The presence of TAA in cancer cells is a mandatory requirement for activation of effector CTLs. TAA can be divided into four different groups: (i) differentiation antigens, which are lineage-specific and expressed in tumor as well as in normal cells from which the tumors arise; (ii) overexpressed antigens, which are broadly expressed in many normal tissues, but present in tumor cells at higher levels; (iii) tumor-specific antigens, usually typical for individual tumors, resulting from genetic alterations; (iv) cancer–testis antigens (CTA) that are expressed in various types of malignant human tumors and are restricted in normal tissues to germ cells of the testis, with occasional expression in female reproductive organs (73). CTA are particularly susceptible to epigenetic regulation. They include melanoma-associated antigen (MAGE), NY-ESO-1, and SSX gene families as well as the GAGE/PAGE/XAGE superfamily. MAGE, GAGE, BAGE, SSX, and LAGE-1/NY-ESO-1 are frequently methylated and down-regulated in tumor cells. The CTA family also includes P1A antigen, one of the best known murine TAA, which is a homolog of human MAGE (74). P1A is an endogenous protein, initially identified in chemically induced mast cell-mastocytoma 815 (75). As a classical CTA, P1A is not expressed in normal tissues, but expressed only in placenta and testis. P1A epitopes are presented to T lymphocytes through MHC H2-k2d and may induce strong specific response of CTLs (76). Similar to human MAGE gene family, in several murine tumors P1A is not expressed as a result of methylation of the promoter regions (77).

# Immunoregulatory Effects of Drugs Targeting Epigenetic Mechanisms

Drugs targeting epigenetic mechanisms can modulate expression of multiple genes including tumor suppressor genes, oncogenes, tumor associated antigens, as well as molecules involved in antigen presentation, co-stimulatory signaling and cytokines. Among different classes of genes described as epigenetically regulated, these encoding TAA are undoubtedly essential for T cell activation and tumor recognition by immune response. The expression of CTA can be restored by a number of hypomethylating agents (78, 79). Over 20 years ago, it was demonstrated that 5-aza-2′-deoxicitidyne (5-aza-dC) up-regulates MAGE-1 expression in tumor, but not in normal cells, and leads to HLA-A1-restricted lysis of tumor cells by CTLs (78). Further studies revealed that a variety of other CTA can be induced by either 5-aza-dC or other inhibitors of DNMTs (80–82). Methyltransferase inhibitors can also induce expression of MHC class I molecules. These effects result from the impact on both MHC genes as well as from regulation of virtually all components of the APM, including TAP1 and 2, proteasome subunits (81, 83). Moreover, antigen presentation can be augmented by up-regulation of type I and II interferons, their receptors, and components of the IFN-signaling pathways (84). Importantly, the effects of methyltransferase inhibitors are long-lasting as CTA are

detectable for next several weeks after treatment and they are recognized by antigen-specific CD8+ T cells (77, 85, 86). Also histone deacetylase inhibitors (HDACi) may affect expression of TAA, but mostly by increasing or restoring expression level of proteins involved in antigen presentation. For example, trichostatin A (TCA) was shown to up-regulate or induce expression of TAP-1, TAP-2, tapasin, and LMP-2 in murine tumor cell lines (87, 88). Moreover, the TCA-mediated increase in MHC levels results in activation of adaptive immune response and inhibition of tumor growth in mice (88). Additionally, HDACi increase the expression of MHC class II and co-stimulatory molecules in human and mouse melanoma and trophoblast cell lines (89, 90). TCA, by means of activation of the pIII-CIITA promoter in neoplastic cells and induction of MHC class II expression, was also found to augment CD4+ T cells proliferation (91). However, the result of their action is complex, since they down-regulate one antigen, Muc1, and up-regulate another, NY-ESO-1, at the same time (92). Based on these findings, a great deal of interest has been generated in investigation of epigenetic therapy influence on antitumor immune response.

It was also demonstrated that HDACi can affect polarization of naive CD4+ T cells toward Th1 and Th2 subsets. Vorinostat by inhibiting STAT6 and TARC may impair the functions of CD4+ T cells, shifting the balance toward Th1 response (93). Also hypomethylating agents increase production of cytokines, including IL-2, TNF, and IFN-γ (94, 95). Epigenetic treatment facilitates killing of tumor cells by NK cells or CTLs through up-regulation of TRAIL-R2 and FAS in transformed cells (96, 97). Furthermore, enhancement of NK-mediated tumor cell death was also induced by TCA by up-regulation of UL-16-binding protein expression (a ligand for cytotoxicity NKG2D receptor) (98).

# Tumor Cell Recognition by Immune System After Photodynamic Therapy Enhanced by Epigenetic Treatment

The still elusive goal for effective cancer immunotherapy is to overcome tumor escape mechanisms and to trigger development of systemic adaptive antitumor immune response allowing for the control of distant metastases. As described above, PDT is an effective local treatment that induces acute local inflammatory response. However, development of systemic adaptive immune response after PDT strongly depends on the efficacy of presentation and recognition of tumor antigens by the immune cells (1). Various approaches have been examined to accelerate the priming phase of immune response after PDT. Induction of antitumor activity depends on activation of CD8+ T cells and administration of immature DCs into the PDT-treated tumors resulted in effective activation of T and NK cells (24). Also, the PDT effectiveness was improved by administration of adjuvants, such as glycated chitosan (99). Additionally, the role of expression level of MHC class I in PDT was evaluated in the treatment of patients with vulval intraepithelial neoplasia (VIN). In VIN lesions that respond to PDT, significant increase of CD8+ infiltration after treatment was observed when compared to nonresponders. Interestingly, none of responding VIN patients showed any evidence of MHC class I down-regulation, whereas all of the cases of VIN lacking of MHC I failed to respond to PDT (100).

One of the approaches to increase immunogenicity of tumor cells focused on their genetic modification to enhance activation of CTLs by DCs. Introduction of foreign antigen, such as green fluorescence protein (GFP) to radiation-induced fibrosarcoma cells was observed to induce strong tumor-specific immune response allowing for long-term tumor control. Re-challenge experiments revealed that survived mice developed resistance to GFP-positive cells (101). These data are in line with another study demonstrating that the presence of β-galactosidase antigen in tumor cells is able to increase immunogenicity allowing PDT to elicit strong antitumor effects and long-term immunity to reinoculated tumor cells (102). In this vein, the same authors have transfected tumor cells with a gene encoding P1A, a model CTA in the mouse. The presence of this antigen led to effective antitumor immune response that only developed when PDT was used and was found sufficient to prevent tumor growth when tumor cells were re-inoculated (74). Moreover, PDT was shown to enhance systemic immune responses to tumors in patients with Basal cell carcinoma. The immune recognition of cancer cell antigen – Hip 1 – was improved by PDT treatment (103).

Considering that 5-aza-dC restores expression of CTA including P1A as well as MHC class I molecules (77), we sought to evaluate the antitumor effects of the combined treatment

to the release of TAA that are further phagocytosed by attracted to the tumor

involving PDT and administration of 5-aza-dC. We have observed that treatment with 5-aza-dC alone restores expression of MHC class I molecules as well as induces expression of P1A antigen in four different murine tumor models and two strains of mice (104). Antitumor effects of 5-aza-dC were rather insignificant when used alone. However, when we combined 5-aza-dC with PDT, we observed prolonged complete antitumor responses in mice with EMT6 mammary tumors and CT26 colon adenocarcinomas and significant prolongation of survival in mice bearing 4T1 mammary tumors and LLC lung carcinomas. The antitumor effects of the combination treatment were strongly dependent on the presence of CD8+ CTLs as their depletion with monoclonal antibodies almost completely abrogated antitumor effects. On the other hand, CD4+ T cells played only a supportive role. We have also observed that the combined treatment led to expansion of IL-17-producing CD4+ T cells, which are known to stimulate CD8+. Intriguingly, pentamer staining for P1A-specific CD8+ T cell population revealed no significant changes in draining LNs and spleens between experimental groups. Moreover, all mice treated with PDT and 5-aza-dC that remained long-term tumor-free have rejected re-inoculated tumor cells even if the cells were P1A-negative. These findings suggest that the presence of P1A is not essential for the maintenance of long-lasting antitumor immunity. It is possible that 5-aza-dC combined with PDT may lead to increased expression and release of TAA in the PDT-treated microenvironment. Together with PDT-induced inflammation and the release of DAMPs, the combination treatment would confer better antigen presentation of P1A. Improved tumor recognition by immune cells can further expand the repertoire of antigen-specific T cells thereby increasing the

cells homing to the tumor site in order to destroy residual tumor cells.

immunogenic outcome of PDT. Thus, it can be hypothesized that PDT of tumors with TAAs up-regulated by 5-aza-dC can induce concomitant immunity to other subdominant and possibly weakly immunogenic antigens and facilitate epitope spreading. Immunity to the latter can sustain antitumor activity in mice. To summarize, our findings demonstrate that treatment combining 5-aza-dC with PDT leads to local cytotoxic effects accompanied by the release of induced TAAs allowing for development of antitumor immune response and long-term survival (**Figure 2**). Based on these findings, we hypothesize that inhibition of DNA methylation could unleash stronger immune response against TAA in tumor-bearing mice undergoing PDT.

