Facts

  • Accelerated progresses and discoveries in the field of oncology, immunology, and virology have made it possible to translate several emerging immunostimulatory strategies to treat malignant cancers towards promising clinical benefits.

  • Profound understanding of the process of immunogenic cell death (ICD) induction by different ICD inducers such as certain chemotherapeutic agents and oncolytic viruses has highlighted the importance of immunological antitumor effects and proposed novel anticancer therapies.

  • The execution of different types of programmed cell death (PCD), including apoptosis, autophagy, necroptosis, and pyroptosis, which are driven by a plethora of stimuli, was recently found to be regulated by orchestrated interactions among them, and importantly, some of these types of PCD exhibit an ICD property.

  • Tumors and cancer cells treated with certain chemotherapeutic agents and oncolytic viruses can undergo ICD and release tumor-associated antigens (TAAs) accompanied by diverse danger-associated molecular patterns (DAMPs) and inflammatory cytokines to restore the tumor microenvironment and incite TAA-specific antitumor immunity.

Open Questions

  • What are the recent advances in the development of anticancer immunotherapeutic modalities in clinical settings?

  • In response to diversified ICD inducers, how are DAMPs such as CRT, high-mobility group protein B1 (HMGB1), and ATP expressed by or released from the dying cancer cells?

  • How do the diverse types of PCD differentially induce ICD to mount an efficient antitumor immunity?

  • What are the prerequisites for an ideal ICD inducer to obtain an optimum level of ICD for long-lasting antitumor effects?

  • It is vital to understand the molecular mechanisms of how ICD inducers, for example, infection with oncolytic viruses and resultant DAMPs, affect the host immune system. Can manipulation of ICD induction and/or combined strategies synergize with current or emerging oncolytic virotherapies?

The concept of immunogenic cell death (ICD) has recently been introduced to describe dying cancer cells that release endogenous danger molecules, the so-called damage-associated molecular patterns (DAMPs), after the exposure to certain cytotoxic agents to be recognized by antigen-presenting cells (APCs) such as dendritic cells (DCs) followed by formation of T-cell-mediated adaptive immunity.1 Although it has long been considered that apoptotic cell death is tolerogenic, DAMPs have also been found to be released from cells undergoing apoptosis, providing a promising anticancer efficacy.2, 3, 4 Therefore, comprehension of ICD induction gradually increases its significance, particularly in the field of cancer immunotherapy.

Overall prognosis of advanced cancer patients still remains dismal, thus making it imminent for oncologists to develop novel anticancer strategies. Recently, sipuleucel-T (Provenge, Dendreon, Seattle, WA, USA), indicated for patients with metastatic castration-resistant prostate cancer, received FDA’s approval as the first therapeutic cancer vaccine.5 In addition, extensive Phase II clinical trials have demonstrated that the oncolytic herpes simplex virus talimogene laherparepvec (T-Vec, Amgen Inc., Thousand Oaks, CA, USA)6 and vaccinia virus JX-547 (Pexa-Vec, Jennerex Biotherapeutics, Inc., San Francisco, CA, USA),7 both of which carry the gene encoding the immunostimulatory cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF), hold great promise for the treatment of advanced cancer patients. Furthermore, cytotoxic T-cell responses directed against oncolytic virus-infected cancer cells have been identified as an essential factor in the process of destruction of cancer.8 Moreover, proinflammatory cytokines generated in the virus-infected cancer cells can restore the immunosuppressive tumor microenvironment.9, 10, 11 Thus, oncolytic viruses are recently viewed as anticancer immunotherapeutic agents. These backgrounds make it imperative to update the molecular pathways and/or cellular constituents that regulate ICD.

