*4.4. Immunogenic Cell Death: A Merge Point of Nanomedicine and Immunotherapy*

The maintenance of homeostasis in the human body involves the continuous replacement of different cell compartments, which does not activate the immune system under normal conditions. Instead, the death of some pathogen-infected cells may generate a strong immune response, further establishing a long-term immune memory. Conventionally, only the "self/non-self" model was used to differentiate homeostatic from pathogen-associated cell death, respectively. However, in the 1990s it was demonstrated that some endogenous entities were capable of initiating an immune response in certain circumstances. This means that there would be another factor other than antigenicity that would determine the immunogenic capacity of the different forms of cell death [251].

The Microbe-Associated Molecular Patterns, called MAMPs, are detected by multiple cells of the innate immune system, such as monocytes, macrophages and DC, before the pathogens activate the adaptive response [252]. MAMPs function as adjuvants, interacting with PRRs, which allow the establishment of the first line of defense while generating favourable conditions to initiate the specific immune response [253]. Signalling through PRRs is of great importance, since these receptors are activated by Damage-Associated Molecular Patterns (DAMPs) (Table 1). DAMPs are produced by cells that are in the process of dying and act as adjuvants, informing the body of the danger situation [254]. Under normal conditions, these DAMPs do not activate the adaptive immune response, but when the dying cells are highly antigenic, this occurs, as new antigenic epitopes ("called neo-epitopes) are detected that have not previously produced tolerance. These neo-epitopes can be expressed from microbial genes or from mutated host genes, as in the case of oncogenesis [255]. Therefore, the other factor determining cell death immunogenicity is adjuvancy, which involves MAMPs and DAMPs [251]. Thus, tumours with a high mutational load respond better to some types of immunotherapy, such as immune checkpoint inhibitors (ICIs), than tumours with a low number of somatic mutations [156].


**Table 1.** Molecules that act as Damage-Associated Molecular Pattern (DAMPs), associated pattern recognition receptors (PRRs) and described biological functions.

Caption: ANXA1, annexin A1; APC, antigen-presenting cell; CALR, calreticulin; CDS, cytosolic DNA sensor; CXCL10, CXC-chemokine ligand 10; CXCR3, CXC-chemokine receptor 3; ds, double-stranded; FPR1, formyl peptide receptor 1; HMGB1, high-mobility group box 1; HSP70, heat shock protein 70 kDa; HSP90, heat shock protein 90 kDa; IFN, interferon; IFNAR, interferon α/β-receptor; LRP1, LDL receptor related protein 1; P2RX7, purinergic receptor P2X7; P2RY2, purinergic receptor P2Y2; RLR, RIG-I-like receptor; ss, single-stranded; TLR, Toll-like recept.

In the past, cell death was classified only in apoptosis as a physiological process and in necrosis as a pathological and immunogenic process. Nowadays, it is now known that these differences between the two processes are not as clear, as regulated forms of necrosis are involved in tissue development and homeostasis, and apoptotic cells can trigger antigen-specific immune responses [251]. ICD is therefore a type of immune-stimulatory apoptosis that is characterised by the ability of dying cells to elicit powerful adaptive immune responses to altered auto-antigens/neo-epitope derived from tumour cells in the case of cancer [256]. Based on a specific panel of multiple DAMPs four types of ICDs have been described (Figure 13):


tumour cells express neoantigens that are different from their own and are therefore not subject to tolerance [251,258].


**Figure 13.** Schematic representation of Immunogenic Cell Death (ICD) classification and their associated DAMPs.

The characteristics of the ICD, such as the exposure of CRT and other ER proteins on the cell surface, the release of HMGB1 or the secretion of ATP, allow for the prediction of the capacity of anti-cancer drugs to stimulate therapeutic immune responses by ICD [265].

