Pyroptosis, metabolism, and tumor immune microenvironment

2.1.1 Inflammasomes and caspase-1

The inflammasomes are large multiprotein complexes which usually consist of pattern recognition receptors (PRRs), inflammatory caspases (caspase-1) and sometimes an adaptor protein, like apoptosis-associated speck-like protein containing a CARD (ASC).^60-62 Pyroptosis mediated by the inflammasomes is commonly categorized as the canonical caspase-1-dependent inflammasome pathway.⁶² Generally, as sensors to various dangers, the PRRs (NLRP1, NLRP3, NLRC4, NLRP6, NLRP7, NLRP9b, NLRP12, pyrin, and AIM2) first recognize a variety of stimulates, then activate pro-caspase-1 cleavage and ASC recruitment to assemble inflammasomes.^63-65 Activated caspase-1 can cleave pyroptosis executor gasdemin D (GSDMD) proteins (Asp275 site) to free the N-terminal (NT) domain and generate nonselective pores on the cell membrane.^{3, 55, 66, 67} At the same time, caspase-1 cleaves and activates the precursors of IL-1β and IL-18 to become mature IL-1β and IL-18. The latter together with other cellular contents then is released from the pores, leading to cell pyroptosis.^{55, 60, 68} In the case of NLRP3, the most common PRRs can be activated by a wide range of factors, including viruses, bacterial toxins, fungi, parasite, nucleic acids, crystalline substrates, some drugs, silica particles, long-chain saturated fatty acids, reactive oxygen species (ROS) and various endogenous damage signals.^69-72 Notably, there is a two-step procedure for NLRP3 inflammasome activation, including the priming step by various activators and the activating step through K⁺/Ca²⁺ efflux, mitochondria and lysosome-related damages and etc.⁷³ Also, NLRP1 responds to the Bacillus anthracis lethal toxin, Toxoplasma gondii, which activates assembly of the NLRP1 inflammasome and induces pyroptosis.^74-76 Various NLR family apoptosis inhibitory protein (NAIP) proteins are essential for activation of the NLRC4 inflammasome, and human NAIPs directly link with flagellin and the type-3 secretion system (T3SS) proteins. Comparably, NAIPs in mice can recognize the needle and inner rods of T3SS and flagellin.^77-80 Then, NAIPs recruit NLRC4 and induce its oligomerization to form the NLRC4 inflammasome.^{81, 82} Non-NLRs, like AIM2, are activated by cytosolic double-stranded DNA.^83-87 Normally, pyrin is stimulated by the inactivation of small GTPase in the Rho family. For the Rho-glucosylation activity of Clostridium difficile (C. difficile) toxin B (TcdB), Clostridium botulinum ADP-ribosylating C3 toxin, and Vibrio parahaemolyticus VopS, some specific mutations like S208A and S242R, as well as the Yersinia pestis GTPase-activating protein (YopE) and cysteine protease (YopT) can all activate the pyrin inflammasome.^88-92 Other inflammasomes have been reviewed by others and not listed here.

2.1.2 Caspase-4/5/11

In contrast, the non-canonical inflammasome pathway is independent of inflammasomes and directly activated by bacterial lipopolysaccharide (LPS) to cleave precursors of human caspase-4, caspase-5, and mouse caspase-11.^{56-58, 93, 94} Then, the activated caspases can cleave GSDMs to rupture the plasma membrane, promote K⁺ efflux, and induce pyroptosis.^{58, 95} Sometimes, the procedure is also linked to activation of the NLRP3/caspase-1 pathway, which ultimately promotes the maturation and release of IL-1β and IL-18, other than caspase-4/5/11.^{3, 57, 58, 96} Besides, the pore formation does not always target the cytoplasmic membrane, and other membranes inside the cell, especially the mitochondrial membrane, are also involved.⁹⁷ During inflammatory lung injury, the LPS-mediated capspase-11/GSDMD-NT pathway can induce the formation of mitochondrial pores and the release of mitochondrial DNA (mtDNA) into endothelial cells (ECs). Then, the mtDNA activates the cyclic GMP-AMP synthase/yes-associated protein signaling pathway to damage endothelial regeneration.⁹⁸

