The mechanisms of immunogenicity underlying mild heat-shock (mHS) treatment < 42°C of tumor cells are largely attributed to the action of heat-shock proteins; however, little is known about the immunogenicity of tumor cells undergoing severe cytotoxic heat-shock treatment (sHS > 43°C). Here, we found that sHS, but not mHS (42°C), induces immunogenic cell death in human cancer cell lines as defined by the induction of ER stress response and ROS generation, cell surface exposure of calreticulin, HSP70 and HSP90, decrease of cell surface CD47, release of ATP and HMGB1. Only sHS-treated tumor cells were efficiently killed and phagocytosed by dendritic cells (DCs), which was partially dependent on cell surface calreticulin. DCs loaded with mHS or sHS-treated tumor cells displayed similar level of maturation and stimulated IFNγ-producing CD8+ T cells without any additional adjuvants in vitro. However, only DCs loaded with sHS-treated tumor cells stimulated antigen-specific CD4+ T cells and induced higher CD8+ T-cell activation and proliferation. sHS-treated murine cells also exposed calreticulin, HSP70 and HSP90 and activated higher DC maturation than mHS treated cells. Vaccination with sHS-treated tumor cells elicited protective immunity in mice. In this study, we defined specific conditions for the sHS treatment of human lung and ovarian tumor cells to arrive at optimal ratio between effective cell death, immunogenicity and content of tumor antigens for immunotherapeutic vaccine generation.

Department of Immunology 2nd Faculty of Medicine and University Hospital Motol Charles University Prague Czech Republic

Laboratory of Tumor Immunology Institute of Microbiology of the ASCR v v i Prague Czech Republic

Sotio a s Prague Czech Republic

Zobrazit více v PubMed

Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T, Felix R, Riess H. The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol 2002; 43:33-56; PMID:12098606; https://doi.org/10.1016/S1040-8428(01)00179-2 PubMed DOI

Mentre P, Hamraoui L, Hui Bon Hoa G, Debey P. Pressure-sensitivity of endoplasmic reticulum membrane and nucleolus as revealed by electron microscopy. Cell Mol Biol 1999; 45:353-62; PMID:10386792; https://doi.org/10.1088/1742-6596/121/1/112003 PubMed DOI

Dudek AM, Garg AD, Krysko DV, De Ruysscher D, Agostinis P. Inducers of immunogenic cancer cell death. Cytokine Growth Factor Rev 2013; 24:319-33; PMID:23391812; https://doi.org/10.1016/j.cytogfr.2013.01.005 PubMed DOI

Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Ann Rev Immunol 2013; 31:51-72; PMID:23157435; https://doi.org/10.1146/annurev-immunol-032712-100008 PubMed DOI

Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer 2012; 12:860-75; PMID:23151605; https://doi.org/10.1038/nrc3380 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:e968434; PMID:25964865; https://doi.org/10.4161/21624011.2014.968434 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; 13:54-61; PMID:17187072; https://doi.org/10.1038/nm1523 PubMed DOI

Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukocyte biol 2007; 81:1-5; PMID:17032697; http://doi.org/10.1189/jlb.0306164 PubMed DOI

Matzinger P. The danger model: a renewed sense of self. Science 2002; 296:301-5; PMID:11951032; https://doi.org/10.1126/science.1071059 PubMed DOI

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; 109:4839-45; PMID:17299090; https://doi.org/10.1182/blood-2006-10-054221 PubMed DOI PMC

Martins I, Tesniere A, Kepp O, Michaud M, Schlemmer F, Senovilla L, Séror C, Métivier D, Perfettini JL, Zitvogel L, et al.. Chemotherapy induces ATP release from tumor cells. Cell Cycle 2009; 8:3723-8; PMID:19855167; https://doi.org/10.4161/cc.8.22.10026 PubMed DOI

Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002; 418:191-5; PMID:12110890; https://doi.org/10.1038/nature00858 PubMed DOI

Tesniere A, Panaretakis T, Kepp O, Apetoh L, Ghiringhelli F, Zitvogel L, Kroemer G. Molecular characteristics of immunogenic cancer cell death. Cell Death Differ 2008; 15:3-12; PMID:18007663; https://doi.org/10.1038/sj.cdd.4402269 PubMed DOI

Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, Michaud M, Sukkurwala AQ, Menger L, Zitvogel L, Kroemer G. Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metastasis Rev 2011; 30:61-9; PMID:21249425; https://doi.org/10.1007/s10555-011-9273-4 PubMed DOI

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; 135:1165-77; PMID:24500981; https://doi.org/10.1002/ijc.28766 PubMed DOI

Roti Roti JL. Cellular responses to hyperthermia (40–46°C): cell killing and molecular events. Int J Hyperthermia 2008; 24:3-15; PMID:18214765; https://doi.org/10.1080/02656730701769841 PubMed DOI

Milleron RS, Bratton SB. 'Heated' debates in apoptosis. Cell Mol Life Sci 2007; 64:2329-33; PMID:17572850; https://doi.org/10.1007/s00018-007-7135-6 PubMed DOI PMC

Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995; 269:1585-8; PMID:7545313; https://doi.org/10.1126/science.7545313 PubMed DOI

Melcher A, Todryk S, Hardwick N, Ford M, Jacobson M, Vile RG. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nat Med 1998; 4:581-7; PMID:9585232; https://doi.org/10.1038/nm0598-581 PubMed DOI

Frey B, Weiss EM, Rubner Y, Wunderlich R, Ott OJ, Sauer R, Fietkau R, Gaipl US. Old and new facts about hyperthermia-induced modulations of the immune system. Int J Hyperthermia 2012; 28:528-42; PMID:22690925; https://doi.org/10.3109/02656736.2012.677933 PubMed DOI

Shi H, Cao T, Connolly JE, Monnet L, Bennett L, Chapel S, Bagnis C, Mannoni P, Davoust J, Palucka AK et al.. Hyperthermia enhances CTL cross-priming. J Immunol 2006; 176:2134-41; PMID:18539499; https://doi.org/10.4049/jimmunol.176.4.2134 PubMed DOI

Zhang HG, Mehta K, Cohen P, Guha C. Hyperthermia on immune regulation: a temperature's story. Cancer Lett 2008; 271:191-204; PMID:18597930; https://doi.org/10.1016/j.canlet.2008.05.026 PubMed DOI

Feng H, Zeng Y, Graner MW, Katsanis E. Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells. Blood 2002; 100:4108-15; PMID:12393401; https://doi.org/10.1182/blood-2002-05-1389 PubMed DOI

Feng H, Zeng Y, Graner MW, Likhacheva A, Katsanis E. Exogenous stress proteins enhance the immunogenicity of apoptotic tumor cells and stimulate antitumor immunity. Blood 2003; 101:245-52; PMID:12393411; https://doi.org/10.1182/blood-2002-05-1580 PubMed DOI

Clark PR, Menoret A. The inducible Hsp70 as a marker of tumor immunogenicity. Cell Stress Chaperones 2001; 6:121-5; PMID:11599573; https://doi.org/10.1379/1466-1268(2001)006<0121:TIHAAM>2.0.CO;2 PubMed DOI PMC

Mise K, Kan N, Okino T, Nakanishi M, Satoh K, Teramura Y, Yamasaki S, Ohgaki K, Tobe T. Effect of heat treatment on tumor cells and antitumor effector cells. Cancer Res 1990; 50:6199-202; PMID:2400985; http://doi.org/10.11501/3057478 PubMed DOI

Takahashi T, Mitsuhashi N, Sakurai H, Niibe H. Modifications of tumor-associated antigen expression on human lung cancer cells by hyperthermia and cytokine. Anticancer Res 1995; 15:2601-6; PMID:8669832; https://doi.org/10.1186/1756-9966-27-5 PubMed DOI

Wong JY, Mivechi NF, Paxton RJ, Williams LE, Beatty BG, Beatty JD, Shively JE. The effects of hyperthermia on tumor carcinoembryonic antigen expression. Int J Radiat Oncol Biol Phys 1989; 17:803-8; PMID:2674083; https://doi.org/10.1016/0360-3016(89)90070-9 PubMed DOI

Zerbini A, Pilli M, Penna A, Pelosi G, Schianchi C, Molinari A, Schivazappa S, Zibera C, Fagnoni FF, Ferrari C et al.. Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses. Cancer Res 2006; 66:1139-46; PMID:16424051; https://doi.org/10.1158/0008-5472.CAN-05-2244 PubMed DOI

Ito A, Honda H, Kobayashi T. Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of “heat-controlled necrosis” with heat shock protein expression. Cancer Immunol Immunother 2006; 55:320-8; PMID:16133113; https://doi.org/10.1007/s00262-005-0049-y PubMed DOI PMC

Hu R, Ma S, Li H, Ke X, Wang G, Wei D, Wang W. Effect of magnetic fluid hyperthermia on lung cancer nodules in a murine model. Oncol Lett 2011; 2:1161-4; PMID:22848282; https://doi.org/10.3892/ol.2011.379 PubMed DOI PMC

Wang H, Zhang L, Shi Y, Javidiparsijani S, Wang G, Li X, Ouyang W, Zhou J, Zhao L, Wang X et al.. Abscopal antitumor immune effects of magnet-mediated hyperthermia at a high therapeutic temperature on Walker-256 carcinosarcomas in rats. Oncol Lett 2014; 7:764-70; PMID:24527084; https://doi.org/10.3892/ol.2014.1803 PubMed DOI PMC

Yu Z, Geng J, Zhang M, Zhou Y, Fan Q, Chen J. Treatment of osteosarcoma with microwave thermal ablation to induce immunogenic cell death. Oncotarget 2014; 5:6526-39; PMID:25153727; https://doi.org/10.18632/oncotarget.2310 PubMed DOI PMC

Brusa D, Migliore E, Garetto S, Simone M, Matera L. Immunogenicity of 56°C and UVC-treated prostate cancer is associated with release of HSP70 and HMGB1 from necrotic cells. Prostate 2009; 69:1343-52; PMID:19496055; https://doi.org/10.1002/pros.20981 PubMed DOI

