Pyroptosis: A promising biomarker for predicting colorectal cancer prognosis and enhancing immunotherapy efficacy

Abstract

In this editorial, we comment on the article by Zhu et al published in the recent issue of the World Journal of Clinical Oncology. We focus specifically on the characteristics and mechanisms of pyroptosis and the impact of changes in the tumor immune microenvironment (TIME) on cancer prognosis. Pyroptosis is a distinct form of programmed cell death; its occurrence can change the TIME and regulate the growth and spread of tumors and therefore is significantly correlated with cancer prognosis. Previous research has demonstrated that pyroptosis-related genes can be used in prognostic models for various types of cancer. These models enhance the mechanistic understanding of tumor evolution and serve as valuable guides for clinical treatment decision-making. Nevertheless, further studies are required to thoroughly understand the function of pyroptosis within the TIME and to assess its mode of action. Such studies should reveal new tumor therapeutic targets and more successful tumor immunotherapy strategies.

Key Words: Pyroptosis; Colorectal cancer liver metastases; Organoid model; Immunotherapy; Prognostic biomarker

Core Tip: Pyroptosis plays a vital role in tumor immunotherapy by triggering robust inflammatory responses and significantly inhibiting tumors. Pyroptosis results in the release of copious amounts of inflammatory cytokines and tumor-associated antigens that stimulate antigen-presenting cells, thereby initiating adaptive immune responses. Therefore, inducing pyroptosis is a promising novel immunotherapy approach for tumors.

INTRODUCTION

Pyroptosis is a form of programmed cell death that was initially misinterpreted as apoptosis due to their similar characteristics, such as caspase dependency, DNA damage, and nuclear condensation[1,2]. However, pyroptosis is a unique inflammatory programmed cell death process distinct from apoptosis[3-10] (Table 1). The term pyroptosis was first introduced in 2001 and originated from the Greek words “pyro” and “ptosis”, which mean “fire” and “falling”, respectively[11]. With the exception of DFNB59, which lacks pore-forming capacity, gasdermin protein family members are responsible for mediating pyroptosis[12-14]. This protein family has two conserved domains: The N-terminal pore-forming domain (PFD) and the C-terminal repressor domain (RD)[15]. At rest, gasdermin proteins preserve their oligomeric structure via PFD-RD interactions, with the latter curbing the cytotoxic potential of the former. Upon cellular exposure to external or internal stimuli, these proteins are cleaved by specific caspases (caspase-1/4/5/11) or granzymes[16-20]. During this process, the N-terminal PFD dissociates from the C-terminal RD, leading to the oligomerization of PFD. This event results in the formation of pores on the cell membrane, thereby enabling the release of inflammatory molecules and inducing cell swelling, culminating in pyroptosis[21].

Table 1 Differences between pyroptosis and apoptosis.

	Apoptosis[5-9]	Pyroptosis[1,2,19-21]
Definition	The orderly death of cells autonomously controlled by genes in order to maintain a stable internal environment	An inflammatory programmed cell death triggered by various pathological stimuli, such as stroke, heart attack, cancer, and microbial infection
Trigger factor	Gene regulation under physiological conditions	Pathological stimulation
Cell morphological changes	The cell size is usually reduced, the cell membrane structure is intact, and the organelles are intact	The cell size is usually enlarged and cell morphology is deformed, the cell membrane is broken, and the organelles are deformed
Marker events	Nucleoprotein, cytoskeleton, and protein crosslinking are destroyed, phagocyte ligands are expressed, and apoptotic bodies are formed	The nucleus is concentrated and pyrosome is formed
DNA change	DNA is degraded into fragments of 180-200 bp and its integral multiples	DNA is randomly degraded
Molecular mechanism	Caspase-2/3/6/7/8/9/10, in which caspase-3 is the main regulator of apoptosis	Human caspase-1/4/5; murine caspase-1/11
Detection indexes	Early apoptosis detection indicators: Annexin V and JC-1; metaphase apoptosis detection index: Caspase; late apoptotic detection index: TUNEL staining	No specific detection indicators, but the cell morphology could be observed by scanning electron microscopy and TUNEL staining, GSDMD immunofluorescence staining could be used to detect the expression levels of pyro related genes or proteins (caspase-1/4/5/11, etc.), and the levels of inflammatory factors (IL-1β, IL-18, etc.) could be detected by ELISA. CCK-8 assay can be used to determine cell viability
Dependency	The endogenous pathway (also called the mitochondrial pathway) is based on caspase-9	Classical pathway: Based on caspase-1; nonclassical pathway: Based on caspase-4/5/11