### References

# Author Contributions

MW, AM, and JG reviewed relevant literature. MW, AM, and JG drafted the manuscript. JG revised the manuscript and supervised MW and AM. MW and AM designed the figure. All authors read and approved the final manuscript.

# Acknowledgments

This work was supported by a grant from the European Commission 7th Framework Program: fp7-regpot-2012-ct2012- 316254-bastion (JG).

granzyme B, chemotherapeutic drugs, and gamma irradiation. *J Immunol* (1998) **161**:3936–42.

*Eur J Immunol* (1998) **28**:4010–9. doi:10.1002/(SICI)1521-4141(199812)28: 12<4010::AID-IMMU4010>3.0.CO;2-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Wachowska, Muchowicz and Golab. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Edited by:*

*Fabrizio Mattei, Istituto Superiore di Sanità, Italy*

#### *Reviewed by:*

*Luis De La Cruz-Merino, Hospital Universitario Virgen Macarena, Spain Carlos Alfaro, Clínica Universidad de Navarra, Spain*

#### *\*Correspondence:*

*Abhishek D. Garg<EMAIL_ADDRESS><EMAIL_ADDRESS>Patrizia Agostinis<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology*

*Received: 17 September 2015 Accepted: 02 November 2015 Published: 20 November 2015*

#### *Citation:*

*Garg AD, Galluzzi L, Apetoh L, Baert T, Birge RB, Bravo-San Pedro JM, Breckpot K, Brough D, Chaurio R, Cirone M, Coosemans A, Coulie PG, De Ruysscher D, Dini L, de Witte P, Dudek-Peric AM, Faggioni A, Fucikova J, Gaipl US, Golab J, Gougeon M-L, Hamblin MR, Hemminki A, Herrmann M, Hodge JW, Kepp O, Kroemer G, Krysko DV, Land WG, Madeo F, Manfredi AA, Mattarollo SR, Maueroder C, Merendino N, Multhoff G, Pabst T, Ricci J-E, Riganti C, Romano E, Rufo N, Smyth MJ, Sonnemann J, Spisek R, Stagg J, Vacchelli E, Vandenabeele P, Vandenberk L, Van den Eynde BJ, Van Gool S, Velotti F, Zitvogel L and Agostinis P (2015) Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death. Front. Immunol. 6:588. doi: 10.3389/fimmu.2015.00588*

# Molecular and translational Classifications of DaMPs in immunogenic Cell Death

*Abhishek D. Garg1 \*, Lorenzo Galluzzi2,3,4,5,6 , Lionel Apetoh7,8,9 , Thais Baert10,11 , Raymond B. Birge12 , José Manuel Bravo-San Pedro2,3,4,5,6 , Karine Breckpot13 , David Brough14 , Ricardo Chaurio15 , Mara Cirone16 , An Coosemans10,11 , Pierre G. Coulie17 , Dirk De Ruysscher18 , Luciana Dini19 , Peter de Witte20 , Aleksandra M. Dudek-Peric1 , Alberto Faggioni21 , Jitka Fucikova22,23 , Udo S. Gaipl24 , Jakub Golab25 , Marie-Lise Gougeon26 , Michael R. Hamblin27 , Akseli Hemminki28,29,30 , Martin Herrmann15 , James W. Hodge31 , Oliver Kepp2,3,4,5,32 , Guido Kroemer2,3,4,5,32,33,34 , Dmitri V. Krysko35,36 , Walter G. Land37 , Frank Madeo38,39 , Angelo A. Manfredi40 , Stephen R. Mattarollo41 , Christian Maueroder15 , Nicolò Merendino42 , Gabriele Multhoff43 , Thomas Pabst44 , Jean-Ehrland Ricci45 , Chiara Riganti46 , Erminia Romano1 , Nicole Rufo1 , Mark J. Smyth47,48 , Jürgen Sonnemann49 , Radek Spisek22,23 , John Stagg50 , Erika Vacchelli2,3,4,5,6 , Peter Vandenabeele35,36 , Lien Vandenberk51 , Benoit J. Van den Eynde52 , Stefaan Van Gool51 , Francesca Velotti53 , Laurence Zitvogel6,54,55,56 and Patrizia Agostinis1 \**

*1Cell Death Research and Therapy Laboratory, Department of Cellular Molecular Medicine, KU Leuven – University of Leuven, Leuven, Belgium, 2Equipe 11 Labellisée Ligue Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France, 3U1138, INSERM, Paris, France, 4Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 5Université Pierre et Marie Curie, Paris, France, 6Gustave Roussy Comprehensive Cancer Institute, Villejuif, France, 7U866, INSERM, Dijon, France, 8 Faculté de Médecine, Université de Bourgogne, Dijon, France, 9Centre Georges François Leclerc, Dijon, France, 10Department of Gynaecology and Obstetrics, UZ Leuven, Leuven, Belgium, 11 Laboratory of Gynaecologic Oncology, Department of Oncology, Leuven Cancer Institute, KU Leuven, Leuven, Belgium, 12Department of Microbiology, Biochemistry, and Molecular Genetics, University Hospital Cancer Center, Rutgers Cancer Institute of New Jersey, New Jersey Medical School, Newark, NJ, USA, 13 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Jette, Belgium, 14 Faculty of Life Sciences, University of Manchester, Manchester, UK, 15Department of Internal Medicine 3 – Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nurnberg, Erlangen, Germany, 16Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, 17de Duve Institute, Université Catholique de Louvain, Brussels, Belgium, 18Department of Radiation Oncology, University Hospitals Leuven, KU Leuven – University of Leuven, Leuven, Belgium, 19Department of Biological and Environmental Science and Technology, University of Salento, Salento, Italy, 20 Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven – University of Leuven, Leuven, Belgium, 21Sapienza University of Rome, Rome, Italy, 22SOTIO, Prague, Czech Republic, 23Department of Immunology, 2nd Faculty of Medicine, University Hospital Motol, Charles University, Prague, Czech Republic, 24Department of Radiation Oncology, Universitätsklinikum Erlangen, Erlangen, Germany, 25Department of Immunology, Medical University of Warsaw, Warsaw, Poland, 26Biotherapy and Vaccine Unit, Institut Pasteur, Paris, France, 27Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA, 28Cancer Gene Therapy Group, Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland, 29Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland, 30 TILT Biotherapeutics Ltd., Helsinki, Finland, 31Recombinant Vaccine Group, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA, 32Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France, 33Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France, 34Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden, 35Molecular Signaling and Cell Death Unit, Inflammation Research Center, VIB, Ghent, Belgium, 36Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, 37Molecular ImmunoRheumatology, INSERM UMRS1109, Laboratory of Excellence Transplantex, University of Strasbourg, Strasbourg, France, 38 Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria, 39BioTechMed Graz, Graz, Austria,*

**Abbreviations:** DAMP, damage-associated molecular pattern; DC, dendritic cell; ER, endoplasmic reticulum; GEMM, genetically engineered murine model; HSP, heat shock protein; Hyp, hypericin; ICD, immunogenic cell death; NDV, Newcastle disease virotherapy; PDT, photodynamic therapy; ROS, reactive oxygen species.