Here, we review the progress of research on ICD, emphasizing how apoptotic, autophagic, and necroptotic cell death, called type 1, 2, and 3 PCD, respectively, are induced by various ICD inducers to achieve successful antitumor immunity. These multiple modes can be categorized by describing initiating events, intermediated changes, terminal cellular events, and their immunological responses, which are summarized in Table 1. In the later section, we outline the characteristics of anticancer agents and oncolytic viruses and how they induce diversified forms of cell death and interact with host’s immune system.

Table 1 Comparison of multiple forms of programmed cell death and necrosis
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Apoptotic Cell Death as ICD

From ten million to billions of cells die per day as a consequence of normal tissue turnover,12 which are vital for organisms to retain homeostasis.13, 14 Therefore, the existence of multiple modes of cell death in nature is not surprising. Apoptosis, type 1 PCD, is a specialized form of cell death, characterized by typical morphological changes, including chromatin condensation, nuclear fragmentation, and membrane blebbing (Table 1).15 Apoptosis occurs ubiquitously in normal tissues and causes ‘quiet’ cell death that uses phosphatidylserine (PS) as an ‘eat-me’ signal to be quickly recognized by peripheral APCs. Although apoptotic cell death has been historically considered to be non-immunogenic,16 recent studies unraveled that several antineoplastic agents, including doxorubicin,1, 17 oxaliplatin,18, 19 cisplatin,20 and irradiation,21, 22, 23 can trigger immunogenic apoptosis.2 Mechanistically, the immunogenic apoptotic bodies induced by exposure to doxorubicin are sensed by APCs through their TLR-2/TLR-9-MyD88 signaling pathways.17

DAMPs: as Effectors in ICD

The primary conceptual theory of the pattern recognition of pathogen-associated molecular patterns (PAMPs), such as viral or bacterial components, has failed to fully explain the consequence of immunogenicity. Thus, the secondary concept of DAMPs has been proposed, which could provoke an immune response.24 Released DAMPs as hallmarks of ICD consisted of adenosine triphosphate (ATP), high-mobility group protein B1 (HMBG1), and exposed molecules on the outer membrane of dying cells such as CRT (ecto-CRT), heat-shock proteins (Hsp90 and Hsp70), and endoplasmic reticulum (ER) sessile proteins.25, 2627 The excretion of DAMPs was considered to occur during necrosis under inflammatory and/or pathological conditions. However, DAMPs have recently been reported to be produced from apoptotic cancer cells treated with chemotherapy1, 18 or radiotherapy.21

ICD Inducers

ICD inducers include multiple anticancer therapeutic modalities. It has been recently proposed that they can be classified into two categories, type I or II ICD inducers, based on their distinct actions to induce ER stress leading to apoptotic cell death (Tables 2 and 3).27, 28 The majority of ICD inducers such as chemotherapeutic agents (mitoxantrone,29 anthracyclines,2, 30, 31 oxaliplatin,18, 19 and cyclophosphamide32), shikonin,33, 34 the proteasome inhibitor bortezomib,35 and 7A7 (an epidermal growth factor receptor-specific antibody),36 cardiac glycosides,37 and the histone deacetylase inhibitor (vorinostat)38 are categorized as type I ICD inducers that primarily target cytosolic proteins, plasma membranes, or nucleic proteins. They also induce ER stress via collateral effects. Bortezomib, cardiac glycosides, and shikonin effectively impede protumorigenic cytokine signaling.27 Shikonin has been found to induce type 1 or 3 PCD, which is determined by caspase-8 activation as the ‘decision-making switch’.39 On the other hand, type II ICD inducers, which preferentially target the ER, include hypericin-based photodynamic therapy (PDT)40, 41, 42 and oncolytic coxsackievirus B3 (CVB3).9 Hypericin-based PDT is an anticancer therapy that utilizes hypericin to induce reactive oxygen species (ROS) in the vicinity of the ER.43 Cancer cell infection with oncolytic viruses produce large amounts of viral proteins, which inevitably cause ER stress and ROS production to promote viral replication.44, 45 The quality and/or quantity of ER stress linking ROS triggered by ICD inducers may determine the ICD properties. Indeed, the previous finding that rigorous ROS-mediated ER stress augmented the release of DAMPs revealed an unrecognized role of RNA-dependent protein kinase (PKR)-like ER kinase (PERK) as a constituent of mitochondria-associated ER membranes to exert ROS-mediated mitochondrial apoptosis.40, 41, 46 These observations indicate the superiority of type II ICD inducers with respect to immunological antitumor efficacies. However, further investigations to elucidate the precise interconnection between the ER stress and ROS production will be required to optimize antitumor immune responses.

