garg, A. B., Kaczmarek, A., Krysko, O., Agostinis, P. & Vandenabeele, P. Immunogenic cell death and DAMPS in cancer therapy. Nat. Rev. Cancer 12, 860–875 (2012).
Garg, D., Krysko, D. V., Verfaillie, T., Kaczmarck, A., Ferreira, G. B., Marysael, T. et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 31, 1062–1079 (2012).
Yatim, N., Cullen, S. & Albert, M. L. Dying cells actively regulate adaptive immune responses. Cell Death and Immunity 17, 262–275 (2017).
Cattaneo, M., Lotti, L. V., S. Martin, S., Alessio, M., Conti, A., Bach, A. et al. Secretion of novel SEL 1L endogenous variants is promoted by ER stress/UPR via endosomes and shed vesicles in human cancer cells. PloS1 6, 172 (2011).
Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev Mol Cell Biol 8, 519–529 (2007).
Walter, P. & Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).
Senft, D. & Ronai, Z. A. UPR, autophagy, and mitochondrial crosstalk underlies the ER stress response. Trends Biochem. Sci. 40, 141–148 (2015).
Shimodaira, Takahashi, S., Kinouchi, Y., Endo, K., Shiga, H., Kakuta, Y. et al. Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α) in human colon cancer cells. Biochem. Biophys. Res. Commun. 445, 524–533 (2014).
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2011).
Tanida, I., Takashi, U. & Kominami, E. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 503–518 (2004).
Mizushima, N., Yoshimorin, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).
Gardai, S. J., McPhillips, K. A., Frasch, S. C., Janssen, W. J., Starefeldt, A., Murphy-Ullrich, J. E. et al. Cell-surface calreticulin initiates clearance of viable apoptotic cells through trans-activation of LPR on the phagocyte. Cell 123, 321–334 (2005).
Panarektakis, T., Kepp, O., Brockmeier, U., Tesniere, A., Bjorklund, A. C., Chapman, D. C. et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 28, 578–590 (2009).
L. Mallucci thanks Professor Tony Ng for interest and support and for reviewing the manuscript and Professor Richard Trembath for support. L. Mallucci and V. Wells thanks Dr Juergen Moll for comments on the manuscript. R. Mariani-Costantini and L.V. Lotti thank Professor Fabio Pulcinelli for the gift of ATP reagents and Mr Sandro Valia for assistance with art work.
The authors declare no competing interests.
Ethics approval and consent to participate
We confirm that that the donation of blood cells by human subjects complies with the Declaration of Helsinki. Donors gave their informed consent. Approval was given by the Comitato Etico Policlinico Umberto Primo Roma.
The work was supported by internal funding from King’s College London, Universita’ di Roma La Sapienza, and the G.d’Annunzio University Chieti.
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Cirone, M., Lotti, L.V., Granato, M. et al. Sourcing the immune system to induce immunogenic cell death in Kras-colorectal cancer cells. Br J Cancer 121, 768–775 (2019) doi:10.1038/s41416-019-0561-z