*40 IRRCS Istituto Scientifico San Raffaele, Università Vita-Salute San Raffaele, Milan, Italy, 41 Translational Research Institute, University of Queensland Diamantina Institute, University of Queensland, Wooloongabba, QLD, Australia, 42 Laboratory of Cellular and Molecular Nutrition, Department of Ecological and Biological Sciences, Tuscia University, Viterbo, Italy, 43Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, 44Department of Medical Oncology, University Hospital, Bern, Switzerland, 45 INSERM, U1065, Université de Nice-Sophia-Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Équipe "Contrôle Métabolique des Morts Cellulaires", Nice, France, 46Department of Oncology, University of Turin, Turin, Italy, 47 Immunology in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Insitute, Herston, QLD, Australia, 48School of Medicine, University of Queensland, Herston, QLD, Australia, 49Department of Paediatric Haematology and Oncology, Children's Clinic, Jena University Hospital, Jena, Germany, 50Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Institut du Cancer de Montréal, Faculté de Pharmacie, Université de Montréal, Montreal, QC, Canada, 51 Laboratory of Pediatric Immunology, Department of Microbiology and Immunology, KU Leuven – University of Leuven, Leuven, Belgium, 52 Ludwig Institute for Cancer Research, de Duve Institute, Université Catholique de Louvain, Brussels, Belgium, 53Department of Ecological and Biological Sciences, Tuscia University, Viterbo, Italy, 54University of Paris Sud, Le Kremlin-Bicêtre, France, 55U1015, INSERM, Villejuif, France, 56Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 507, Villejuif, France*

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

Keywords: anti-tumor immunity, immunogenicity, immunotherapy, molecular medicine, oncoimmunology, patient prognosis, translational medicine

# INTRODUCTION AND HISTORICAL BACKGROUND

Augmenting the immunogenicity of cancer cells to improve the efficacy of cancer therapy is a paradigm that has gained significant momentum over the past 5 years (1–5). Researchers have realized that besides therapeutically exploiting innate or adaptive immune cells directly (e.g., through dendritic cell (DC)-based vaccines or adoptive T-cell transfer) and/or improving the effector functions of T cells (through checkpoint-blocking therapies), cancer cells also need to be made immunogenic (1, 4, 6, 7). This has diverted attention toward studying the interface between stressed or dying cancer cells and the immune system, in the hope of efficiently exploiting it for therapeutic purposes (1).

Early indications regarding immune system-driven tumor control emerged in the eighteenth century, when feverish infections in cancer patients were circumstantially associated with tumor remission (8). The first evidence that immunotherapy can be applied to achieve tumor regression emerged from the work of William Coley, who in the 1890s achieved tumor regression in some sarcoma/lymphoma patients upon the intra-tumoral injection of streptococcal cultures (provided by Robert Koch) (8, 9). In the following 43 years, Coley injected nearly 900 (mostly sarcoma) patients with his bacterial preparation (achieving a cure rate >10%), which later became known as "Coley's toxin" (8, 10). However, the Coley's toxin came under intense scrutiny owing to an elevated toxicity and some difficulties in reproducing remission rates (8). Eventually, the first experimental evidence that virus-unrelated tumors can indeed be recognized by the host immune system emerged in the 1940s, and by the 1960s, coupled with the discovery of T cells, it was proposed that the human immune system may also react against tumors (11). The ability of anticancer therapies to enhance the immunogenic potential of malignant cells gained some appreciation by the 1970s (12–14). It was recognized that if specific treatments are applied (e.g., radiotherapy, the bacillus Calmette–Guerin, or some chemotherapeutics), the immunogenicity of malignant cells increases enough to induce durable anti-tumor immunity (12–14). By the 1980s, researchers started to report more specific observations regarding the therapeutic impact of cancer cell immunogenicity, e.g., the ability of curative hyperthermia to cause the (heat-shock based) generation of circumstantial anti-tumor immunity (15), the fact that the immunogenicity of cancer cells influences patient prognosis after radiotherapy (16), and the increase in tumor immunogenicity due to hydrostatic pressure (17). However, these early studies (especially those published before the 1980s) had several issues linked to a lack in consensus. For instance, due to early controversies on the existence of tumor-associated antigens (TAAs) (11), the target of tumor-specific immune responses was unclear, and the mechanism of action of some therapies came under scrutiny. Moreover, such therapies could operate by directly modulating immune effector cells rather than improving the immunogenic potential of tumors (18). In particular, the death of cancer cells exposed to therapy was never suspected to drive anti-tumor immunity, since it was considered to be a relatively "silent" process in terms of immunogenicity (19). Moreover, the classical "self/non-self " theory was unable to explain the possibility that dying cancer cells could elicit an immune response (20).

By the early 1990s, the molecular characterization of mice and human TAAs clarified the entities targeted by anti-tumor immune responses (11). Similarly, the so-called "danger theory" started to emerge, challenging the classical model of "self/nonself " immune recognition, especially in a diseased or damaged tissue (20, 21). This model proposed that the immune recognition is not restricted to "non-self " entities, but rather discriminates between "dangerous" and "safe" entities, irrespective of source (20–22). Indeed, "dangerous" entities include pathogens as well as injured, infected, diseased and necrotic tissues, or cells undergoing non-physiological cell death which emit danger signals (or alarmins) with pro-inflammatory activity (21, 22). These danger signals are now collectively referred to as "damage-associated molecular patterns" (DAMPs) (23). DAMPs are endogenous molecules that are concealed intracellularly in normal conditions, but are exposed or released upon stress, injury, cell death, thereby becoming able to bind cognate receptors on immune cells (3, 24–27). **Table 1** summarizes the most prominent DAMPs characterized to date and their mode of emission, the cell death pathway they are associated with, and their known cognate receptors. It is important to consider that not all DAMPs may act as immunogenic danger signals. Several DAMPs exist that are crucial for the maintenance of tissue homeostasis, and the avoidance of auto-immune responses, as they exert immunosuppressive effects, including phosphatidylserine (PS), annexin A1 (ANXA1), death domain 1α (DD1α), B-cell CLL/lymphoma 2 (BCL2) and some extracellular matrix-derived molecules (**Table 1**). Accordingly, the blockade of these anti-inflammatory DAMPs accentuates the immunogenic potential of dying cells, or renders immunogenic otherwise tolerogenic forms of cell death (28, 29). Moreover, some danger signals are not always involved in the immunogenicity of cell death, but act as "bystanders." This is the case for heat shock protein 90 kDa alpha (cytosolic), class A member 1 (HSP90AA1, best known as HSP90) exposed on the cell surface after melphalan treatment (30). Last (but not least), several DAMPs may be subjected to post-translational modifications (e.g., oxidation, reduction, citrullination) that may potentially neutralize, increase, or change their immunogenic properties (31, 32) – a process that is still incompletely understood.

Despite these advances, the overall role of regulated cell death (RCD) (97) in augmenting cancer immunogenicity remained obscure. Initial observations involving the immunogenicity of cell death in the efficacy of cancer therapy were published between 1998 and 2004, when it was proposed that the non-apoptotic demise of malignant cells (within the context of the so-called "immunogenic death") could be associated with the emission of the danger signal heat shock 70 kDa protein 1A (HSPA1A, best known as HSP70) (**Table 1**), enhancing the immunogenic potential of dying cancer cells *in vivo* (98, 99). The dogmatic view that only necrotic or non-apoptotic (as postulated by the "immunogenic death" concept) cancer cells are characterized by an elevated immunogenic potential started to be questioned by a series of studies published between 2005 and 2007 (41, 70, 100, 101). These publications outlined that cancer cells undergoing apoptosis in response to specific anticancer therapies are immunogenic [a subroutine termed immunogenic cell death (ICD)], as long as they emit precise DAMPs in a spatiotemporally defined fashion (26, 102, 103). Cells succumbing to ICD are sufficient for the elicitation of durable anti-tumor immune responses (1, 26, 53, 102, 104). ICD is indeed paralleled by the redirection and emission of DAMPs, owing to the stimulation of distinct danger signaling pathways occurring in synchrony with cell death signaling (103). **Table 2** summarizes the main signaling pathways that play a role in the trafficking and emission of DAMPs. ICD-associated DAMPs and other immunostimulatory factors released by cells destined to undergo ICD favor the establishment of a productive interface between dying cancer cells and innate immune cells (like DCs or macrophages), thereby leading to the initiation of a therapeutically relevant adaptive immune response (**Figure 1**) (102, 105). In some contexts, DAMPs may regulate the function of specific innate immune cell subsets, e.g., following anthracycline treatment, extracellular adenosine triphosphate (ATP) assists in recruitment and differentiation of CD11c+Cd11b+Ly6Chigh cells into CD11c+CD86+MHCII+ DCs (106); similarly, necrosis associated F-actin exposure activates an immune response by directing the dead cell debris to specifically CD8α+ DCs (59, 107). Indeed, DCs and other antigen-presenting cells exposed to cancer cells succumbing to ICD can then prime CD4+ T cells (and polarize them into TH1, TH17, or TH1/TH17 like phenotype), CD8+ cytotoxic T lymphocytes (CTLs) and γδ T lymphocytes against one or several TAAs (**Figure 1**) (102). Of note, residual cancer cells that survive ICD inducers can also show some enduring immunogenic characteristics that make them susceptible to immunological control by CTLs (108–110).