2.1.3 Apoptotic caspases

In addition to inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11), apoptosis-related caspases (caspase-3, caspase-6, caspase-8) are also involved in pyroptosis. Caspase-3 can trigger gasdemin E (GSDME)-dependent pyroptosis with tumor necrosis factor alpha (TNF-α) treatment, chemotherapy drugs, and the high-expression of GSDME.^{6, 36} The effector protein YopJ from Yersinia can activate the RIPK1/caspase-8-dependent GSDMD cleavage, resulting in pyroptotic cell death.^{42, 99-101} Under hypoxia, macrophage-derived TNF-α can induce pyroptosis through the caspase-8/ gasdemin C (GSDMC) pathway, in a process which requires nuclear programmed death-ligand 1 (PD-L1). Furthermore, it is reported that antibiotic chemotherapy drugs also induce caspase-8/GSDMC-mediated pyroptosis in breast cancer.⁴⁶ Generally, LPS usually induces caspase-4/5/11-related pyroptosis; however, a recent report has shown caspase-8-mediated pyroptosis in LPS-activated macrophages. The process could be blocked by long isoform cFLIPL via promoting complex II formation.¹⁰² Recently, another apoptotic caspase, caspase-6, was found to regulate the pyroptosis pathway by enhancing the interaction between receptor-interacting serine/threonine protein kinase 3 and Z-DNA-binding protein 1, therefore, activating NLRP3/caspase-1-mediated pyroptotic signaling.⁵⁰ Although there is no direct evidence that caspase-9 can cleave GSDMs, it can cleave and activate caspase-3, thus linking with GSDME indirectly.¹⁰³ The roles of other caspases in pyroptosis pathway may be revealed by further and more in-depth studies.¹⁰⁴

2.1.4 Other enzymes

Moreover, GSDMs are cleaved beyond different caspases. Granzyme family members including GZMA and GZMB are also proved to activate GSDM B (GSDMB) and GSDME, respectively.^{6, 45} Then, in neutrophils and monocytes, neutrophil elastase (ELANE) and Cathepsin G (CatG) cleave GSDMD to generate active GSDMD-NT fragments and induce pyroptosis.^{40, 44}

2.2.1 GSDMA

GSDMA is the first GSDM family member, which was originally named GSDM1; its gene is located on human chromosome 17q21.1, while the Gsdma1, Gsdma2, and Gsdma3 genes are clustered on mouse chromosome 11D. GSDMA is primarily detected in the upper gastrointestinal tract, epithelium of the skin, and the lung,^{126, 142} while it is generally silenced in gastric cancer cells.¹⁹ GSDMA is upregulated by transforming growth factor-beta to induce apoptosis in pit cells of the gastric epithelium.¹¹¹

The molecular mechanism of GSDMA is not well known until now. It may play roles in stress responses due to its expression in the suprabasal layer, differentiating cells and the epidermis outside the tumor.¹⁴³ Asthma-associated polymorphisms at the locus 17q12-21 mediate the expression and methylation of the GSDMA gene in CD4⁺ T cells.¹⁰⁸ In turn, the GSDMA polymorphism (rs3859192 on chr17q21) is strongly linked with asthma susceptibility and intermediate phenotypes in asthma.^{142, 144} Also, the GSDMA variant (rs3894194) also modulates systemic sclerosis (SSc) susceptibility in monocyte-derived macrophages (MDMs).¹⁰⁹

Among the three Gsdma genes in mice, mutations of Gsdma3 are hotspots, which not only cause alopecia during the formation of hair follicle-associated sebaceous glands, as well as the transition from catagen to telogen at the final period of hair follicle morphogenesis,²³ but display obvious phenotypes in epidermal hyperplasia and progressive hair loss.¹⁴⁵ Gain-of-function mutations in the CT domains of GSDMA3 reveal the function of its NT fragments. The GSDMA3 Y344H mutant protein and NT domain induce autophagy with mitochondria and ROS generation.^{146, 147} GSDMA3 regulates mitochondrial homeostasis by impinging on the mitochondria via heat shock protein 90 (HSP90), inducing oxidative stress and mitochondrial permeability transition.¹¹⁰

2.2.2 GSDMB

GSDMB is found on human chromosome 17q21.1, while no Gsdmb gene has been identified in mice. Human GSDMB has six isoforms (Q8TAX9, UniProt). There are differences in the length and sequence of the linkers between the CT and NT domains of isoforms 1–4 and isoform 6, whereas there is no NT domain in isoform 5.¹⁰⁶ GSDMB is increasingly expressed in multiple tissues, especially the upper gastrointestinal epithelium,¹⁴⁸ liver and colon epithelium,¹⁴⁸ airway epithelial cells,¹⁴⁹ neuroendocrine cells,¹⁴⁸ and immune cells (T cells).^{45, 115, 150}