Extensive scholarly research has revealed a strong correlation between pyroptosis and the onset and spread of numerous cancers[22-24]. Chronic inflammatory infiltration increases the risk of cancer. In particular, pyroptosis-induced release of cytokines such as IL-1 and IL-18 might increase the risk of carcinogenesis and metastasis[25]. Nevertheless, Li et al[26] reported that pyroptosis impedes tumor progression through the induction of tumor cell death and activation of the immune system. The concurrent release of cytokines during pyroptosis not only spurs the activation of various immune cells but also modulates the tumor immune microenvironment (TIME) by interacting with key immune checkpoints such as PD-1/PD-L1, indicating promising implications for tumor immunotherapy. The intricate mechanisms by which pyroptosis can stimulate an immune response while simultaneously mitigating its tumor-promoting effects remain a subject for further research. However, current evidence indicates that the manifestations of pyroptosis are closely associated with the onset and progression of colorectal cancer (CRC) and other types of tumors, underscoring the significant potential of these biological markers for evaluating therapeutic outcomes and predicting patient prognoses in the field of oncology. For instance, Dihlmann et al[27] reported that AIM2 inflammasome-mediated pyroptosis is the underlying mechanism of CRC progression. Another study revealed that gasdermin C, an oncogene that is not present in normal colorectal tissues, is upregulated due to inactivating mutations in TGFBR2 in CRC tissues[28].

In summary, pyroptosis triggers inflammation that activates macrophages and boosts T-cell-mediated antitumor immunity, thereby regulating the TIME to effectively suppress tumor growth and spread (Figure 1)[26,29]. Current research focuses on inhibiting tumor progression through the regulation of pyroptosis and its combination with chemotherapy and immunotherapy. In addition, pyroptosis biomarkers are significant for predicting the efficacy and prognosis of CRC and other cancers and could serve as potential therapeutic targets.

Figure 1 Role of pyroptosis in regulating the tumor immune microenvironment and tumor growth. In the canonical pathway, inflammasomes recognize exogenous pathogens and endogenous damage. The intracellular sensor proteins NLRP1, NLRP3, NLRC4, AIM2, Pyrin, and NLRC4 recruit ASC and then activate caspase-1. In the noncanonical pathway, lipopolysaccharide can activate caspase-4/5/11. Activated caspase-1/4/5/11 can cleave GSDMD to release N-GSDMD and pro-IL-1β/18, promoting their maturation. On one hand, N-GSDMD can form nonselective pores, further causing water influx, lysis, and death. Additionally, IL-1β and IL-18 can be released via these pores. On the other hand, N-GSDM, IL-1β, and IL-18 can affect immune cells such as cancer cells in the tumor immune microenvironment, tumor-associated macrophages, CD8+ T cells, and NK cells. Therefore, pyroptosis is a complex regulatory network that acts as a bridge between the immune system and tumors and as a double-edged sword in the tumor immune microenvironment. In this way, it influences tumorigenesis, leading to protumor effects and antitumor effects. LPS: Lipopolysaccharide; TIME: Tumor immune microenvironment.

