**3. Immunogenic Cell Death**

As defined by the Nomenclature Committee on Cell Death, ICD is a modality of RCD [28,29]. This implies that ICD activation depends on signaling transduction programs, and can thus be triggered or modulated by drugs and genetic components [28].

ICD differs from other RCD types, such as necroptosis, ferroptosis and pyroptosis, not only by the conditions under which it is triggered, but also by the fact that it activates an adaptive immune response in immunocompetent syngeneic hosts against antigens expressed by the dying cell [28]. The cells undergoing ICD exhibit morphological and molecular hallmarks of apoptosis with a well-defined pattern of release and exposure of DAMPs [29]. Thus, cells at ICD exhibit the two conditions necessary for eliciting an adaptive immune response: antigenicity and adjuvanticity [28,29].

DAMPs form a group of different molecules that normally perform structural and metabolic functions in living cells unrelated to their immune functions during ICD [30]. However, when emitted with a specific temporospatial profile during ICD, DAMPs are able to trigger or boost antigen-specific immune responses [31,32]. The activation of pattern

recognition receptors (PRR) by DAMPs results in the maturation of dendritic cells (DCs) and the activation of CD4<sup>+</sup> and CD8<sup>+</sup> T cells [33], as represented in Figure 1. Different aspects of the activation of adaptive immune responses are fulfilled by the different DAMPs involved in ICD. (DCs) and the activation of CD4+ and CD8+ T cells [33], as represented in Figure 1. Different aspects of the activation of adaptive immune responses are fulfilled by the different DAMPs involved in ICD.

DAMPs form a group of different molecules that normally perform structural and metabolic functions in living cells unrelated to their immune functions during ICD [30]. However, when emitted with a specific temporospatial profile during ICD, DAMPs are able to trigger or boost antigen-specific immune responses [31,32]. The activation of pattern recognition receptors (PRR) by DAMPs results in the maturation of dendritic cells

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**Figure 1.** Engagement of adaptive immune response after immunogenic cell death in a tumor**.**  Chemotherapy, radiotherapy, and photodynamic therapy can induce immunogenic cell death (ICD), which is a programmed cell death accompanied by the exposure of damage-associated molecular patterns (DAMPs). This can occur, for instance, as a consequence of oxidative stress in the endoplasmic reticulum. Some DAMPs, such as heat-shock protein (HSP)70, HSP90, and calreticulin are exposed on the plasma membrane, while others such as adenosine triphosphate (ATP), high mobility group box 1 protein (HMGB1), C-X-C motif chemokine ligand 10 (CXCL10) and annexin A1 (ANXA-1) are released to the extracellular medium. DAMPs then activate pattern recognition receptors of dendritic cells (DCs) and other antigen-presenting cells. This culminates in the maturation of the DCs and in the recruitment and activation of T cells. In this way, ICD can trigger or boost an adaptive antitumor immune response. The time profile of the release and exposure, as well as the specific actions performed **Figure 1.** Engagement of adaptive immune response after immunogenic cell death in a tumor. Chemotherapy, radiotherapy, and photodynamic therapy can induce immunogenic cell death (ICD), which is a programmed cell death accompanied by the exposure of damage-associated molecular patterns (DAMPs). This can occur, for instance, as a consequence of oxidative stress in the endoplasmic reticulum. Some DAMPs, such as heat-shock protein (HSP)70, HSP90, and calreticulin are exposed on the plasma membrane, while others such as adenosine triphosphate (ATP), high mobility group box 1 protein (HMGB1), C-X-C motif chemokine ligand 10 (CXCL10) and annexin A1 (ANXA-1) are released to the extracellular medium. DAMPs then activate pattern recognition receptors of dendritic cells (DCs) and other antigen-presenting cells. This culminates in the maturation of the DCs and in the recruitment and activation of T cells. In this way, ICD can trigger or boost an adaptive antitumor immune response.