Highly expressed GSDMB is also linked to tumor aggressiveness in several types of cancers, such as gastric, hepatic, colon, and cervical cancer,^{124, 148, 151} as well human epidermal growth factor receptor 2 (HER2)-positive breast cancer with poor prognosis,^{106, 118, 152, 153} and pancreatic cancer susceptibility (false discovery rate ≤ 0.05),¹¹⁷ implying the role of GSDMB as an oncogene and potential attractive therapeutic target in metastasis, poor prognosis, and drug resistance to anti-HER2 therapy.

Interferon-gamma (IFN-γ) upregulates the expression of GSDMB, lymphocyte-derived GZMA cleave GSDMB between the CT and NT linkers at the Lys²⁴⁴ site, leading to pyroptosis in GSDMB-positive cells.⁴⁵ Caspase-1 cleaves GSDMB at the aspartate 236 (D236) position, inducing pyroptosis in airway epithelial cells.¹⁴⁹ Like GSDMD, GSDMB also could be cleaved by apoptotic caspase-3, 6, 7 within the NT domain, therefore, losing pyroptotic ability,¹⁰⁶ indicating the dependence of GSDM function on specific structural forms.

Genome-wide sequencing and functional genes indicate that single nucleotide polymorphisms of GSDMB are related to the increased susceptibility of ulcerative colitis and Crohn's disease, as well as asthma exacerbations and antiviral pathways.^{106, 112, 149} Analogous to GSDMA, GSDMB is also linked to childhood-onset asthma,¹⁵⁴ while GSDMB (rs9303277) in CD4⁺ T cells is an SSc-associated susceptibility locus.¹¹⁵ In addition, GSDMB is included in the Type 1 and 2 diabetes (T1D, T2D) islet expression quantitative trait loci interaction network.¹¹⁴

2.2.3 GSDMC

GSDMC is originally isolated from melanoma cells, so it is known as a melanoma-derived leucine zipper extranuclear factor (MLZE), whose gene is located on human chromosome 8q24.21. The homolog of GSDMC in mice is composed of four members, Gsdmc 1, Gsdmc 2, Gsdmc 3, and Gsdmc 4, which are clustered on mouse chromosome 15 (15D1).¹²⁶ The GSDMC is preferentially found in the epithelium of the gastrointestinal tract and skin,¹²⁶ spleen,²⁰ and metastatic melanoma cells,²⁰ as well as lung and colorectal cancer (CRC) tissues.^{125, 155}

Ultraviolet irradiation can induce the expression of GSDMC mediated by the calcium/calcineurin/nuclear factor of activated T-cells, the cytoplasmic 1 (NFATc1) pathway and the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/matrix metalloproteinase 1 pathway in human skin keratinocytes.^{156, 157} Genome-wide association studies show that GSDMC is associated with chronic back pain,¹⁵⁸ Mycoplasma hyopneumoniae (M. hyo) Ab variations in pigs,¹⁵⁹ lumbar spinal stenosis,¹²² multi-type methylation in subchondral bone cartilage and osteoarthritis-related cartilage,^{160, 161} sciatica derived from lumbar disc herniation,¹⁶² and monocyte counts.¹⁶³

GSDMC functions as an oncogene, mediating varieties of progressive processes involving types of tumors. GSDMC may serve as a significant therapeutic target in CRC patients as it promotes tumor cell proliferation in CRC by reducing activation of the transforming growth factor β receptor type II.¹²⁵ GSDMC is hypomethylated in lung adenocarcinoma (LUAD) cells, and its upregulation implies poor prognosis in LUAD patients.¹⁵⁵ Also, GSDMC is linked to metastasis in malignant melanoma,^{123, 164} and the high expression of GSDMC is related to the poor survival of breast cancer. Upon hypoxia, the GSDMC gene is transcriptionally activated for nPD-L1/p-Y705-signal transducer and activator of transcription 3 (STAT3) complex binding to the STAT3-binding site in the promoter region of GSDMC. Experiments in vivo have shown that GSDMC, along with nuclear PD-L1 and caspase-8, is required in the tumor necrosis induced by TNF-α which are derived from macrophages.⁴⁶ GSDMC variants at the chromosome 8q24 locus also modulate genetic risk to several cancers, including prostate cancer.^{165, 166} However, GSDMC, whose expression is suppressed, can inhibit the proliferation of gastric and esophageal cancer cells. It is suggested that GSDMC has the potential to serve as a tumor suppressor.¹²⁴ Therefore, the roles of GSDMC might serve as an organ-specific feature, and its function in carcinogenesis is not very clear, virtually.