FIGURE 1 | The molecular complexity of immunogenic cell death in cancer. Cancer cells undergoing immunogenic cell death (ICD) emit danger signals for establishing a productive interface with components of the host immune system, including dendritic cells (DCs). DCs exposed to cancer cells succumbing to ICD "prime" the adaptive arm of the immune system, consisting of various effector T-cell populations, which in turn targets therapy-resistant cancer cells. Various molecules are critical for the execution of these processes. The molecular network of ICD-relevant proteins was build using the STRING modeling database (http:// string-db.org/) (126).

# IMMUNOGENIC CELL DEATH INDUCERS

Over the past few years, a number of single-agent ICD inducers have been discovered, encompassing conventional chemotherapeutics, targeted anticancer agents and various other biological and physicochemical therapies (18, 102, 104, 127). **Table 3** summarizes single-agent ICD inducers characterized so far, as per consensus guidelines (104), and the spectra of DAMPs and other immunostimulatory signals associated with them. For combinatorial therapeutic strategies capable of achieving ICD, readers may want to refer to other recent publications (18, 128, 129). It is clear that a general structure–function relationship capable of clustering all existing ICD inducers and predicting new ones does not exist (130), an issue that makes discovering new ICD-inducing therapies based on cheminformatic analyses challenging, if not impossible. A peculiar characteristic of most, if not all, ICD inducers is their ability to induce reactive oxygen species (ROS)-based/ associated endoplasmic reticulum (ER) stress, as first delineated for anthracyclines (30, 34, 35, 42, 123, 131–133). This peculiarity was exploited for the targeted discovery of hypericin-based photodynamic therapy (Hyp-PDT) – a therapeutic modality that can trigger ICD through the induction of ROS that target the ER (35, 116, 134). Along with an ever more precise characterization of the links between ROS, ER stress, and ICD induction (135, 136), it became clear that the more "focused" ER stress is, the higher the probability of inducing ICD (3, 26, 53, 137). These observations paved way for a classification system based on how ICD inducers engage ER stress for cell death and danger signaling (3, 26, 53, 138). Based on this classification, Type I ICD inducers are defined as anticancer agents that act on non-ER proteins for the induction of cell death, but promote collateral ER stress for danger signaling, thereby operating on multiple targets (3, 26, 53), while Type II ICD inducers are anticancer agents that target the ER for both cell death induction and danger signaling (3, 26, 53). **Table 4** summarizes the classification of current ICD inducers into Type I and Type II, and their cell death/danger signaling targets. Such a classification suggest that while Type I ICD inducers can be discovered through various approaches (e.g., DAMP-based drug screening platforms) (130, 139), putative Type II ICD inducers can be characterized rapidly on the basis of their ability to selectively or predominantly target the ER. Recent findings comforted the purpose and usefulness of this classification system, as two novel Type II ICD inducers [i.e., PtII N-heterocyclic carbene complex (140) and Newcastle disease virotherapy (NDV) (43)] were identified based on the notion that they induce predominant ROS-based ER stress (138). Nevertheless, as more ICD inducers and features are discovered, this classification system is expected to evolve or be substituted by a more refined one.

Since its discovery, a plethora of molecular and immunological components responsible for ICD have been discovered (**Figure 1**) (26, 102, 188). **Table 5** summarizes the molecular and immunological determinants of ICD characterized so far, as well as the models of ICD in which they operate (in a positive, negative or dispensable manner). Anthracyclines and oxaliplatin are the most common ICD inducers employed in experimental settings, followed by Hyp-PDT. According to current understanding, cancer cell-associated determinants of ICD can be subdivided into those that are common to all ICD inducers (i.e., "core" signaling components), and those that operate in an ICD inducer-dependent manner (i.e., "private" signaling components) (26, 189). Thus, eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3, best known as PERK) and the ER-to-Golgi secretory machinery are considered "core" signaling components on the cancer cell side (26, 102). Similarly, from the immune system side, a general role for (IFNγ-producing) CD4+ and CD8+ T cells has been confirmed for most, if not all, ICD inducers (**Table 5**). Interestingly, some components that are required for ICD induction by some agents (like autophagy for anthracyclines and oxaliplatin) (190) might be either dispensable for ICD induction by other agents, e.g., autophagy for NDV (43) and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), caspase-8 (CASP8) activation or cytosolic Ca2+ levels for Hyp-PDT (35); or even negatively regulate ICD in some settings, e.g., autophagy in case of Hyp-PDT (34) (**Table 5**). Thus, it will be important to expand our molecular knowledge of ICD to as many experimental settings as possible.

# IMMUNOGENIC CELL DEATH FROM BENCH TO BEDSIDE

The relevance of ICD has been verified in a number of rodent models, with a variety of chemical and physicochemical ICD inducers (26, 102). **Table 6** summarizes the most prominent mouse or rat models used so far for the characterization and study of ICD. For the moment, ICD has been mostly investigated in heterotopic syngeneic subcutaneous models (195). Within such models, inter-species differences (mouse *versus* rats), inter-strain differences (among BALB/c, C57BL/6, C3H and KMF mice), and inter-cell line differences, as well as differences in therapeutic setups (prophylactic *versus* curative) have been amply accounted for (**Table 6**). Nevertheless, there is predominance in the use of cancer cells derived from carcinogen-induced tumors and transplanted subcutaneously (**Table 6**). In very few cases, ICD has been characterized in either orthotopic (for NDV) or spontaneous (for anthracyclines) tumor murine models (**Table 6**). This has been questioned as a prominent Achilles' heel of ICD research (195). While this criticism is valid, it has to be recognized that no rodent model is perfect at all immunological levels (196).

As a recent systematic review summarized (196), heterotopic murine models suffer from a number of caveats, including the inability to recapitulate the early interaction between transformed cells and the immune system and the incompatibility between the cancer type and the site-of-transplantation (196). Orthotopic murine models are useful as they overcome the cancer cell-tissue type incompatibility issue (196). While genetically engineered tumor murine models (GEMMs) overcome most of the issues mentioned above, they come with their own set of shortcomings, including a limited genetic mosaicism, a low tumor heterogeneity, a lack of well-defined immunogenic TAAs, the presence of unintended "passenger" genetic modifications, and a reduced mutational spectrum (196). Many of these parameters are critical for responses to immunotherapy/ICD. For instance, the lack of well-defined immunogenic TAAs was the reason why preliminary results obtained in spontaneously developing murine tumors disputed the very existence of TAAs (11). Similarly, a high mutational spectrum (which produces considerable amounts of neo-antigens) has been found to be mandatory for the clinical efficacy of checkpoint blockers (209). Last (but not least), laboratory rodent models in general are associated with some critical issues, including the fact that a high level of inbreeding (which produces a number of shortcomings e.g., homozygous recessive defects) reduces the general immunological fitness, responsiveness and diversity in these models (196, 210, 211). Moreover, numerous immunological differences between mouse and humans tend to affect the translational relevance of the findings obtained (26, 211, 212). Also, the time frames of tumor growth rates between rodent models and humans are relatively divergent (196, 213, 214). This further complicates clinical translation of immunotherapeutic paradigms since the level of immunosurveillance and immunoediting experienced by human tumors can be much higher than any rodent tumor model.