Table 2 Classification of type I ICD inducers determined by their major targets to provoke antitumor responses
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Table 3 Classification of type II ICD inducers determined by their major targets to provoke antitumor responses
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Calreticulin Exposure

In response to specific chemotherapeutic agents, oncolytic viruses, and vorinostat, ecto-CRT has been found only on cells succumbing to immunogenic apoptosis.2, 9, 38 This ‘eat-me’ signal promotes phagocytosis by DCs, thereby facilitating their tumor antigen presentation and incitement of TAA-specific cytotoxic T cells.2, 47 It has been shown that blockade of CRT inhibits phagocytosis of anthracycline-treated tumor cells by DCs and impairs their immunogenicity in mice.2, 47 In general, CRT exposure during ICD is an earlier process occurring within a few hours than PS externalization.48 The ecto-CRT induction capacity of ICD inducers has been shown to depend on the properties of ER stress and ROS production.2, 37, 49 Cancer cells can induce ecto-CRT followed by disturbance of the ER structure with GADD34 activation and PERK phosphorylation. It has been shown that depletion of PERK abolishes anthracycline-driven ecto-CRT and immunogenicity of cellular death (ER stress module),19 and that caspase-8 acts upstream of apoptotic proteins Bax and Bak, and subsequent cleavage of its substrate Bap31 (apoptotic module) is indispensable for ecto-CRT induction.19 Furthermore, a direct interaction between ecto-CRT and ERp57 was shown to be required for their cotranslocation to the cell surface (Figure 1).29 Unlike the release of HMGB1 and ATP, ecto-CRT could be one of the determinants that distinguishes between immunogenic and non-immunogenic cell death.47

Figure 1
figure 1

Oncolytic virus (CVB3) infection-triggered cancer cell death induces innate immune cell-mediated antitumor immunity. Intratumoral CVB3 infection-activated natural killer (NK) cells and granulocytes with enhanced expression of CD107a, a cytolytic degranulation marker, have been found to contribute to substantial antitumor effects as evidenced by NK cell and granulocyte depletion assays. Upon CVB3 infection, tumor cells can partially induce ecto-CRT on human tumor cells during early apoptosis, whereas majority of other viruses subvert ICD by circumventing ecto-CRT induction, and followed by robust release of DAMPs, including ATP and HMGB1, during later stages of cell death, which facilitates maturation of DCs via binding to Toll-like receptor 4 (TLR4)/RAGE and P2 × 7R, respectively. Viral genomes and/or viral progenies also stimulate DCs for their activation. Mature DCs may then efficiently phagocytose TAAs simultaneously released from dying cells and ultimately cross-present them to CD8+ T cells with the support with CD4+ T cells to elicit substantial antitumor immunity. Although ATP secretion relies on autophagic machinery, the other forms of cancer cell death, such as autophagic cell death and necroptosis, triggered by CVB3 infection have not yet been fully investigated