2.2.4 GSDMD

GSDMD is located on human chromosome 8q24.3, while Gsdmd is found on mouse chromosome 15D3-E1. GSDMD is first identified as the efficient substrate of inflammatory caspases among the GSDM family members.²⁸ In 2015, GSDMD was shown to be the real executor of inflammasome-induced pyroptosis.^{3, 33} Since the key turning point, the functional role and molecular features have been gradually revealed. GSDMD is expressed in various cells, including the gastrointestinal epithelium, placenta, and immune cells, especially macrophages and dendritic cells (DCs), as well as cancers, such as esophageal and gastric cancer, melanoma, pancreatic cancer, prostate cancer, salivary gland tumor, and Jurkat T, and Ramos B cancer cell lines.^{24, 124, 167, 168}

Although GSDMD is the first effector protein to be found in pyroptosis and has been studied the most frequently, the perspicuous regulatory mechanism is not well known. IFN regulatory transcription factor-2 (IRF2) is found to transcriptionally regulate GSDMD expression.¹⁶⁹ Given that IRF2 is reported as a transcription factor which regulates IFN and IFN-inducible genes, and the down-regulation of IRF2 in CRC cells is associated with immune suppression and immunotherapy resistance,¹⁷⁰ the IRF2-GSDMD regulatory axis may link GSDMD to immune activity in the tumor microenvironment (TME).

GSDMD is found to be cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-4/5/11. However, the way in which GSDMD is recognized specifically by these caspases is not clear. Until recently, crystal structural analysis of caspase-1-GSDMD-C complex and caspase-4/11-GSDMD-C complex has revealed the specific recognition mode.⁵¹ In contrast to other GSDM members, there have been no specific disease-related mutants of GSDMD reported, which implies that GSDMD plays a broad-spectrum role in hosts.

The pyroptosis pathway mediated by GSDMD can also be limited in different ways. For example, disulfiram is reported to specifically inhibit pore formation by modification at the Cys191/Cys192 sites of GSDMD in human/mouse, but this does not disturb the processing of IL-1β and GSDMD, thereby blocking IL-1β release and GSDMD-mediated pyroptosis.⁴⁹ In contrast, mafenide is reported to prevent the cleavage of GSDMD by binding to the Asp275 site of GSDMD directly, reducing NT fragments and weakening pyroptosis.¹⁷¹ Mouse models have showed that GSDMD exerts the effect in many inflammatory disorders, such as endotoxemia and sepsis, experimental autoimmune encephalomyelitis, Familial Mediterranean Fever, and macular degeneration and neonatal onset multisystem inflammatory disease.^{32, 132, 133, 172, 173}

2.2.5 GSDME/DFNA5

GSDME, also known as DFNA5, is located on human chromosome 7p15.3, while Gsdme, also known as Dfna5, is found on mouse chromosome 6B2.3. GSDME mutation was first identified to cause an autosomal dominant hearing impairment.^{18, 174, 175} GSDME is mostly expressed in the cochlea, placenta, brain, female reproductive tract (especially the placenta), kidney, heart, and muscle.³⁶ In contrast to non-tumoral tissues, expression of GSDME is limited in most cancers including gastric cancer, melanoma, CRC, invasive breast cancer, and other human cancers.^{36, 138} GSDME suppression in cancer cells is most dependent on the DNA methylation of the GSDME promoter.^{138, 164, 175} The inactivation of GSDME conforms to the clinical and animal results that lower the expression of GSDME and is associated with a poor 5-year survival and enhanced metastases in breast cancer.^{6, 164, 175} All of this suggests that GSDME might be a tumor suppressor and worth in-depth exploration. However, there are also various loss-of-function (LOF) mutations of GSDMD in different cancers, although this is less common than the epigenetic suppression of GSDME. Thus, LOF mutations together with the epigenetic suppression of GSDME might be two arch strategies developed by cancer cells to escape GSDME-mediated tumor suppression.^{36, 164, 175} The transcription of GSDME may be regulated by p53, which is also inactivated in many cancer cells, as virtually nothing is known about the exact regulatory details.¹⁷⁶ Generally, caspase-3 is reported to specifically cleave GSDME, leading to GSDME-mediated pyroptotic cell death when viral infection or chemotherapy drugs are used. Given that GSDME is also expressed in normal cells and tissues, chemotherapy drug-induced pyroptosis might also cause damage to the host,^{36, 177, 178} while 2-bromopalmitate might inhibit chemotherapy-induced pyroptosis.¹⁷⁸