by the DAMPs, orchestrates the attraction, phagocytic activity and maturation of APCs in the tumor bed. The simple presence of a single DAMP or just a couple of them in the vicinity of tumor cells is generally not enough for the initiation or boosting of a cytotoxic, effective anticancer immune response. The absence of calreticulin or ANXA1, for example, is known to severely limit immune responses against tumor cells [34–36]. Moreover, the tumor microenvironment must permit immune cells to be activated and to perform their roles properly in order for DAMPs to exert their immunoadjuvant effects. Specific conditions are known to induce ICD, such as chemotherapy [37–39], The time profile of the release and exposure, as well as the specific actions performed by the DAMPs, orchestrates the attraction, phagocytic activity and maturation of APCs in the tumor bed. The simple presence of a single DAMP or just a couple of them in the vicinity of tumor cells is generally not enough for the initiation or boosting of a cytotoxic, effective anticancer immune response. The absence of calreticulin or ANXA1, for example, is known to severely limit immune responses against tumor cells [34–36]. Moreover, the tumor microenvironment must permit immune cells to be activated and to perform their roles properly in order for DAMPs to exert their immunoadjuvant effects.

radiotherapy [40,41] and PDT [26,27,42–45]. Parameters such as the protocol of application and the drug used in these treatments are crucial to determine whether ICD is induced or not. In the case of chemotherapy, for instance, ICD can be induced by drugs such as mitoxantrone, oxaliplatin and cyclophosphamide, but not by cisplatin, etoposide and Specific conditions are known to induce ICD, such as chemotherapy [37–39], radiotherapy [40,41] and PDT [26,27,42–45]. Parameters such as the protocol of application and the drug used in these treatments are crucial to determine whether ICD is induced or not. In the case of chemotherapy, for instance, ICD can be induced by drugs such as mitoxantrone, oxaliplatin and cyclophosphamide, but not by cisplatin, etoposide and mitomycin C. In the case of PDT, the type and concentration of the photosensitizer, as well as the irradiation regimen, are key factors [26,27].

### **4. Endoplasmic Reticulum Stress 4. Endoplasmic Reticulum Stress**

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as the irradiation regimen, are key factors [26,27].

Different ICD-inducers act as stressors of the endoplasmic reticulum (ER) of target cells [31,33,46]. Indeed, the ER has been linked by many studies to programmed cell death [46–48]. Stressing conditions, such as oxidative stress and hyperthermia, can impair the folding of proteins. Unfolded proteins bind the luminal ER chaperone GRP78/BiP (Figure 2), triggering the activation of three ER transmembrane proteins: inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). The downstream cellular events following the activation of these ER sensors can either restore normal ER metabolism or induce cell death [48]. Different ICD-inducers act as stressors of the endoplasmic reticulum (ER) of target cells [31,33,46]. Indeed, the ER has been linked by many studies to programmed cell death [46–48]. Stressing conditions, such as oxidative stress and hyperthermia, can impair the folding of proteins. Unfolded proteins bind the luminal ER chaperone GRP78/BiP (Figure 2), triggering the activation of three ER transmembrane proteins: inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). The downstream cellular events following the activation of these ER sensors can either restore normal ER metabolism or induce cell death [48].

mitomycin C. In the case of PDT, the type and concentration of the photosensitizer, as well

**Figure 2.** Activation of responses to protein malformation. These three pathways—IRE1α (**a**), ATF6 (**b**) and PERK (**c**)—are activated in unfolded protein response (UPR) to cope with disturbances in protein folding and to restore endoplasmic reticulum homeostasis after stress. The phosphorylation of eIF2α is a hallmark of immunogenic cell death. Legend: ROS: reactive oxygen species; BiP: Binding immunoglobulin protein; ER: endoplasmic reticulum; IRE1α: inositol-requiring enzyme 1α; XBP1: X box-binding protein 1 mRNA; XBP1S: spliced X box-binding protein 1 mRNA; ATF6: activating transcription factor 6; PERK: protein kinase RNA-like endoplasmic reticulum kinase; ATF4: activating transcription factor 4; S1P: site-1 protease; S2P: site-1 protease; eIF2α: eukaryotic initiation factor 2α. **Figure 2.** Activation of responses to protein malformation. These three pathways—IRE1α (**a**), ATF6 (**b**) and PERK (**c**)—are activated in unfolded protein response (UPR) to cope with disturbances in protein folding and to restore endoplasmic reticulum homeostasis after stress. The phosphorylation of eIF2α is a hallmark of immunogenic cell death. Legend: ROS: reactive oxygen species; BiP: Binding immunoglobulin protein; ER: endoplasmic reticulum; IRE1α: inositol-requiring enzyme 1α; XBP1: X box-binding protein 1 mRNA; XBP1S: spliced X box-binding protein 1 mRNA; ATF6: activating transcription factor 6; PERK: protein kinase RNA-like endoplasmic reticulum kinase; ATF4: activating transcription factor 4; S1P: site-1 protease; S2P: site-1 protease; eIF2α: eukaryotic initiation factor 2α.