INFLUENCE OF THE TIME IN PROMOTING IMMUNE EVASION, TUMOR PROGRESSION, AND IMMUNOTHERAPY

The TIME encompasses cellular and acellular components, including cancer cells and immune cells such as T cells, B cells, NK cells, dendritic cells, myeloid-derived suppressor cells, and macrophages, among others. The review by de Visser et al[30] highlights the role of primary immune cells within the TIME and their influence on the effectiveness of tumor immunotherapy. The overall proportion, characteristics, and activation status of Th1 and Tc cells within the TIME are major factors in assessing recurrence and/or metastasis risk in CRC. B cells exert antitumor effects through antigen presentation to T cells, antibody production, and cytokine secretion. Additionally, these proteins promote tumor progression by releasing factors that cause inflammation, immunosuppression, angiogenesis, and complement activation. These dual roles make B cells both protective and oncogenic within the cancer milieu[31,32]. Tumor-associated macrophages also exert both pro- and antitumorigenic effects in the TIME, where they secrete vascular endothelial growth factor and facilitate new blood vessel formation[33,34]. Vessels and the extracellular matrix are also nonnegligible components of the TIME. Vessels are vital for nourishing tumors and maintaining tumor perfusion. They also serve as conduits for immune cells, facilitating their movement from the original tumor location to other areas, where they can contribute to immunological responses. The extracellular matrix consists of proteins, including collagen, fibronectin, elastin, and laminin. The specific composition and organization of this matrix, where tumor cells reside, can influence how immune cells interact with tumors. In certain cases, the matrix may form a physical barrier that impairs the effectiveness of antitumor immune cells.

In the recent issue of the World Journal of Clinical Oncology, Zhu et al[35] published an interesting paper titled “Identification and validation of a pyroptosis-related prognostic model for CRC based on bulk and single-cell RNA sequencing data”. The authors developed a pyroptosis-related prognostic model for CRC, confirming its correlation with immune infiltration. Specifically, the researchers accessed CRC patient data from The Cancer Genome Atlas (TCGA) database and the single-cell RNA sequencing dataset GSE178341 from the Gene Expression Omnibus to extract genes linked to pyroptosis. They further performed graphene oxide enrichment analysis on the set of pyroptosis-related genes and analyzed their differential expression. A total of 178 key genes derived from the scRNA-seq and TCGA datasets were obtained, and 8 differently expressed genes related to prognosis, CHMP2B, SDHB, BST2, UBE2D2, GJA1, AIM2, PDCD6IP, and SEZ6L2, were eventually identified. These genes were used to establish the prognostic model utilizing univariate Cox analysis and the LASSO Cox regression algorithm, and its efficacy was verified by receiver operating characteristic analysis. Additionally, the differences in immune-related functions between the high- and low-risk groups were compared using the CIBERSORTx algorithm to investigate the relationship between immune infiltration and CRC. Overall, this study may hold promise for enhancing clinical management and immunotherapy strategies for CRC patients.

In recent years, the use of three-dimensional organoid models for comprehensive evaluation of the TIME has attracted increasing amounts of attention. Neal et al[28] described an organoid methodology that may inspire subsequent research and drug discovery for studies by Zhu et al[35]. Specifically, they successfully generated patient-derived organoids (PDOs) from 100 human biopsies of surgically resected primary and metastatic tumor tissues, including the intestine, stomach, and pancreas, by plating mechanically dissociated tissue fragments in type I collagen matrix air-liquid interface (ALI) culture. They utilized WENR base medium to expand and serially passage mechanically processed tumor fragments as ALI organoids and successfully established PDOs, including CRC. This organoid model offers two main advantages: These PDOs preserve the tumor architecture and stroma expressing SMA and vimentin, indicating faithful recapitulation of the parental tumor histology, with a 73% success rate across different tumor types after one month of culture. Additionally, PDOs preserve the TIME and diverse integrated immune elements, including T cells, B cells, NK cells, and macrophages. Simultaneous analysis of gene expression and the immune repertoire in single cells suggested that the T-cell receptor repertoire is highly conserved between tumors and their corresponding PDOs. Despite these advancements, several questions remain unanswered in the study by Neal et al[36]. The exact mechanisms underlying the roles of CHMP2B and PDCD6IP, which are newly proposed prognostic biomarkers for CRC, in pyroptosis and tumors remain to be elucidated. Moreover, translating bioinformatic insights into improvements in TIME organoid model development is crucial for validating research findings and advancing personalized immunotherapy for CRC. The current investigation confirmed the predictive significance of pyroptosis-related genes and revealed that compared with other traditional prognostic models, the authors’ pyroptosis-related risk model could provide insight into personalized immunotherapy for CRC patients.

