High hydrostatic pressure (HHP) promotes key characteristics of immunogenic cell death (ICD), in thus far resembling immunogenic chemotherapy and ionizing irradiation. Here, we demonstrate that cancer cells succumbing to HHP induce CD4+ and CD8+ T cell-dependent protective immunity in vivo. Moreover, we show that cell death induction by HHP relies on the overproduction of reactive oxygen species (ROS), causing rapid establishment of the integrated stress response, eIF2α phosphorylation by PERK, and sequential caspase-2, -8 and -3 activation. Non-phosphorylatable eIF2α, depletion of PERK, caspase-2 or -8 compromised calreticulin exposure by cancer cells succumbing to HHP but could not inhibit death. Interestingly, the phagocytosis of HHP-treated malignant cells by dendritic cells was suppressed by the knockdown of caspase-2 in the former. Thus, caspase-2 mediates a key function in the interaction between dying cancer cells and antigen presenting cells. Our results indicate that the ROS→PERK→eIF2α→caspase-2 signaling pathway is central for the perception of HHP-driven cell death as immunogenic.

Cell Death Research and Therapy Unit Department of Cellular and Molecular Medicine KU Leuven University of Leuven Leuven Belgium

Centre de Recherche en Cancérologie Nantes Angers INSERM U892 Nantes France

Laboratory of Tumor Immunology Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Sotio Prague Czech Republic

Sotio Prague Czech Republic; 2nd Medical Faculty and University Hospital Motol Charles University Prague Czech Republic

Zobrazit více v PubMed

Galluzzi L, Vacchelli E, Bravo-San Pedro JM, Buque A, Senovilla L, Baracco EE, Bloy N, Castoldi F, Abastado JP, Agostinis P et al.. Classification of current anticancer immunotherapies. Oncotarget 2014; 5(24):12472-508; PMID:25537519; http://dx.doi.org/10.18632/oncotarget.2998 PubMed DOI PMC

Fucikova J, Kralikova P, Fialova A, Brtnicky T, Rob L, Bartunkova J, Spísek R. Human tumor cells killed by anthracyclines induce a tumor-specific immune response. Cancer Res 2011; 71(14):4821-33; PMID:21602432; http://dx.doi.org/10.1158/0008-5472.CAN-11-0950 PubMed DOI

Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F, Aymeric L, Michaud M, Apetoh L, Barault L et al.. Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene 2010; 29(4):482-91; PMID:19881547; http://dx.doi.org/10.1038/onc.2009.356 PubMed DOI

Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P, Zitvogel L, Kroemer G. Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC light-induced apoptosis. Cell Death Differ 2007; 14(10):1848-50; PMID:17657249; http://dx.doi.org/10.1038/sj.cdd.4402201 PubMed DOI

Koks C, Garg AD, Ehrhardt M, Riva M, Vandenberk L, Boon L, De Vleeschouwer S, Agostinis P, Graf N, Van Gool SW. Newcastle disease virotherapy induces long-term survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death. Int J Cancer 2014; 136(5):E313-25; PMID:25208916; http://dx.doi.org/10.1002/ijc.29202 PubMed DOI

Vacchelli E, Eggermont A, Sautes-Fridman C, Galon J, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: oncolytic viruses for cancer therapy. Oncoimmunology 2013; 2(6):e24612; PMID:23894720; http://dx.doi.org/10.4161/onci.24612 PubMed DOI PMC

Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, Rubio N, Firczuk M, Mathieu C, Roebroek AJ et al.. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J 2012; 31(5):1062-79; PMID:22252128; http://dx.doi.org/10.1038/emboj.2011.497 PubMed DOI PMC

Garg AD, Vandenberk L, Koks C, Verschuere T, Boon L, Van Gool SW, Agostinis P. Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell-driven rejection of high-grade glioma. Sci Transl Med 2016; 8(328):328ra327; PMID:26936504; http://dx.doi.org/2594162110.1126/scitranslmed.aae0105 PubMed DOI

Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P, Apetoh L, Aranda F, Barnaba V, Bloy N et al.. Consensus guidelines for the detection of immunogenic cell death. Oncoimmunology 2014; 3(9):e955691; PMID:25941621; http://dx.doi.org/10.4161/21624011.2014.955691 PubMed DOI PMC

Garg AD, Galluzzi L, Apetoh L, Baert T, Birge RB, Bravo-San Pedro JM, Breckpot K, Brough D, Chaurio R, Cirone M et al.. Molecular and translational classifications of DAMPs in immunogenic cell death. Front Immunol 2015; 6: 588; PMID:26635802; http://dx.doi.org/10.3389/fimmu.2015.00588 PubMed DOI PMC

Cao C, Han Y, Ren Y, Wang Y. Mitoxantrone-mediated apoptotic B16-F1 cells induce specific anti-tumor immune response. Cell Mol Immunol 2009; 6(6):469-75; PMID:20003823; http://dx.doi.org/10.1038/cmi.2009.59 PubMed DOI PMC

Spisek R, Charalambous A, Mazumder A, Vesole DH, Jagannath S, Dhodapkar MV. Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications. Blood 2007; 109(11):4839-45; PMID:17299090; http://dx.doi.org/10.1182/blood-2006-10-054221 PubMed DOI PMC

Garg AD, Elsen S, Krysko DV, Vandenabeele P, de Witte P, Agostinis P. Resistance to anticancer vaccination effect is controlled by a cancer cell-autonomous phenotype that disrupts immunogenic phagocytic removal. Oncotarget 2015; 6(29):26841-60; PMID:26314964; http://dx.doi.org/10.18632/oncotarget.4754 PubMed DOI PMC

Martins I, Kepp O, Galluzzi L, Senovilla L, Schlemmer F, Adjemian S, Menger L, Michaud M, Zitvogel L, Kroemer G. Surface-exposed calreticulin in the interaction between dying cells and phagocytes. Ann NY Acad Sci 2010; 1209: 77-82; PMID:20958319; http://dx.doi.org/1916515110.1111/j.1749-6632.2010.05740.x PubMed DOI

Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, Durchschlag M, Joza N, Pierron G, van Endert P et al.. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J 2009; 28(5):578-90; PMID:19165151; http://dx.doi.org/10.1038/emboj.2009.1 PubMed DOI PMC

Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N et al.. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13(1):54-61; PMID:17187072; http://dx.doi.org/10.1038/nm1523 PubMed DOI

van Vliet AR, Martin S, Garg AD, Agostinis P. The PERKs of damage-associated molecular patterns mediating cancer immunogenicity: from sensor to the plasma membrane and beyond. Semin Cancer Biol 2015; 33: 74-85; PMID:25882379; http://dx.doi.org/10.1016/j.semcancer.2015.03.010 PubMed DOI

Garg AD, Dudek AM, Ferreira GB, Verfaillie T, Vandenabeele P, Krysko DV, Mathieu C, Agostinis P. ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death. Autophagy 2013; 9(9):1292-307; PMID:23800749; http://dx.doi.org/10.4161/auto.25399 PubMed DOI

Galluzzi L, Kepp O, Kroemer G. Enlightening the impact of immunogenic cell death in photodynamic cancer therapy. EMBO J 2012; 31(5):1055-7; PMID:22252132; http://dx.doi.org/10.1038/emboj.2012.2 PubMed DOI PMC

Fucikova J, Moserova I, Truxova I, Hermanova I, Vancurova I, Partlova S, Fialova A, Sojka L, Cartron PF, Houska M et al.. High hydrostatic pressure induces immunogenic cell death in human tumor cells. Int J Cancer 2014; 135(5):1165-77; PMID:24500981; http://dx.doi.org/10.1002/ijc.28766 PubMed DOI

Adkins I, Fucikova J, Garg AD, Agostinis P, Spisek R. Physical modalities inducing immunogenic tumor cell death for cancer immunotherapy. Oncoimmunology 2014; 3(12):e968434; PMID:25964865; http://dx.doi.org/10.4161/21624011.2014.968434 PubMed DOI PMC