In summary, it would be ideal to test ICD across as many different rodent models as possible, in order to determine the features that can be exploited for therapeutic purposes in humans. Moreover, if ICD fails in a specific experimental model, active effort should be made to characterize the mechanisms behind such failure, since resistance phenotypes can have profound clinical implications. This emerges from various studies summarized in **Table 7**. Indeed, several ICD resistance mechanisms exist operating at both the cancer cell and the immune system level, which have been characterized in different experimental models. Several of these resistance mechanisms have also been identified in cancer patients, thereby justifying further studies along these lines **Table 7**.

A considerable amounts of clinical findings support the relevance of ICD or ICD-related signatures in (at least subsets of) cancer patients. As summarized in **Table 8**, various ICD-linked (specific) parameters have been associated with the prognosis of cancer patients treated with clinically relevant ICD inducers (like anthracyclines, oxaliplatin, paclitaxel, or radiotherapy). Moreover, it is becoming clear that ICD-related or ICD-derived (immunological) genetic signatures (e.g., a *MX1*-centered metagene, a *CXCR3*-*PRF1*-*CASP1*-centered metagene, an *ASAH1*-centered metagene) can be positively associated with good prognosis in patients affected by various neoplasms, including breast, lung, and ovarian malignancies (141, 188, 220). These observations indicate that ICD or ICD-relevant parameters may have prognostic or predictive relevance in at least a subset of cancer patients. It will be important to characterize new and more specific ICD-associated parameters linked to patient prognosis as well as biomarkers that may predict improved disease outcome in cancer patient treated with ICD inducers. Of note, considering the current clinical experience with immunotherapies (209, 221), the patients with an increased likelihood to benefit from ICD inducers are probably those that display pre-existing (baseline) immune reactivity against cancer cells (220, 222, 223). This may depend on the ability of ICD to reboot and/or revive pre-existing TAA-directed immunity rather to prime *de novo* immune reactivity (5, 191, 224). In future, it would be crucial to characterize biomarkers that allow clinicians to delineate patients with reduced baseline immune reactivity against malignant cells so that proper combinatorial therapies involving ICD inducers can be implemented.

# CONFRONTING THE CLINICAL REALITIES OF ANTI-TUMOR IMMUNITY

It is well-established that the response of cancer patients to immunotherapy relies on the activity of effector T cells [that employ their T-cell receptors (TCRs) for recognizing TAAs]. However, these TAA-targeting T cells may also constitute obstacles for effective anti-tumor immunity (234). As opposed to T lymphocytes recognizing pathogen-associated antigens (PAAs) (**Figure 2**), indeed, T cells directed against some TAAs (derived from nonmutated proteins that are source of self or near-to-self antigens) are developmentally subjected to negative selection in the thymus and peripheral lymphoid organs (234, 235) (**Figure 2**). As a result, T cells bearing TCRs with high affinity for self antigens (including some TAAs) are clonally deleted to avoid auto-immunity (234–237) (**Figure 2**). However, some "leakiness" in this process allows TAA-specific T cells possessing TCRs with low affinity to escape deletion (234, 236, 237) and persist, although at low precursor frequencies (238) (**Figure 2**). Unfortunately, as compared to PAA-specific T cells, which bear high-affinity TCRs (**Figure 2**), TAA-specific T cells exhibit limited effector and memory functions (234, 239). Coupled with the tendency of progressing tumors to generate a highly immunosuppressive microenvironment, this renders the insurgence of lifelong protective immunity nearly impossible (234). Of note, central and peripheral tolerance may not affect T cells reactive toward neo-tumor-specific antigens (neo-TSAs) e.g., tumor-specific neo-antigens that are generated *de novo* in the course of tumor progression because of mutational events (240, 241). However, the extent to which such neo-TSAs can elicit consistent "immunodominant" T cell reactivity is still a matter of investigation (240, 241). Nevertheless, in this context, inefficient T-cell stimulation can be overcome through the ICDbased improvement of effector T-cell functions (102). ICD can be further combined with checkpoint-blocking therapies, which

FIGURE 2 | Population dynamics of antigen-specific T cells during an immune response to infection or cancer. (A) T cells capable of putatively recognizing non-self, pathogen-associated antigens (PAAs) are not exposed to negative selection in the thymus or peripheral organs like lymph nodes. This allows for the constitutive presence of T lymphocytes bearing high-affinity T-cell receptor (TCR) in naïve conditions. Upon infection, these cells undergo robust expansion and acquire potent effector functions, hence driving an immune response that clears the pathogen and PAAs. Finally, PAA-specific T cells undergo contraction along with the establishment of immunological memory. To a limited extent, T cells reacting against PAAs expressed by virus-induced tumors may exhibit similar (although not identical) responses. (B) T cells that may recognize self or close-to-self antigens expressed by virus-unrelated malignancies undergo robust negative selection in the thymus and lymph nodes. Thus, all putative T lymphocytes bearing a high-affinity TCR against tumor-associated antigens (TAAs) are eliminated. However, some leakiness in this process allows for the persistence of TAA-specific T lymphocytes with low-affinity TCR, although at very low precursor frequencies. This is one of the reasons why in some individuals immunosurveillance at some stage fails to impede tumor progression. As malignant lesions progress, the amount of TAAs increases, causing a weak rise in TAA-specific T cells. However, tumor progression is generally coupled with the establishment of robust immunosuppressive networks that potently inhibit such TAA-targeting T cells. In this context, the administration of immunogenic cell death (ICD) according to a schedule that does not lead to lymphodepletion can favor the stimulation of TAA-targeting T cells and (re) instate immunosurveillance. Combining ICD inducers with checkpointblocking agents may further boost TAA-targeting immune responses. However, these treatments may not ensure the lifelong persistence of TAA-recognizing T cells, some of which are susceptible to elimination through tolerance mechanisms. Anticancer vaccines may counteract, at least to some extent, such loss. The figure was partly inspired from Baitsch et al. (234).

potently reverse immunosuppression (209, 242). However, the lifelong maintenance of anti-tumor T cells remains a particularly hard challenge.

In the clinical reality, anticancer agents are administered to patients in a limited number of cycles. Even if these therapeutic regimens may attain optimal efficacy in terms of ICD induction, they are unlikely to ensure the lifelong persistence of TAAdirected T cells with low-affinity TCR (234, 243). This probably reflects the contraction of TAA-targeting T cells occurring once the immunostimulatory stimulus provided by ICD ceases, owing to peripheral tolerance mechanisms (234). Clinically, it may not be feasible to administer ICD inducers repeatedly over time, since many of them can cause lymphopenia (which negatively affects disease outcome), or are associated with other side effects (244). It has been proposed that active immunization with ICDbased anticancer vaccines (which are associated with robust immunogenicity) given in a repetitive manner may achieve this goal (**Figure 2**) (234, 243, 245). Thus, it will be important to test whether the long-term administration of ICD-based anticancer vaccines can sustain the effector function of TAA-specific T cells bearing low-affinity TCRs, hence, ensuring lifelong disease-free survival. Of note, in the case of hematological malignancies, this issue could be overcome upon the adoptive transfer of CTLs expressing chimeric antigen receptors (CARs) (1). However, whether CAR-expressing CTLs generate protective immunological memory in the absence of considerable side effects remains to be determined. Moreover, the use of this therapeutic strategy against solid malignancies is relatively challenging owing to lack of well-defined "unique" TAAs (1, 246).

# CONCLUSION

The model of ICD has been considerably refined since the initial identification of a cell death modality manifesting apoptotic features but able to induce an adaptive immune response. This model strives to integrate several phenomena observed throughout the second half of the twentieth century in one therapeutically relevant platform. However, as discussed above, several challenges still need to be addressed. First, comprehensive testing should be performed in advanced experimental settings like GEMMs or orthotopic tumor models. Second, ICD resistance mechanisms should be characterized with precision. Third, various issues linked to the successful translation of ICD to cancer therapy will have to be resolved, including (but not limited to) treatment

# REFERENCES

schedules, dosages, and combinatorial strategies. This translational drive also needs to be coupled with effective strategies for the discovery of new and effective ICD inducers. Drug screening programs are often complicated by the possibility of false-positive (due to bystander presence of DAMPs) (30) or false-negative (due to limited number of biomarkers used for screening) hits. This issue can only be ironed out by discovering new and common regulators of ICD, and integrating them into existing screening platforms. Last, but not least, it will be important to identify new ICD-related/derived biomarkers that can be used to improve current protocols of patient stratification and clinical decision making. We are positive that all these objectives are at reach.