In the past decade, remarkable progress has been made in immunotherapy for CRC, which could be a successful approach for overcoming the limitations of initial diagnoses and current treatments. Yu et al[37] recently reviewed new immune-based therapies for primary and metastatic CRC. There are three major classes of immunotherapy: Immune checkpoint inhibitors (ICIs), adoptive cell transfer therapy, and tumor vaccines. Immunotherapies aimed at treating stage IV cancers using ICIs have been shown to greatly improve patient outcomes in mismatch repair-deficient CRC patients, but the use of adoptive cell transfer therapy and tumor vaccines has not yet been widely applied. In addition, monoclonal antibodies have demonstrated efficacy in treating a wide range of tumors, largely because of their favorable safety profile and ability to precisely target tumor cells. Despite these advantages, the use of monoclonal antibodies is currently limited by their high cost. Furthermore, their exquisite specificity, while beneficial in many respects, may not be optimal for combating heterogeneous tumor cell populations, including cancer stem cells, which can contribute to tumor resistance and recurrence. Thus, it is necessary to gain more insight into the appropriate and new biomarkers that will provide accurate and reliable diagnostic and prognostic information regarding the influence of the immune system on CRC. Targeting pyroptosis may reveal the potential of developing immunotherapies that treat microsatellite-stable CRC, which accounts for the majority of all CRC cases.

CLINICAL IMPLICATIONS

CRC and liver metastases from CRC are important clinically relevant issues for at least three reasons. First, CRC is the major cause of death in both men and women worldwide and in both developed and undeveloped countries. It is diagnosed more frequently due to the progressive aging of the population, obesity, and sedentarism. Second, the majority of patients die from metastatic liver disease, and only a few of the patients with colorectal liver metastases are candidates for liver resection. Third, immunotherapy, chemotherapy, and radiotherapy can potentially increase the survival time of patients with unresectable colorectal liver metastases. Extensive results have shown that CRC responds poorly to ICIs, partly because of the immunosuppressive tumor microenvironment in immunologically cold tumors, and widespread chemoresistance has led to great concern regarding their efficacy, safety, and tolerability. Studies on pyroptosis in immunotherapy have the potential to transform the way that CRC and other metastatic tumors are treated and indicate more diverse treatment approaches for CRC.

CONCLUSION

In recent years, research efforts to identify biomarkers and develop prognostic models for CRC have intensified due to their potential for improving outcome prediction. Although various strategies are being pursued, there is currently no active immunotherapy available for treating metastatic or recurrent CRC, underscoring the urgent need for more effective prognostic and predictive biomarkers. The recommendations for these patients are as follows: (1) Sufficient physical activity and weight reduction can improve cancer outcomes[38]; (2) Chemotherapy and surgical resection for patients with limited metastatic disease can play an important, and sometimes curative, role in the treatment of select patients with mCRC[39,40]; and (3) The use of 5-fluorouracil can improve survival; alternatively, aspirin can be used (indicated by a large number of epidemiological and experimental studies to be effective in the treatment of CRC). Clinicians should also recognize that combination treatment regimens of standard drugs with newer agents have been shown to improve overall survival, disease-free survival, time to progression, and quality of life compared to standard drugs alone in patients with advanced CRC. For instance, oxaliplatin plus intravenous bolus fluorouracil and leucovorin has been shown to be superior to intravenous bolus fluorouracil and leucovorin in terms of disease-free survival[41].

ACKNOWLEDGEMENTS

This study benefited from the cooperation of each researcher, whose contributions are sincerely appreciated.