Adkins I, Koberle M, Grobner S, Autenrieth SE, Bohn E, Borgmann S, Autenrieth IB. Y. enterocolitica inhibits antigen degradation in dendritic cells. Microbes Infect 2008; 10:798-806; PMID:18539499; https://doi.org/10.1016/j.micinf.2008.04.014 PubMed DOI

Adkins I, Kamanova J, Kocourkova A, Svedova M, Tomala J, Janova H, Masin J, Chladkova B, Bumba L, Kovar M et al.. Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells. PloS One 2014; 9:e104064; PMID:25084094; https://doi.org/10.1371/journal.pone.0104064 PubMed DOI PMC

Hradilova N, Sadilkova L, Palata O, Mysikova D, Mrazkova H, Lischke R, Spisek R, Adkins I. Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy. PLoS One 2017; 12:e0171539; PMID:28187172; https://doi.org/10.1371/journal.pone.0171539 PubMed DOI PMC

Grobner S, Adkins I, Schulz S, Richter K, Borgmann S, Wesselborg S, Ruckdeschel K, Micheau O, Autenrieth IB. Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells. Apoptosis 2007; 12:1813-25; PMID:17624595; https://doi.org/10.1007/s10495-007-0100-x PubMed DOI

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; 71:4821-33; PMID:21602432; https://doi.org/10.1158/0008-5472.CAN-11-0950 PubMed DOI

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; 31:1062-79; PMID:22252128; https://doi.org/10.1038/emboj.2011.497 PubMed DOI PMC

Garg AD, Krysko DV, Vandenabeele P, Agostinis P. Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin. Cancer Immunol Immunother 2012; 61:215-21; PMID:22193987; https://doi.org/10.1007/s00262-011-1184-2 PubMed DOI PMC

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; 28:578-90; PMID:19165151; https://doi.org/10.1038/emboj.2009.1 PubMed DOI PMC

Tufi R, Panaretakis T, Bianchi K, Criollo A, Fazi B, Di Sano F, Tesniere A, Kepp O, Paterlini-Brechot P, Zitvogel L et al.. Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin. Cell Death Differ 2008; 15:274-82; PMID:18034188; https://doi.org/10.1038/sj.cdd.4402275 PubMed DOI

Shellman YG, Howe WR, Miller LA, Goldstein NB, Pacheco TR, Mahajan RL, LaRue SM, Norris DA. Hyperthermia induces endoplasmic reticulum-mediated apoptosis in melanoma and non-melanoma skin cancer cells. J Invest Dermatol 2008; 128:949-56; PMID:17989736; https://doi.org/10.1038/sj.jid.5701114 PubMed DOI

Chen T, Guo J, Han C, Yang M, Cao X. Heat shock protein 70, released from heat-stressed tumor cells, initiates antitumor immunity by inducing tumor cell chemokine production and activating dendritic cells via TLR4 pathway. J Immunol 2009; 182:1449-59; https://doi.org/10.4049/jimmunol.182.3.1449 PubMed DOI

Srivastava PK, Udono H, Blachere NE, Li Z. Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 1994; 39:93-8; PMID:8276462; https://doi.org/10.1007/BF00188611 PubMed DOI

Zappasodi R, Pupa SM, Ghedini GC, Bongarzone I, Magni M, Cabras AD, Colombo MP, Carlo-Stella C, Gianni AM, Di Nicola M. Improved clinical outcome in indolent B-cell lymphoma patients vaccinated with autologous tumor cells experiencing immunogenic death. Cancer Res 2010; 70:9062-72; PMID:20884630; https://doi.org/10.1158/0008-5472.CAN-10-1825 PubMed DOI

Bettaieb A, Averill-Bates DA. Thermotolerance induced at a mild temperature of 40°C alleviates heat shock-induced ER stress and apoptosis in HeLa cells. Biochim Biophys Acta 2015; 1853:52-62; PMID:25260982; https://doi.org/10.1016/j.bbamcr.2014.09.016 PubMed DOI

Dutta S, Chiu YC, Probert AW, Wang KK. Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death. Biol Chem 2002; 383:785-91; PMID:12108543; https://doi.org/10.1515/BC.2002.082 PubMed DOI

Vanags DM, Porn-Ares MI, Coppola S, Burgess DH, Orrenius S. Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J Biol Chem 1996; 271:31075-85; PMID:8940103; https://doi.org/10.1074/jbc.271.49.31075 PubMed DOI

Martins I, Michaud M, Sukkurwala AQ, Adjemian S, Ma Y, Shen S, Kepp O, Menger L, Vacchelli E, Galluzzi L et al.. Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death. Autophagy 2012; 8:413-5; PMID:22361584; https://doi.org/10.4161/auto.19009 PubMed DOI

Adkins I, Koberle M, Grobner S, Bohn E, Autenrieth IB, Borgmann S. Yersinia outer proteins E, H, P, and T differentially target the cytoskeleton and inhibit phagocytic capacity of dendritic cells. Int J Med Microbiol 2007; 297:235-44; PMID:17462949; https://doi.org/10.1016/j.ijmm.2007.02.005 PubMed DOI

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