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HMGB1

HMGB1, one of the DAMPs, is a DNA-binding protein originally known as a nuclear non-histone chromatin-binding protein.50 Although extracellular HMGB1 had been deemed to be released mainly from the nucleus during necrosis,42 it was found to be excreted from cells undergoing late stage of apoptosis and autophagy.30, 51 HMGB1 inhibition in cancers undergoing immunogenic apoptosis impaired their ability to incite antitumor immunity in a prophylactic vaccination model.30 HMGB1 initiates potent inflammation by stimulating the production of proinflammatory cytokines52 from APCs via its binding to different surface receptors including receptor for advanced glycation end-products (RAGE), TLR2, TLR4, TLR9, and TIM3 (Figure 1).53, 54 Importantly, the binding of HMGB1 to TLR4 on APCs was required to suppress tumor development, which is consistent with clinical study showing that breast cancer patients harboring a single-nucleotide polymorphism (Asp299Gly) in the TLR4 gene undergo an early relapse after anthracycline treatment.30, 55, 56 In contrast, secreted HMGB1 could induce a protumor inflammation to facilitate tumor progression.57 In addition, HMGB1 expression is significantly associated with overall survival of patients with bladder cancer.58 As HMGB1 is an intrinsic sensor of oxidative stress,59 the immunomodulatory properties of HMGB1 might be determined by its redox status.60, 61 Indeed, reduced HMGB1 production from dying cells was shown to trigger the immunogenic DCs, whereas oxidized HMGB1 during apoptosis fails.51 As the extracellular space is usually oxidative under physiological conditions but is unpredictably variable under pathogenic conditions,62 the unstable redox status of the tumor microenvironment might account for these inconsistent findings. However, the observation that the tumor microenvironment tends to be pro-oxidative63 implies that a therapeutic approach using antioxidants to decrease ROS production would be favorable to stimulate antitumor immunity. Importantly, many anticancer agents, including chemotherapy,30 radiation,22 or oncolytic viruses,9, 64, 65 have been shown to induce HMGB1 release from cancer cells, highlighting the significance of further addressing the mechanism of how these modalities affect the redox status of HMGB1.

Adenosine Triphosphate

Extracellular ATP released from apoptotic cells is another important factor in ICD induction. ATP signaling recognized by P2Y2 receptors on phagocytes as a ‘find-me’ signal enables them to migrate into inflamed sites.66 Indeed, ATP released from cancer cells treated with chemotherapeutic agents is essential for effective antitumor immune responses.67 In addition, small interfering RNA-mediated inhibition of autophagic machinery abolishes ATP release from chemotherapy-treated tumor cells and mitigates the antitumor response.68 Radiotherapy triggers ATP release from dying tumor cells through its interaction with the P2 × 7 purinergic receptor,69 possibly resulting in the activation of the NLRP3–ASC–inflammasome axis and subsequent secretion of IL-1β.70

We and others recently showed that oncolytic viruses induce secretion of extracellular ATP from human cancer cells (Figure 1).9, 65 Unlike ecto-CRT induction, the release of ATP and HMGB1 is triggered by a range of death-inducing stimuli, and is not restricted to induction in apoptotic cell death.47 Although ATP production is required for efficient vaccinia virus production71 and facilitates HIV infection through its interaction with P2Y2 receptors,72 there is little knowledge of how oncolytic viruses provoke ATP release.

Autophagic Cell Death

Autophagy physiologically has catabolic roles, particularly in cell survival.73 However, persistent autophagy causes a caspase-independent form of cell death that is, morphologically defined as autophagic cell death (ACD), termed as type 2 PCD, through lysosomal proteinase-regulated elimination of cellular organelles.74, 75 Autophagy sometimes directs itself to cellular death, either in cooperation with apoptosis or as a back-up system, and thus is deemed as a cellular program with a ‘double-faced’ role.