# AUTHOR CONTRIBUTIONS

ADG did the literature study, data collection, as well as conceived and wrote the manuscript. PA provided senior supervision and guidance, conceived the paper, helped in writing, and critically revised the manuscript. LG improved and edited the manuscript. JMBSP helped with the preparation of figures. All authors participated in the critical reading of the manuscript (wherever applicable), approved content and conclusions, as well as helped in ensuring the accuracy of cited literature.

# ACKNOWLEDGMENTS

We would like to explicitly declare that this manuscript does not aim to describe guidelines for the fields of ICD and DAMP research. Rather, it is meant to be a comprehensive classification and review of relevant literature expressing consensus discussions, opinions, and conclusions endorsed and/or supported by a number of researchers and clinicians investigating ICD and DAMPs. We would also like to acknowledge the following colleagues for their support, reading and/or positive appraisal of this manuscript: Wee Han Ang, Vincenzo Barnaba, Marco E. Bianchi, Karin de Visser, Sandra O. Gollnick, Peter Henson, Polly Matzinger, Marek Michalak, Kodi Ravichandran, and Andrew Thorburn. ADG is a recipient of the FWO postdoctoral fellowship 2013. This work was supported by grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen; G.0661.09, G.0728.10 and G.0584.12N) and KU Leuven (GOA/11/009) to PA; This paper presents research results of the IAP7/32, funded by the Interuniversity Attraction Poles Programme, initiated by the Belgian State, Science Policy Office.

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**Conflict of Interest Statement:** Akseli Hemminki is shareholder in Targovax AG and TILT Biotherapeutics Ltd. The remaining authors have no conflict of interest to declare.

*Copyright © 2015 Garg, Galluzzi, Apetoh, Baert, Birge, Bravo-San Pedro, Breckpot, Brough, Chaurio, Cirone, Coosemans, Coulie, De Ruysscher, Dini, de Witte, Dudek-Peric, Faggioni, Fucikova, Gaipl, Golab, Gougeon, Hamblin, Hemminki, Herrmann, Hodge, Kepp, Kroemer, Krysko, Land, Madeo, Manfredi, Mattarollo, Maueroder, Merendino, Multhoff, Pabst, Ricci, Riganti, Romano, Rufo, Smyth, Sonnemann, Spisek, Stagg, Vacchelli, Vandenabeele, Vandenberk, Van den Eynde, Van Gool, Velotti, Zitvogel and Agostinis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# TABLES

TABLE 1 | A list of prominent damage-associated molecular patterns (DAMPs) associated with cell death pathways or extracellular matrix.

*(Continued)*

#### TABLE 1 | Continued

*CD, cluster of differentiation; CLEC9A, C-type lectin domain family 9, member A; CPS-1, carbamoyl-phosphate synthase 1, mitochondrial; CXCR3, C-X-C motif receptor 3; FEEL-1/ CLEVER-1, fasciclin EGF-like/common lymphatic endothelial and vascular endothelial receptor-1; FPR-1, formyl peptides receptor-1; G2A, G2 accumulation; HMGB1, high-mobility group box 1; HSP, heat shock proteins; ICD, immunogenic cell death; IL, interleukin; LPG, leucine-rich alpha-2-glycoprotein-1; MFG-E8, milk fat globule-egf factor 8 protein; Mit DNA, mitochondrial DNA; P2XR, P2X receptor; P2YR, P2Y receptor; RAGE, receptor for advanced glycation endproducts; SREC-1, scavenger receptor class f member 1; TFAM, mitochondrial transcription factor A; TIM, transmembrane immunoglobulin and mucin domain; TLR, toll-like receptor(s).*

*Glossary (5, 19, 97): (1) Necrosis: primary necrosis is a form of cell death that can occur in a regulated or accidental manner, characterized by cellular swelling and rapid breakdown of the plasma membrane; (2) Necroptosis: necroptosis is a form of regulated cell death (RCD) manifesting with necrotic morphology and controlled by a signaling cascade involving (among other proteins) RIPK1, RIPK3, and MLKL; (3) Apoptosis: apoptosis is a form of RCD largely dependent on caspases activity and morphologically characterized by cell shrinkage, membrane blebbing, formation of apoptotic bodies, chromatin condensation, and systematic DNA fragmentation; (4) Secondary Necrosis: Secondary necrosis is a terminal process experienced by late-apoptotic cells if they are not cleared by phagocytes in time, and is characterized by general spill-over of apoptotic cellular contents. "?" Unclear or not determined yet.*

#### TABLE 2 | Danger signaling pathways characterized as traffickers of DAMPs.

*"*+*" denotes ability to positively regulate trafficking; "*−*" denotes ability to negatively regulate trafficking; "0" denotes confirmation of no role in regulation of trafficking and "?" denotes that the role in regulating the trafficking is unknown; "*+*/0" denotes positive or no role in regulation of trafficking in an inducer-dependent fashion; "*−*/0" denotes negative or no role in regulation of trafficking in an inducer-dependent fashion.*

*ATP, adenosine triphosphate; CRT, calreticulin; DT-EGF, epidermal growth factor receptor-targeted diphtheria toxin; ER, endoplasmic reticulum; HMGB1, high-mobility group box 1 protein; HSP, heat shock protein; LRP1, low-density lipoprotein receptor-related protein 1; ROS, reactive oxygen species.*

#### TABLE 3 | A list of prominent single-agent immunogenic cell death (ICD) inducers in cancer and their specific associations with danger signaling and other immunostimulatory signaling.

*(Continued)*

#### TABLE 3 | Continued

*CRT or CALR, calreticulin; CTLs, cytotoxic T lymphocytes; DAMPs, damage-associated molecular patterns; EGFR, epidermal growth factor receptor; GZMB, granzyme B; HMGB1, high-mobility group box-1 protein; HSP, heat shock protein; ICD, immunogenic cell death; IFN, interferon; MDSC, myeloid-derived suppressor cells; OAMPs, oxidation-associated molecular patterns; OS, overall survival; PFS, progression-free survival.*

*Important note: It is worth noting that recently various promising candidate therapies have emerged that induce in vitro DAMPs relevant for ICD, e.g., Rose Bengal-based PDT (168), Docosahexaenoic acid (169), and Capsaicin (170, 171). Such agents may emerge as potent inducers of ICD in future, however, in order to establish them as inducers of ICD-like immunogenicity, it is imperative to confirm their (i.e., cancer cells treated with these agents) ability to stimulate T cells (in vitro or in vivo) and/or induce anti-cancer vaccination effect, in vivo, as per the consensus guidelines (104).*

*Glossary: In the current setting, it is crucial to differentiate between the meanings of the words, "immunogenic" and "immunogenicity" as they are not supposed to have interchangeable meanings. Immunogenic, derives from the word immunogen, which refers to any substance that can elicit an immune response; this includes, whole cells or organisms (eukaryotic or prokaryotic), specific cellular entities or specific proteins (e.g., antigens) (172). On the other hand, immunogenicity is a much more specific terms that is closer to antigenicity in operational sense, since it refers to the ability of a specific entity (e.g., an antigen or an epitope) to be recognized by the immune system through binding interactions with T or B cells, which may or may not result in an overt immunological response (4, 11).*

*"?" Unclear or not determined yet.*

*"#" Unconfirmed anti-tumour immune responses in adaptive immune system-competent.*

#### TABLE 4 | Classification of ICD inducers into Type I and Type II based on their ER or non-ER-targeting *modus operandi*.

*EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; ICD, immunogenic cell death; PDT, photodynamic therapy; ROS, reactive oxygen species. "?" Unclear or not determined yet.*

#### TABLE 5 | A list of molecular and immunological components crucial for regulation of ICD.

*(Continued)*

TABLE 5 | Continued

*ATG, autophagy-related protein; BECN1, beclin-1; CD, cluster of differentiation; CRT, calreticulin; CXCL, C-X-C ligand; CXCR, C-X-C motif receptor; eIF2, eukaryotic initiation factor 2; ER, endoplasmic reticulum; ERp57, endoplasmic reticulum protein 57; HMGB1, high-mobility group box 1; HSP, heat shock protein; Hyp-PDT, hypericin-based photodynamic therapy; ICD, immunogenic cell death; IFN, interferon; IL, interleukin; KO MEFs, knock-out murine embryonic fibroblasts; LAMP, lysosome-associated membrane glycoprotein; LRP1, low-density lipoprotein receptor-related protein 1; MBC, methyl-*β*-cyclodextrin; NLRP3, NOD-like receptor family, pyrin domain containing 3; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PI3K, phosphoinositide 3-kinase; PRF, perforin; TLR, toll-like receptor; TNF, tumor necrosis factor.*

#### TABLE 6 | A list of prominent preclinical mice or rat models used for analysis of ICD.

*(Continued)*

#### TABLE 6 | Continued

*DC, dendritic cell; ICD, immunogenic cell death; PDT, photodynamic therapy.*

#### TABLE 7 | Existence of intrinsic or naturally occurring resistance to ICD in experimental cancer models.

*CD, cluster of differentiation; CRT or CALR, calreticulin; DC, dendritic cells; ER, endoplasmic reticulum; HMGB1, high-mobility group box-1 protein; HSP, heat shock protein; Hyp-PDT, hypericin-photodynamic therapy; ICD, immunogenic cell death; IL, interleukin; MDR, multiple drug-resistance; NO, nitric oxide; NOS, nitric oxide synthase; PD-L1, programed cell death protein ligand 1; PTX, paclitaxel; RT, radiotherapy; TLR, toll-like receptor.*

#### TABLE 8 | A list of clinical observations supporting the existence of ICD in cancer patients.

*CRT or CALR, calreticulin; HMGB1, high-mobility group box-1 protein; Hyp-PDT, hypericin-photodynamic therapy; ICD, immunogenic cell death; IFN, interferon; OS, overall survival; PFS, progression-free survival; TLR, toll-like receptor.*


**11. Pre-hospital cooling**

It has been recommended that the target therapeutic temperature should be reached as soon as possible (Safar, 2002). In successfully resuscitated out-of-hospital cardiac arrest (OHCA) patients, pre-hospital initiation of cooling appears to be the method of choice. However, initiation of cooling in the field is not a simple shift of in-hospital procedure to pre-hospital area. A pre-hospital emergency team frequently operates in demanding conditions and is engaged in the process of cardiopulmonary resuscitation, early post-cardiac arrest stabilisation and decision on transport direction. Therefore, pre-hospital cooling calls for a very simple, efficient and safe cooling method and its usage should not cause a transport delay. A background of regional hospitals practicing TH is essential to ensure cooling continuity.

A few studies demonstrating the efficacy and safety of this strategy, predominantly for the RIVA method, have been published (Kim et al., 2007; Kämäräinen et al., 2008). When performed properly, it can decrease BT by >1.4 °C during transport. Moreover, a dose of 10 – 20 ml/kg of cold normal saline can contribute to hemodynamic stabilization. In addition, this way of cooling exhibits an excelent safety profile with minimal risk of volume overload. Considering its simplicity and low costs, the RIVA method is the first choice for pre-hospital cooling (Figure 6).

A major barrier for a wide recommendation of pre-hospital cooling in successfully resuscitated OHCA patients is the absence of clear evidence of the outcome benefit. Apparently, improved survival is not related to pre-hospital hypothermia itself but to the close coupling of PTH with its in-hospital continuation (Škulec et al., 2010; Castrén et al., 2010). In any case, a large randomized trial is required.

Considering the pros and cons, at present, in the setting of proper collaboration of the emergency medical service and the target in-hospital facility, it is justifiable to implement prehospital cooling even in the absence of unambiguous evidence to support this practice.

Other possibilities suitable for pre-hospital cooling include surface cooling via manufactured cooling pads and intanasal local brain cooling.

Therapeutic Hypothermia in Cardiac Arrest Survivors 87

More or less, implementation has increased over the years. Nevertheless, there are a lot of challenges and room for improvement. Firstly, effort should be focused on enhancing the frequency of TH application in every ICU. Secondly, unavailability of cooling techniques should not be a barrier for TH implementation, it is reliable to start with low-cost simple conventional cooling methods (RIVA, surface cooling). Thirdly, growth of TH implementation has to progress hand in hand with the quality of provided care. The method must be protocol-guided, respecting clear indications and contraindications and applying

complex neuroprotective and cardioprotective post-cardiac arrest care.

resuscitation (also called suspended reanimation).

proved.

clinical studies.

**14. References**

2791.

S351.

**13. Alternative thermoregulation strategies in cardiac arrest survivors**

There are two alternative thermoregulation neuroprotective strategies other than postcardiac arrest cooling: intra-arrest cooling and a principle of emergency preservation and

Animals experiments showed that early intra-arrest initiation of cooling resulted in significantly better neurological outcome than cooling after return of spontaneous circulation (Abella et al., 2004; Kuboyama et al., 1993). This concept was also evaluated in a few clinical studies. RIVA method was very effective but clinical outcome was inconclusive (Kämäräinen et al., 2008; Bruel et al., 2008). More promising seems to be the method of intranasal cooling. The recent PRINCE study revealed its safety, effectivity and suggested outcome improvement (Castrén et al., 2010). A large outcome trial is ongoing. Nevertheless, it must be stressed that intra-arrest cooling intervenes in standard cardiopulmonary resuscitation while cooling after return of spontaneous circulation does not. Therefore, intaarrest cooling should be reserved only for clinical studies, until the clinical benefit is clearly

Emergency preservation and resuscitation is a novel approach supposed to treat victims of exsanguination cardiac arrest. Management is to be as follows: a cold aortic flush is used to induce deep hypothermic preservation in the field followed by transport to hospital facility for emergent surgical treatment and delayed resuscitation with cardiopulmonary bypass (Drabek, 2007). This approach has been tested in animal experiments and stands at door of

Abella BS, Zhao D, Alvarado J, Hamann K, Vanden Hoek TL, Becker LB. (2004). Intra-arrest

Arrich, J. (2007). European Resuscitation Council Hypothermia After Cardiac Arrest

Atwood C, Eisenberg MS, Herlitz J, Rea TD. (2005). Incidence of EMS-treated out-of-hospital

Aufderheide TP, Lurie KG. (2004). Death by hyperventilation: a common and life-

cardiac arrest. *Crit Care Med*,35:1041–1047.

cardiac arrest in Europe. *Resuscitation*,67:75-80.

cooling improves outcomes in a murine cardiac arrest model. *Circulation*,109:2786-

Registry Study Group. Clinical application of mild therapeutic hypothermia after

threatening problem during cardiopulmonary resuscitation. *Crit Care Med*,32:S345-

Fig. 6. Refrigerator in the ambulance car with supply of cold saline.

### **12. Worldwide implementation of therapeutic hypothermia**

Nine years have elapsed since randomized clinical studies allowing the use of TH were published. During this period, in-hospital TH utilisation has varied from 8 to 95 % in different countries until now (figure 7) (Škulec et al., 2010).

Fig. 7. TH implementation in different countries in the last years. USA…United States of America, D…Germany, GB…Great Britain, AUS…Australia, FIN…Finland, CA…Canada, CZ…Czech republic, PL…Poland, SCOT…Scotland, NL…Netherlands, TH…therapeutic mild hypothermia

Nine years have elapsed since randomized clinical studies allowing the use of TH were published. During this period, in-hospital TH utilisation has varied from 8 to 95 % in

Fig. 6. Refrigerator in the ambulance car with supply of cold saline.

**12. Worldwide implementation of therapeutic hypothermia**

different countries until now (figure 7) (Škulec et al., 2010).

Fig. 7. TH implementation in different countries in the last years.

NL…Netherlands, TH…therapeutic mild hypothermia

USA…United States of America, D…Germany, GB…Great Britain, AUS…Australia, FIN…Finland, CA…Canada, CZ…Czech republic, PL…Poland, SCOT…Scotland,

More or less, implementation has increased over the years. Nevertheless, there are a lot of challenges and room for improvement. Firstly, effort should be focused on enhancing the frequency of TH application in every ICU. Secondly, unavailability of cooling techniques should not be a barrier for TH implementation, it is reliable to start with low-cost simple conventional cooling methods (RIVA, surface cooling). Thirdly, growth of TH implementation has to progress hand in hand with the quality of provided care. The method must be protocol-guided, respecting clear indications and contraindications and applying complex neuroprotective and cardioprotective post-cardiac arrest care.