The activated IRE1α dimerizes and is autophosphorylated, becoming able to catalyze the unconventional, cytosolic splicing of the mRNA for the transcription factor X boxbinding protein 1 (uXBP1) to sXBP1, which is then translated. The XBP1 is a key protein in UPR, activating the transcription of different genes that can reinstate normal ER metabolism [49]. The PERK protein also dimerizes and auto-phosphorylates, activating its kinase domain to phosphorylate the eukaryotic initiation factor 2α (eIF2α) [50,51]. The phosphorylation of eIF2α is the key event in the integrated stress response (ISR), which is a part of the ER stress response, and is common to ICD induced by different cytotoxicants The activated IRE1α dimerizes and is autophosphorylated, becoming able to catalyze the unconventional, cytosolic splicing of the mRNA for the transcription factor X box-binding protein 1 (uXBP1) to sXBP1, which is then translated. The XBP1 is a key protein in UPR, activating the transcription of different genes that can reinstate normal ER metabolism [49]. The PERK protein also dimerizes and auto-phosphorylates, activating its kinase domain to phosphorylate the eukaryotic initiation factor 2α (eIF2α) [50,51]. The phosphorylation of eIF2α is the key event in the integrated stress response (ISR), which is a part of the ER stress response, and is common to ICD induced by different cytotoxicants [34]. Thus, the presence of phosphorylated eIF2α is a hallmark of ICD and can be used as a biomarker of ISR in cell cultures and in biological samples [52].

Phosphorylated elF2α triggers the selective translation of the activating transcription factor (AFT4), which activates the expression of genes involved in protein folding, amino acid metabolism and regulation of oxidative stress [53]. Also, ATF6 is translocated to the Golgi apparatus, where it is then cleaved to ATF6N, releasing its transcription factor moiety that activates the transcription of genes for chaperones, XBP1, C/enhancer binding protein-homologous protein (CHOP), and others [48,54].

The UPR is important for cells under stress to survive and restore homeostasis. The activation of this response is often observed in cancer cells, as the tumor is often a stressing environment, with acidic pH and hypoxia, for instance. Thus, the phosphorylation of eIF2α is frequently observed in cancer cells and plays an important role in tumor growth, invasion, and angiogenesis [55]. In this situation, tumors can cope with the constant stress and progress.

However, if the UPR is unable to restore the ER protein folding capacity, then cell death is triggered. In this context, the pro-apoptotic factor CHOP plays a key role [55–57]. The PERK/eIF2α/ATF4 pathway activates the production of CHOP [53], which activates the expression of the BH3-only protein Bim. This leads to the activation of Bax/Bak and the release of cytochrome C to the cytosol, with the consequent induction of apoptosis [56,58]. It is worth mentioning that CHOP also acts on the Bcl2 protein (autophagy activation factor), inhibiting its action [59,60]. IRE1α and ATF6 will also activate CHOP via fragments of XBP1S or ATF6N, thereby triggering apoptosis [60,61].

An intense stress in the ER not only activates apoptosis but also promotes the exposure of ER chaperones, such as calreticulin and HSP70, and other DAMPs involved in ICD [62]. The main DAMPs are discussed in the next section.