Interestingly, the key molecules of autophagy and apoptosis pathways are intricately intertwined with shared several molecules including regulatory genes such as p53 and p19ARF.76 This crosstalk therefore can be viewed as a significant clue to understand the fate of dying cancer cells from therapeutic view points. Although ACD occurs without chromatin condensation but with massive autophagic vacuolization,77 autophagy, often disabled in cancer, has been shown to be required for induction of immunogenicity.68 First, dying cells in embryoid bodies that lack autophagy-related gene are unable to express the ‘eat-me’ signals and secrete lower levels of the ‘come-get-me’ signal, lysophosphatidylcholine.78 Second, autophagy deficiency hinders ATP secretion from dying cancer cells, resulting in the impairment of DC recruitment and formation of adaptive immunity responses (Table 1).68 Third, the inability of autophagy-deficient cancer cells to provoke antitumor immunity after chemotherapy can be reverted by suppression of extracellular ATP-degrading enzymes.68, 79 Therefore, immunogenicity of ACD could be mediated by subtle spatiotemporal alterations in the treated cancer cells.

Novel strategy of autophagy inhibition is therapeutically effective for eliminating apoptosis-resistant cancer cells based on the rationale that growing tumors may harness autophagy as an adaptation to resist therapeutic stresses.80, 81, 82 Hence, more efforts should be made to elucidate the intricate interaction between autophagy inhibition and resulting effects on the immunogenicity.

Necrotic Cell Death and Necroptosis

Necrotic cell death is induced by external factors such as toxins, cancer, infections, and trauma, and is morphologically characterized by cellular swelling, rupture of the plasma membrane, and loss of cytoplasmic contents.83 Understanding the immunogenicity of necrotic cell death is becoming important because it frequently induces robust inflammatory reactions to mount protective immune responses (Table 1).84, 85, 86 Although necrosis has long been viewed as non-PCD, its execution was shown to be controlled by specific signal-transduction pathways and catabolic mechanism.87, 88, 89 This alternative form of necrotic PCD, aptly termed necroptosis (type 3 PCD), is induced by tumor necrosis factor (TNF) receptor signaling that involves activation of the receptor-interacting protein (RIP) family. Upon inhibition of apoptotic pathway by the caspase inhibitor, activation of RIP1 and RIP3 kinase leads to mitochondrial instability and cell death.90, 91 Phosphorylated RIP1 and RIP3 generate a molecular complex called the necrosome, which initiates necroptosis. ROS production under necroptosis has been shown to facilitate TNF-α-induced cell death by sustaining c-Jun N-terminal kinase activation.92 Intriguingly, necroptosis can also be executed via stimulation by apoptosis-inducible ligands such as TNF-α, FasL, or TRAIL (Table 1). Notably, cytotoxic agents are shown to induce necrotic cell death in apoptosis-defective cancer cells,93 probably because necroptosis is principally induced when a cell cannot die via apoptotic pathways.94 On the other hand, conventional therapy-resistant cancer stem cells (CSCs) have a higher antiapoptotic activity than that of their counterparts.95, 96 Therefore, it would be vital to clarify the key machinery of not only the necroptosis induction in cancer cells for CSC-directed therapeutic application but also the resultant immunogenicity to modulate antitumor immunity.

Pyroptosis

Pyroptosis is a recently indentified form of PCD stimulated by microbial infections and non-infectious stimuli such as myocardial infarction and cancer. In contrast to apoptosis, pyroptosis is uniquely mediated by caspase-1 activity triggered by the formation of a cytosolic complex termed the ‘inflammasome’, resulting in highly inflammatory outcomes (Table 1). Pyroptotic cells represent morphological characteristics, some of which are shared with apoptosis and necrosis.97 The function of activated caspase-1 is to cleave proteolytically the proforms of the proinflammatory cytokines, IL-1β and IL-18, to their active forms.97 Although pyroptosis has been intensively studied in the context of bacteria-infected macrophages,98 it can also be triggered in human cancer cells infected with recombinant herpes simplex virus 2 (HSV-2) (Table 4).99 Pyroptotic cancer cells induced by microbial infection have been recently shown to facilitate phagocytosis by macrophages, presumably through their PS exposure and ATP release.100 Accordingly, the caspase-1-dependent generation of proinflammmatory cytokines and other DAMPs could be essential factors to provide a suitable inflammation for ICD induction.