2.2.6 DFNB59/PJVK

DFNB59, also known as Pejvakin (PJVK), is located on human chromosome 2q31.2 and comprises seven exons,¹⁷⁹ while Dfnb59, also known as Pjvk is found on mouse chromosome 2C3. PJVK encoded by DFNB59, is a protein (352 residues) which is distantly related to other GSDM protein family members.^{19, 179} PJVK expression has been found in outer hair cells, as well as supporting cells, hair cells, and cell bodies of spiral ganglion neurons (SGNs) in the inner ear.^{25, 26} PJVK is also critical for protection of the architecture of stereocilia and the function of hair cells and SGNs.^{34, 140, 180-183}

DFNB59, the same as its paralog DFNA5, is also reported to be associated with progressive hearing loss. DFNB59 is first identified as the causal gene of autosomal recessive deafness, which also plays key role during the signal transmit of auditory nerve.²⁵ As a hearing-loss gene, variants of DFNB59 are linked with non-syndromic hearing loss.^{141, 184-187} In addition, case reports have reported that PJVK also plays a role in other hearing-related syndromes like auditory neuropathic spectrum disorder (ANSD) and poor cochlear implants performance.^{25, 188}

4.1.1 Inflammatory mediators

The inflammatory state of the TME has implications for the response to immune checkpoint inhibitor (ICI) therapy. By triggering pyroptosis, a form of ICD, converting these immune “cold” tumors to “hot” tumors may alter the TME and the influx of tumor-infiltrating lymphocytes (TILs). Pyroptotic immune response involves a long process which goes from the release of damage-associated molecular patterns (DAMPs) (e.g. HMGB1, ATP, etc.) and inflammatory cytokines, directly modulating the innate immune response, to enhancing the recruitment of adaptive immune cells along with increased antigen presentation and TLR activation, leading to broader immune activation.^{249, 250} Understanding the complex mechanisms of pyroptosis and how the process alters the TME may have profound effects on future therapeutic strategies in both the up-front and salvage treatment settings.

Which features of pyroptotic tumor cells contribute to activating antitumor immunity is not currently clear. Inflammatory cytokines and immunostimulatory alarmins released during pyroptosis, including IL-1β, IL-18, ATP, and HMGB1, could be prime suspects. During pyroptosis, IL-1β is activated and released from the GSDM pores to exert an influence on the TME. IL-1β signaling could induce DC maturation and monocyte differentiation into DCs and inflammatory macrophages. Then, antigens will be represented to T cells to elicit immune killing. In addition, IL-1β can directly act on cells in the lymphoid compartment to drive antigen-specific cytotoxic CD8⁺ T cell responses, increase Th1 CD4⁺ T cell numbers, and inhibit the differentiation of immunosuppressive T regulatory cells.^{249, 250} Indeed, it has been shown that IL-1β by intra-tumoral injections is sufficient to cause CD8⁺ T cell-mediated tumor regression in models of adenocarcinoma, melanoma, and sarcoma.²⁵¹

IL-18 can strongly induce γ-IFN when it combines with IL-12 or IL-15, which both upregulate the IL-18 receptor levels. Thus, IL-18 exerts an important effect on natural killer (NK) cell recruitment and activation, as well as Th-1 polarization.²⁵²

In addition to IL-1β and IL-18, other types of cytokines such as IL-6, are also released from pyroptotic cells. Accumulating evidence shows that IL6 has a profound impact on the adaptive immune response. For instance, IL-6 signaling can increase T cell immune response to an actively responsive state from a suppressive state, thereby effectively fighting against tumors. Also, IL-6 is also important for enhancing T cell trafficking to lymph nodes and the tumor sites. Then, they have chances to be activated and further execute their cytotoxic effector functions.²⁵³ Additionally, IL6 exerts an influence on increasing antibody production, increasing cytotoxic CD8⁺ T cell differentiation and decreasing regulatory T cell differentiation.^{253, 254}