Mishra R, Karande AA. Endoplasmic reticulum stress-mediated activation of p38 MAPK, caspase-2 and caspase-8 leads to abrin-induced apoptosis. PLoS One 2014; 9(3):e92586; PMID:24664279; http://dx.doi.org/10.1371/journal.pone.0092586 PubMed DOI PMC

Uchibayashi R, Tsuruma K, Inokuchi Y, Shimazawa M, Hara H. Involvement of Bid and caspase-2 in endoplasmic reticulum stress- and oxidative stress-induced retinal ganglion cell death. J Neurosci Res 2011; 89(11):1783-94; PMID:21805492; http://dx.doi.org/10.1002/jnr.22691 PubMed DOI

Upton JP, Austgen K, Nishino M, Coakley KM, Hagen A, Han D, Papa FR, Oakes SA. Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress. Mol Cellular Biol 2008; 28(12):3943-51; PMID:18426910; http://dx.doi.org/10.1128/MCB.00013-08 PubMed DOI PMC

Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson PM. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005; 123(2):321-34; PMID:16239148; http://dx.doi.org/10.1016/j.cell.2005.08.032 PubMed DOI

Blachere NE, Darnell RB, Albert ML. Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation. PLoS Biol 2005; 3(6):e185; PMID:15839733; http://dx.doi.org/10.1371/journal.pbio.0030185 PubMed DOI PMC

Podrazil M, Horvath R, Becht E, Rozkova D, Bilkova P, Sochorova K, Hromadkova H, Kayserova J, Vavrova K, Lastovicka J et al.. Phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer. Oncotarget 2015; 6(20):18192-205; PMID:26078335; http://dx.doi.org/10.18632/oncotarget.4145 PubMed DOI PMC

Zappasodi R, de Braud F, Di Nicola M. Lymphoma immunotherapy: current status. Front Immunol 2015; 6: 448; PMID:26388871; http://dx.doi.org/10.3389/fimmu.2015.00448 PubMed DOI PMC

Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, Yamazaki T, Sukkurwala AQ, Michaud M, Mignot G et al.. Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med 2012; 4(143):143ra199; PMID:22814852; http://dx.doi.org/2115117610.1126/scitranslmed.3003807 PubMed DOI

Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S, Michaud M, Menger L, Gdoura A, Tajeddine N et al.. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 2011; 30(10):1147-58; PMID:21151176; http://dx.doi.org/10.1038/onc.2010.500 PubMed DOI

Miyamoto S, Inoue H, Nakamura T, Yamada M, Sakamoto C, Urata Y, Okazaki T, Marumoto T, Takahashi A, Takayama K et al.. Coxsackievirus B3 is an oncolytic virus with immunostimulatory properties that is active against lung adenocarcinoma. Cancer Res 2012; 72(10):2609-21; PMID:22461509; http://dx.doi.org/10.1158/0008-5472.CAN-11-3185 PubMed DOI

Prasad V, Chandele A, Jagtap JC, Sudheer Kumar P, Shastry P. ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis. Free Radic Biol Med 2006; 41(3):431-42; PMID:16843824; http://dx.doi.org/10.1016/j.freeradbiomed.2006.03.009 PubMed DOI

Sandow JJ, Dorstyn L, O'Reilly LA, Tailler M, Kumar S, Strasser A, Ekert PG. ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis. Cell Death Differ 2014; 21(3):475-80; PMID:24292555; http://dx.doi.org/10.1038/cdd.2013.168 PubMed DOI PMC

Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2: the laymen's view. Cell Death Differ 2009; 16(2):195-207; PMID:19023332; http://dx.doi.org/10.1038/cdd.2008.170 PubMed DOI PMC

Lauber K, Blumenthal SG, Waibel M, Wesselborg S. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell 2004; 14(3):277-87; PMID:15125832; http://dx.doi.org/10.1016/S1097-2765(04)00237-0 PubMed DOI

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

Trial watch: chemotherapy-induced immunogenic cell death in immuno-oncology

Induction of Tolerance and Immunity by Dendritic Cells: Mechanisms and Clinical Applications