Table 4 DNA oncolytic viruses and their differential properties to induce either multiple forms of cell death or antitumor immunity
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DAMPs Induced by Infection with Oncolytic Viruses

Because oncolytic viral infection can produce abundant PAMPs, including viral proteins and nucleic acids, followed by the release of DAMPs and the entire repertoire of TAAs from treated tumors,101 oncolytic virus-triggered ICD may be more effective for induction of antitumor immunity. As viruses have developed sophisticated machineries to evade apoptotic cell death and interfere with ER stress and autophagy responses for their survival,102, 103 ICD may have played an essential role in the everlasting war between viruses and their hosts. We and other groups have found that many oncolytic viruses can induce apoptotic cell death and/or necrosis in cancer cells,9, 104, 105, 106 supporting their immunostimulatory potential to augment antitumor efficacy (Tables 4 and 5).107, 108 CVB3 infection induces multiple DAMPs including ecto-CRT, HMGB1 translocation from nuclei, and ATP release from human lung cancer cells. Importantly, intratumoral CVB3 administration can prominently recruit cytolytic degranulation marker CD107a-mobilized NK cells and granulocytes, and mature DCs into the tumor bed (Figure 1).9, 27 As pathogenic viruses have developed their strategies to subvert ecto-CRT and circumvent ICD induction,109 it is noteworthy that CVB3 infection can induce ecto-CRT accompanied by other DAMPs.9 Furthermore, we demonstrated that both NK cells and granulocytes substantially contributed to the CVB3-mediated antitumor efficacy as evidenced by in vivo depletion assays.9

Table 5 RNA oncolytic viruses and their differential properties to induce either multiple forms of cell death or antitumor immunity
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Upon intratumoral replication of oncolytic viruses, resultant alterations in tumor microenvironment may restore the compromised antitumor immunity, presumably through induction of IFNs and/or cytokines that activate NK cells and APCs.110, 111 Although tumor-infiltrating DCs were impaired at maturation by immunosuppressive IL-10, PGE2, and transforming growth factor β produced from tumor cells,112 unidentified components in the culture media from reovirus-infected cancer cells facilitated maturation of DCs.113

Recent studies delineated that oncolytic viruses such as vaccinia, measles, HSV-2, and adenovirus cause the release of HMGB1.64, 65, 114, 115, 116, 117 Although HMGB1 interacts with viral components and may modulate viral replication,117 the molecular mechanisms of how each oncolytic virus differentially produces these DAMPs remain largely elusive.

Multimodal PCD Induced by Oncolytic Viruses

We showed that approximately 20% of CVB3-mediated cytotoxicity of A549 cells resulted from apoptotic cell death.9 This induction was presumably due to the capacity of CVB3 infection to induce PKR-mediated phosphorylation of eIF2 and caspase-8-mediated activation of proapoptotic mediator, caspase-3 (Figure 1).118, 119 Other DNA and RNA oncolytic viruses have been reported to induce apoptotic cancer cell death (Tables 4 and 5). However, there are only two reports showing that virus-induced ecto-CRT was correlated with enhanced intratumoral infiltrations of immune subpopulations, which accounted for the ‘in vivo’ remarkable antitumor immunity.9, 65

Several studies showed that recombinant oncolytic adenoviruses induced ACD in human malignant glioma cells,120 brain tumor stem cells,121 osteosarcoma cells,105 and lung cancer cells.122 Newcastle disease virus also triggered autophagy in glioma cells to promote its viral replication.123 Reovirus-mediated oncolysis of multiple myeloma was reported to be orchestrated via upregulation of autophagy.124 Because cancer cells are largely refractory to apoptotic inducers but vulnerable to necroptosis,39 overcoming anticancer drug resistance may be achieved by activation of necroptotic rather than apoptotic pathways, where the former might be the intrinsic ‘Achilles heel’ of cancers.125 So far only recombinant adenovirus has been shown to facilitate both necroptotic and apoptotic cell death with a synergistic effect on cancer cells when combined with doxorubicin (Tables 4 and 5).126 In addition, most oncolytic viruses may induce pyroptotic cancer cell death accompanied by abundant proinflammatory cytokines and DAMPs. Accordingly, some oncolytic viruses may induce multimodal ICD, allowing them to be a plausible modality as promising agents of immunotherapy.