As a type of alarmins, extracellular ATP reinforces pyroptosis by triggering of purinergic P2 × 7R signaling in infiltrating immune cells or cancer cells.²⁵⁵ Like apoptosis, pyroptotic cells could release macrophage attractants, “find-me” and “eat-me” signals, the main component of which is ATP. Phagocytosis induction of pyroptotic cells by macrophages using the two signals will ultimately promote CD8⁺ T-cell activation and IFN-γ production.²⁵⁶ For the positive aspects, the release of HMGB1 from dying tumor cells could force host DCs to process and present tumor antigens. Also, HMGB1 signaling can interact with TLR2 and TLR4 receptors on the surface of neutrophils, monocytes, macrophages, DCs, and lymphocytes, leading to the activation of transcription factors NF-κB and AP1, the production of inflammatory cytokines such as IL-6, TNF α, IL-8, and IL-1, and an increase in co-stimulatory molecules required for the cross-priming of antitumor T lymphocytes.^{257, 258}

4.1.2 Lymphocyte cytotoxicity

Pyroptosis is essential for lymphocyte cytotoxicity against tumor cells, and pyroptotic tumor cells induce the TME toward an immunostimulatory state. To some extent, a positive feedback loop is formed in anticancer immunity as for the promotion between pyroptosis and cytotoxic lymphocytes. In the case of GSDME, a tumor suppressor which activates pyroptosis, this enhances antitumor immunity.⁶ It is well known that caspase-3 cleaves GSDME to activate pyroptosis, and the cell killer GZMB has also been reported to activate caspase-independent pyroptosis in target cells by directly cleaving GSDME (after D270) at the same site as caspase-3. Indeed, non-cleavable or non-pore-forming GSDME is unable to perform its tumor-suppressive ability. Interestingly, on the one hand, the antitumor ability of GSDME mainly depends on the killing ability of CD8⁺ T cells, NK cells, granzymes, and perforin. On the other hand, given that immunotherapeutic strategies often aim to improve T cell responses to cancer, the overexpression of GSDME in cancer cells significantly increases the number and function of infiltrating NK cells and antigen-specific CD8⁺ T lymphocytes inside tumors, as well as the expression of effective molecules including GZMB, perforin, IFN-γ, and TNF-α in TILs, together with the phagocytosis of tumor-associated macrophages (TAMs).^{259, 260} GSDME converts more moderate and non-inflammatory apoptotic death into more rapid and inflammatory pyroptotic death that can stimulate an effective antitumor immune response.⁶ It is also reported that GSDMD participates in cell death mediated by cytotoxic T lymphocytes (CTLs) in lung squamous cell carcinoma and LUAD.²⁶¹

4.1.3 Phagocytosis by macrophages

As for GSDMC, it has been shown that the expression of GSDMC under hypoxia is transcriptionally enhanced by p-STAT3 and is physically correlated with PD-L1 and its nuclear translocation. Then, after treatment with TNF-α derived from macrophages, GSDMC is specifically cleaved by caspase-8, liberating the NT domain to form pores on the plasma membrane, thereby inducing pyroptosis. The high expression of GSDMCs is also associated with poor patient survival.⁴⁶ Pyroptosis in macrophages induced by sorafenib could trigger NK cell-mediated cytotoxicity against hepatocellular carcinoma (HCC). Then, pyroptotic macrophages release IL-18 and CCL5 to enhance the chemotaxis and cytotoxicity of NK cells and eliminate HCC by activating antitumor immunity.²⁶²

4.1.4 Pyroptosis in other immune cells

In cellular immunity, CTLs and NK cells kill target cells via the delivery of serine protease granzymes by perforin. Notably, lymphocyte-derived GZMA could cleave GSDMB between CT and NT linker at Lys²⁴⁴ site, resulting in pyroptotic cell death in GSDMB-positive cells killed by NK cells and CTLs.⁴⁵