For example, calreticulin (CRT) is an ER-associated chaperone involved in various functions, such as MHC-I assembly or calcium homeostasis. Tumour cells that undergo chemotherapy-induced cell death expose CRT on their surface, causing CRT to internalize tumour material and present tumour antigens, activating tumour-specific cytotoxic T cells [265]. Tumours that do not properly expose CRT have been shown to have reduced efficacy of chemotherapy, so this immunogenic signal is necessary to obtain good immune responses [266]. In the clinic, CRT exposure is related to patient survival. In patients with non-Hodgkin's lymphoma, the therapeutic benefit of a pulsed DC vaccine with primary lymphoma cells that undergo ICD is correlated with CRT exposure [267]. For patients with acute myeloid leukemia, CRT exposure by tumour cells is known to predict anti-tumour T-cell responses and improve patient survival [268]. Colorectal cancers that do not express CRT have a worse prognosis [269]. Therefore, the expression of CRT affects the immune responses to the cancer in an important way.

Another important factor is that the tumours are competent in autophagy, since these tumours, in response to chemotherapy, recruit macrophages, DC and T lymphocytes more effectively. This is because autophagy is essential for the immunogenic release of ATP by dying cells, which is a potent chemotherapeutic agent [265]. When autophagy is inhibited in cancer, the recruitment of immune effectors to the tumour bed fails, so it may be an escape mechanism from immune surveillance. Inhibiting enzymes that degrade ATP may improve antineoplastic therapies when autophagy is deactivated [270].

In the case of HMGB1, it is a potent pro-inflammatory stimulus whose release can be induced by most antineoplastic agents [271]. HMGB1 activates the release of pro-inflammatory cytokines by monocytes and macrophages. It has been observed that its neutralization with antibodies prevents cross-presentation of tumour antigens by DC in co-culture experiments, therefore its release is a critical determinant of ICD [272].

The induction of ICD in vivo also generates a TME dominated by Th1 and Th17 cytokines [273], which is expected to increase the efficacy of anti-tumour vaccines and therapies designed to reagent TILs, such as ICI.

But once again, these approaches also encounter some obstacles in the tumour cells. Pathogenic viruses and bacteria have developed different mechanisms to prevent the release or detection of DAMPs to escape the immune response. To this end, they express functional orthopaedics of some molecules and inhibitors of different processes that would be necessary for manifest pathogenicity, mainly limiting adjuvancy. The same occurs with tumour cells, which although they present a high antigenicity, control immunogenicity by acting on the adjuvancy, inhibiting the different processes related to the emission of DAMPs, which impairs the efficacy of treatments such as chemotherapy or immunotherapy [251].

Nanomedicine can also act at the level of ICDs and DAMPs (damage associated molecular patterns), with the aim of restraining the immunogenicity of tumour cells. The NPs can be used to enhance the "danger signals" that are released by these tumour cells. Adjuvant-charged NPs are used and placed in the cells suffering from ICD, thus promoting the transmission of these signals [274]. Another application is the targeted delivery of ICD inducers by other NPs, in the form of discs, which allow them to accumulate in the tumour and positively regulate the danger signals, thus enhancing the response of T cells to neo-antigens, tumour-associated antigens and whole tumour cells [275]. Some NPs also have intrinsic properties to induce ICD, such as gold NPs, which release endogenous immune-stimulatory molecules and facilitate DCs activation [276]. NPs combined with chemotherapy and PDT can also be used to induce ICD or to capture TAAs that are released after radiotherapy, with the goal of enhancing T-cell response when treated with ICIs such as anti-PD-L1 [277,278].

Therefore, the immunotherapeutic strategies described above could benefit from the concept of ICD, avoiding some of the drawbacks that occur in the clinic and enhancing an effective immune response, since the different molecules that determine ICD are involved in multiple processes of the immune cycle against cancer. Pre-clinical and clinical studies have already been conducted that could lay the foundations for the design of combined therapies that restore cellular immunogenicity [279,280].

The concept of ICD can also be used to identify biomarkers to predict therapeutic responses in cancer patients. The distinctive features of ICD in tissues need to be identified and correlated with immunological and clinical observations. It is also important to determine what changes in the immune infiltrate of tumours are caused by the ICD and how they affect therapeutic responses.