Strategies to Enhance the Potentials of ICD Induced by Oncolytic Viruses

Besides DAMPs, massive production of type I IFNs (IFN-α/β) upon oncolytic viral infection can be a potent immunomodulator through their indirect immunostimulatory effects on neutrophils and T cells,127, 128 as well as through their direct antiproliferative effects.129 Despite a creation of multimodal ICD by oncolytic viruses to facilitate antitumor immunity, much attention should be paid to the preferential antiviral immunity that might impede direct viral oncolysis-mediated tumor destruction. To avoid this, cyclophosphamide is shown to retard immune removal of oncolytic viruses, enhancing the persistence of viral infection.130 Another promising strategy to overcome antiviral immunity could be potentiating immune responses by gene modification of oncolytic viruses to arm them with immunostimulatory cytokines, such as GM-CSF, IL-2, IL-12, and IL-15. Indeed, the results of clinical trials of the GM-CSF gene-harboring oncolytic vaccinia virus JX-594 and the GM-CSF gene-harboring oncolytic herpes virus talimogene laherparepvec demonstrated that a clinical benefit can be accomplished by combined respective oncolytic activity with the recruitment of immune cells.6, 7, 131 The combination of adoptive T-cell therapy with oncolytic viruses is shown to elicit an increased antitumor effect.131, 132 Collectively, the design of combinatorial therapies of oncolytic viruses with immunotherapeutic modalities may hold the key to mount maximally a multifaceted attack against cancers.

Conclusions

Although mechanism of ICD induction is a very complicated process, we need to elucidate how dying cells become much more stimulatory in shaping antitumor immune responses than was ever expected. Notably, intermediate death processes, including caspase activation, mitochondrial degradation by autophagy, ROS production, and oxidative modification of DAMPs, have been found to fine-tune the balance between antitumor tolerance and immunity, providing implications in manipulation of ICD.

Four forms of PCD, apoptosis, autophagy, necroptosis, and pyroptosis, may jointly decide the fate of cells of malignant cells. However, in terms of immunogenicity, investigations of only apoptotic cell death in cancer cells have just begun. Therefore, further elucidation of determinants of respective PCD-inducing pathway and characterization of resultant ICD should aid to develop novel anticancer strategies. A recent review advocates a list of characteristics for an ideal ICD inducer,27 as follows: (1) efficiently activates apoptosis or necrosis leading to emission of multiple DAMPs and TLR agonists;133, 134 (2) irrelevant in drug-efflux pathways;135 (3) can induce ER stress;134 (4) has negligible suppressive or inhibitory effects on immune cells;136 (5) counteracts immunosuppressive responses;136, 137 and (6) directly targets not only the primary tumor but also metastases.138 No ideal ICD inducer exists, but it is important to seek for ideal combinatorial therapies that could achieve these properties. Of the currently known relevant ICD inducers, those that meet most of these properties include mitoxantrone, hypericin-PDT, and shikonin. However, diverse oncolytic viruses could be the promising ICD inducer as we gain more knowledge about the properties yet to be investigated. Evidently, they can destroy conventional therapy-resistant CSCs,139 possibly through their ability to induce distinctive PCD and/or modification to express genes that target CSC-specific signaling pathways underpinning their cell survival.140

Gaining more detailed insights into the mechanisms of ICD induction, to be perceived by the immune system, will not only ameliorate the development of promising anticancer agents or combinatorial therapies but also offer useful knowledge in various life science fields including virology, immunology, and clinical medicine.