Tumor cells undergoing pyroptosis release IL-1β, IL-18, and various DAMPs, which are signals used to activate and recruit DCs or macrophages to phagocytose the pyroptotic cells and promote their maturation. Then, mature DCs present tumoral antigens to tumor-specific CTLs, killing pyroptotic tumor cells.^{256, 263} Melanoma is known as a “cold” tumor due to the poor responses to ICI therapy; the combination of B-Raf Proto-Oncogene, serine/threonine kinase (BRAF) and Methyl Ethyl Ketone (MEK) inhibitors (BRAFi + MEKi) is an FDA-approved approach to therapy BRAF V600E/K-mutant melanoma. However, the efficacy of the BRAFi + MEKi approach is correlated with immune responses in the TME. Given that pyroptosis could elicit antitumor immunity in the tumor immune microenvironment, the involvement of pyroptosis in ICI treatment is being investigated in new researches. They have found that the treatment of BRAF-mutant melanoma cells with BRAFi + MEKi inhibitors promotes the cleavage of GSDME and the release of HMGB1, all of which are markers of pyroptotic cell death. Interestingly, GSDME-deficient melanoma represents defective HMGB1 release, the reduced infiltration of tumor-associated T cells and activated DCs in response to BRAFi + MEKi treatment, and the more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant diseases show the decreased infiltration of intra-tumoral T cells and the lack of pyroptosis-related markers but still retain sensitivity to pyroptosis-induced chemotherapy.⁴⁷ The data imply that pyroptosis could as an adjunctive therapy to ignite antitumor immune responses for resistant melanoma. In addition, in KRAS-, EGFR-, or ALK-driven lung cancer, upon treatment of diverse small-molecule inhibitors, robust pyroptotic cell death, associated with GSDME cleavage, and the mitochondrial intrinsic apoptotic pathway are elicited and spread in lung cancer cells uniformly, making concession under genotype-specific regimens.²⁶⁴

4.1.5 New application of pyroptosis in antitumor approaches

As tumor treatment is obstructive and complex, the effect of immunotherapy is gaining increasing prominence. However, the immune checkpoint blockade often responds ineffectively to some tumor treatments as for the low inflammatory response among the TME. Thus, the development of new approaches to boost antitumor immunity is critical. Recently, the Phe-BF3 desilylation bio-orthogonal system applied efficiently has been shown to transport desilylation catalyzed by Phe-BF3 with the NP-GSDMA3-mediated delivery into specific mammary tumor cells in mice. The novel technique leads to T cell-dependent tumor progression along with increasing numbers and the function of CD4⁺, CD8⁺, NK cell, and M1 macrophage populations, while decreasing regulatory T cell, M2 macrophage, monocyte, neutrophil, and myeloid-derived suppressor cell (MDSC) populations. As a new application, the system uncovers the antitumor immune potential of pyroptosis, suggesting that a GSDM agonist may improve the efficacy of cancer immunotherapy.¹⁰

Pyroptosis also functions in patients with extrahepatic cholangiocarcinoma (CCA). Tumor-cell-derived microvesicles whose plasma membranes are infused with methotrexate could induce pyroptosis in CCA cells through a GSDME-dependent pathway. Then, intracellular contents released from pyroptotic CCA cells activate patient-derived macrophages to produce pro-inflammatory cytokines, which attract the secondary accumulation of neutrophils to the tumor site. The procedure is relevant to the upregulation of uridine diphosphate glucose and complement C5, leading to degradation of the stromal barrier in the CCA TME. The approach has been proved to relieve biliary obstruction in nearly 25% of CCA patients.²⁶⁵

Another application of pyroptosis is that of a commonly used tumor-targeting nanoliposome loaded with cisplatin, which is applied in drug delivery and administration. Combining decitabine (DAC) with chemotherapy nanodrugs to manage drugs to activate and upregulate the caspase-3/GSDME signal, further triggers pyroptosis in tumor cells, therefore strengthening the immunological effect of chemotherapy with cisplatin in a mouse triple-negative breast cancer model. Also, DAC can demethylate the GSDME gene in tumor cells with specific tumor-bearing mice.²⁶⁶

The chimeric co-stimulatory converting receptor is designed to disturb the PD-1 pathway which enhances the activity of chimeric antigen receptor (CAR)-NK cells to fight solid tumors. Then, the antitumor activity of NK92 cells was enhanced by the neo-complex PD1-NKG2D-41BB receptor, which is mainly dependent on pyroptosis activation.²⁶⁷