#### **13. Alternative thermoregulation strategies in cardiac arrest survivors**

There are two alternative thermoregulation neuroprotective strategies other than postcardiac arrest cooling: intra-arrest cooling and a principle of emergency preservation and resuscitation (also called suspended reanimation).

Animals experiments showed that early intra-arrest initiation of cooling resulted in significantly better neurological outcome than cooling after return of spontaneous circulation (Abella et al., 2004; Kuboyama et al., 1993). This concept was also evaluated in a few clinical studies. RIVA method was very effective but clinical outcome was inconclusive (Kämäräinen et al., 2008; Bruel et al., 2008). More promising seems to be the method of intranasal cooling. The recent PRINCE study revealed its safety, effectivity and suggested outcome improvement (Castrén et al., 2010). A large outcome trial is ongoing. Nevertheless, it must be stressed that intra-arrest cooling intervenes in standard cardiopulmonary resuscitation while cooling after return of spontaneous circulation does not. Therefore, intaarrest cooling should be reserved only for clinical studies, until the clinical benefit is clearly proved.

Emergency preservation and resuscitation is a novel approach supposed to treat victims of exsanguination cardiac arrest. Management is to be as follows: a cold aortic flush is used to induce deep hypothermic preservation in the field followed by transport to hospital facility for emergent surgical treatment and delayed resuscitation with cardiopulmonary bypass (Drabek, 2007). This approach has been tested in animal experiments and stands at door of clinical studies.

#### **14. References**

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**5**

R. Ernesto Oqueli

 *Australia*

*Ballarat Health Services, Victoria*

**Percutaneous Intervention Post Coronary**

**Saphenous Vein Graft Disease – State of the Art**

The success of coronary artery bypass grafting, although the gold standard for the treatment of multivessel coronary artery disease is limited by poor long-term vein graft patency. Despite the superiority of arterial graft patency over that of vein grafts, the multivessel nature of coronary artery disease and ready availability of saphenous veins still result in its use in over 70% of coronary artery bypass graft procedures (Murphy & Angelini, 2004). These thin walled grafts promptly begin to fail with intimal hyperplasia, thrombosis and progressive atherosclerosis when exposed to an abrupt increase in wall stress imparted by

Recurrent ischaemia in patients who have had previous saphenous bypass surgery occurs not only because of attrition of the saphenous vein grafts but also because of progression of

During the first year after bypass surgery up to 15% of venous grafts occlude, between 1 and 6 years the graft attrition rate is 1% to 2% per year, and between 6 and 10 years it is 4% per year. By 10 years after surgery only 60% of vein grafts are patent and only 50% of patent vein grafts are free of significant stenosis. In addition, native coronary artery disease

Reflecting this graft and native vessel attrition, angina recurs in up to 20% of patients during the first year after saphenous vein grafting and in approximately 4% of patients annually

Angiographic studies have shown that 70% to 80% of bypass surgery patients who present with acute coronary syndrome have their culprit lesion located in the saphenous vein graft

Further revascularisation, either reoperative bypass surgery or percutaneous intervention, is required in approximately 4% of patients by 5 years, 19% of patients by 10 years, and 31% of

Both surgical and percutaneous forms of repeat revascularisation have considerable limitations. As compared with initial surgery, reoperation carries a higher mortality rate (3%

coronary artery disease in the native coronary arteries (de Feyter et al., 1993).

progresses in approximately 5% of patients annually (Motwani & Topol, 1998).

**1. Introduction**

systemic arterial pressure (Hiscock et al., 2007).

during the ensuing 5 years (Motwani & Topol 1998).

patients by 12 years after initial bypass surgery.

(Pregowski J et al., 2005).

 **Artery Graft Surgery in Patients with**


**7. Some general guidelines to apply active-alert hypnotic procedure**

There is no big difference between how the linguistic structure is applied in common, relaxed trances, as you may have seen in the approach we share with you, there are some "hypnotic language forms" [24] or "Milton Model" patterns [28, 29], such as analogical marks, interspersal, ambiguities (phonological and punctuation), or lack of a referential index, among others.

In addition, the use of posthypnotic suggestions is key to the construction of a good and effective hypnotic script, since you are looking for a "posthypnotic behavior" [30] integrated into the "normal" behavior of the person [5], and there are some suggestions for their usage [3]:

**129**

dumbbells technique:

*Active-Alert Hypnosis to Achieve Personal, Professional, and Therapeutic Goals*

you [unavoidable fact], you will [suggestion]")

• Say them at different times and with depth in the trance session

• Express it in different language levels (such as metaphorical, explicit, or with

• Express them as a contingent suggestion (the standard form is "next time that

Unestahl [2] states that every time that a posthypnotic suggestion is triggered, a "posthypnotic trance" occurs, providing an opportunity for the person to elicit all the sensations and learning acquired in the hypnotic session and to bring them to a

There is a process in our brain called hippocampal offline replay that allows our memory to consolidate while in a sleep state. However, there is evidence suggesting that this process can occur in a special consciousness state (relaxed awake state) to bring back learned behaviors or sequences of thoughts in a shorter period of time [31, 32]. That process may be the one that will help us to consolidate and accomplish our most important goals through active-alert hypnosis, posthypnotic suggestions,

This did not happen with that half-baked goal of mine about "getting some new shoes" because that instruction was not specific enough to obtain the desired result. My hippocampus just replays "new shoes… new shoes" a lot, without any more details, and that is what I obtained in the end. A better hypnotic suggestion might

Active-alert hypnotic states could be an alternative in some cases where traditional, relaxed-state hypnosis may not be applied. This approach can be used as a tool to guide people to realize how hypnosis could be experienced on a daily basis and as an auto-hypnosis method, making use of posthypnotic trance states.

Reaching a goal requires people to think, to feel, and to act, both consciously and unconsciously. Both active-alert hypnosis and traditional relaxed-state hypnosis help to store goal-seeking information into the unconscious mind in order to encourage the person to reach his/her goals in a nonconscious manner. However, an advantage of active-alert hypnosis is that it seems to increase motivation and energy

According to our experience, this dumbbells technique is an excellent tool helping people to set and achieve their own goals, whatever they are: personal, professional, financial, and health-related ones. Also, in many cases it could produce a flow state characterized by a focused attention and full concentration, merging awareness and action, "freedom" from worries about failure, self-consciousness disappearing, distorted time sense, and auto-rewarding experiences [33]; this flow state leads the person to a higher motivation and energy, augmented self-efficiency

AAH could be very useful in high-performance athletes, as has been shown by Unestahl's research [30]. We have used the dumbbells technique, as a kind of AAH, in several cases of high-performance athletes and sport teams, from amateurs to professionals. The following is a report from a female cyclist who won gold medal in 2018 Pan-American games, who experienced this technique after a session of AAH

have been the following "buy a pair of new and *comfortable* shoes."

as well as increasing the probability of producing a state of flow.

perception, mind clarity, and self-control.

• Use distinctive nonverbal cues to indicate that suggestion's importance.

*DOI: http://dx.doi.org/10.5772/intechopen.92197*

context where they will be useful.

and clearer goal setting.

**8. Conclusion**

ambiguity)

*Active-Alert Hypnosis to Achieve Personal, Professional, and Therapeutic Goals DOI: http://dx.doi.org/10.5772/intechopen.92197*

Unestahl [2] states that every time that a posthypnotic suggestion is triggered, a "posthypnotic trance" occurs, providing an opportunity for the person to elicit all the sensations and learning acquired in the hypnotic session and to bring them to a context where they will be useful.

There is a process in our brain called hippocampal offline replay that allows our memory to consolidate while in a sleep state. However, there is evidence suggesting that this process can occur in a special consciousness state (relaxed awake state) to bring back learned behaviors or sequences of thoughts in a shorter period of time [31, 32]. That process may be the one that will help us to consolidate and accomplish our most important goals through active-alert hypnosis, posthypnotic suggestions, and clearer goal setting.

This did not happen with that half-baked goal of mine about "getting some new shoes" because that instruction was not specific enough to obtain the desired result. My hippocampus just replays "new shoes… new shoes" a lot, without any more details, and that is what I obtained in the end. A better hypnotic suggestion might have been the following "buy a pair of new and *comfortable* shoes."