**5. Damage-Associated Molecular Patterns**

Processes associated with ICD result in the emission of DAMPs, such as HSP70, HSP90, calreticulin, HMGB1, ATP, type I IFN, cancer cell-derived nucleic acids, ANXA1, and others [29,31,63]. The recognition of DAMPs by cells of the immune system triggers intracellular signaling pathways involved in responses to injury [33]. Frequently, many of these DAMPs perform specific functions in cells not related to immunity. Nevertheless, outside the cells, they can activate various immune cell receptors from the family of PRR. They are also capable of potentiating pro-inflammatory effects such as the maturation and activation of antigen-presenting cells, such as dendritic cells and macrophages, which ultimately activate T cells (CD8<sup>+</sup> ), creating an anti-tumor immunity [32].

The DAMPs are emitted by different mechanisms, such as the active exposure on plasma membrane (e.g., calreticulin, HSP70 and HSP90), released as end-stage degradation products (e.g., DNA and RNA), and secreted to the extracellular medium (e.g., ATP, HMGB1, uric acid, IL-1α and other pro-inflammatory cytokines) [33,64].

A well-studied DAMP is calreticulin, a soluble protein found mainly in the ER. Its main functions in this organelle are the buffering of Ca2+ [65], contributing to the homeostasis of this cation, and the lectin-like chaperone activity, essential to the folding of N-glycosylated proteins [66]. During ICD, it is exposed on the plasma membrane even before phosphatidylserine [67] and acts as an eat-me signal, inducing antigen-presenting cells to phagocytose the target cell [68]. Although calreticulin is essential to the immunogenicity of cells undergoing ICD, its presence alone on the cell surface is not enough for starting an immune response [36]. The presence of other DAMPs, therefore, is crucial for the immunogenicity of ICD. Other chaperones exposed on the plasma membrane in ICD are HSPs, which also act as eat-me signals for phagocytes [69,70].

In cancer cells undergoing ICD, type I interferons (IFN-Is) are released by mechanisms involving the detection of endogenous nucleic acids [28], i.e., dsRNA by TLR3 [71], or ds-DNA by cyclic GMP-AMP synthase (cGAS) [72]. The release of this DAMP has been shown to be crucial for the success of the ICD-inducing anthracycline-based chemotherapy [71]. IFN-Is are potent immunostimulatory proteins with significant anticancer effects, and they have been used to treat chronic myeloid leukemia, acute lymphoblastic leukemia, multiple

myeloma, melanoma, Kaposi's sarcoma, and renal cell and bladder carcinoma [73]. It is notable that the nucleic acids released by cancer cells at ICD are also taken up by DCs and other immune cells, triggering potent IFN-I responses [28].

ANXA1 is also among the crucial DAMPs in ICD. This protein is expressed in granules of different cells, such as neutrophils, eosinophils and monocytes, and is found in different organs and tissues, such as the lungs the bone marrow and intestine [74,75]. Like other members of the annexins superfamily, ANXA1 binds phospholipids and can thus affect eicosanoid production [74]. It has an important role in the regulation and resolution of inflammation. Although ANXA1 has been described as a negative regulator of innate immunity, with neutrophils being its main target [74], it is known to be passively released by cells undergoing ICD, then activating the migration of APCs towards the dying cells, facilitating their engulfment and processing [34]. The importance of this DAMP in tumor immunology can be noted by the fact that the low expression of ANXA1 is often associated with poor DCs and T lymphocyte infiltration in the tumor bed and a higher ability of different human cancers to escape immunity [35].

Another DAMP released to the extracellular medium is ATP [43,76]. Extracellular ATP operates as a strong chemoattractant and promotes not only the recruitment of immune cells but also their maturation [77,78]. In addition to serving as one of the main sources of intracellular energy, ATP also acts in the extracellular signaling mechanisms [67]. ATP is secreted in response to the cytotoxic and cytostatic effects of some aggressive agents such as chemotherapy drugs [46]. ATP release occurs during the process of apoptosis, and different mechanisms can lead to the release of ATP from the intracellular to the extracellular environment, such as the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases and autophagy [79–81]. Of these three, opening pannexin 1 channels and autophagy appear to occur in ICD.

During the formation of apoptotic bodies, caspases 3 and/or 7 act in the cleavage in the C-terminal portion leading to the opening and activation of PANX 1, then releasing the ATP into the extracellular region [82]. In autophagy, the cytoplasmic constituents are degraded in double-membrane organelles, and the autophagosomes are subsequently fused with lysosomes, resulting in the degradation of the autophagocytic content by acid hydrolases and recycling towards energy metabolism or anabolic reactions, releasing the ATP for the extracellular portion [83]. ATP binds the purinergic receptors P2RY2 and P2RX7 of DCs, resulting in the influx of K<sup>+</sup> and Ca2+ ions, activating the inflammasome NLRP3, followed by activation of caspase-1, consequently stimulating the proteolytic maturation and secretion of interleukin 1 (IL-1) and interleukin 18 (IL-18), contributing to the immunogenic response [84].

HMGB1 is a non-histone nuclear protein that, once secreted, acts as an essential DAMP in the activation of DCs by ICD [76,85–87]. Its release occurs sometime after apoptosis [63,86]. It can also be released by cells of the innate immune system without the need for cell death in response to pathogens or secreted by cells in response to some damage in the late phase of apoptosis—necrosis [86,88,89]. HMGB1 activates toll-like receptors (TLR)4 and 2, and stimulates the production of pro-inflammatory factors by DCs [90], strongly contributing to the immunogenicity of ICD [33,91].

The exposure of calreticulin and the release of ANXA1, ATP and HMGB1 result in the attraction and maturation of DCs in the tumor microenvironment [91,92]. Once there, the DCs phagocytose the tumor cells and release pro-inflammatory mediators in the tumor, such as IL-1β, IL-18, IFN-γ, among others. Activated DCs also present the tumor antigens, culminating mainly in the activation of CD8<sup>+</sup> cytotoxic T lymphocytes, which are fundamental in the activation of the tumor retraction immune antitumor response [33,43]. The effects of the DAMPs discussed in this section are summarized in Table 1.


**Table 1.** Damage-associated molecular patterns involved in immunogenic cell death.
