PANoptosis and Immune Remodeling in the Tumor Microenvironment

ABSTRACT

In recent years, PANoptosis has garnered increasing attention for its potential to promote inflammation. Nevertheless, several issues, including resistance to conventional treatment and immune evasion, correlate with inflammation in the tumor microenvironment. Controlled by the assembly of NLRP12‐, ZBP1‐, AIM2‐, and RIPK1‐PANoptosome, the newly discovered programmed cell death PANoptosis simultaneously manifests the characteristics of apoptosis, necroptosis, and pyroptosis. This review summarizes the multifaceted characteristics and the molecule determining PANoptosis sensitivity within the microenvironment. It examines the immunomodulatory function of PANoptosis in the tumor microenvironment, including how immune cells mediate PANoptosis in tumor cells and how tumor cells leverage PANoptosis to evade immune response and promote immunosupression. The crucial role of PANoptosis in immune remodeling suggests promising therapeutic strategies for tumors, such as immunotherapy and combination therapy. The induction of PANoptosis may establish a positive feedback loop that promotes immune activation and has a potential to remodel the immunosuppressive TME. Future strategies is expected to provide new directions for more precise, personalized cancer treatment.

The immune remodeling of PANoptosis in tumor microenvironment. The crucial role of PANoptosis in immunity remodeling suggests promising therapeutic strategies, such as conventional therapy, immunotherapy, and nonatechnology therapy. Controlled by the assembly of NLRP12‐, ZBP1‐, AIM2‐, and RIPK1‐PANoptosome, the newly discovered programmed cell death PANoptosis simultaneously manifests the characteristics of apoptosis, necroptosis, and pyroptosis. The active induction of PANoptosis provides a robust positive feedback loop for immune activation and offers a sophisticated strategy to dismantle the immunosuppressive TME. AIM2, absent in melanoma 2; Arg‐1, Arginase‐1; ASC, Apoptosis‐associated speck‐like protein containing a CARD; CASP1/3/6/8, Caspase 1/3 / 6/8; DAMP, Damage‐associated molecular pattern; FADD, Fas‐associated protein with death domain; GSDMD, Gasdermin D; GSDMD‐N, N‐terminal domain of Gasdermin D; IL‐1β/IL‐6/IL‐10, Interleukin 1β/6/10; MLKL, Mixed lineage kinase domain‐like protein; MLKL‐P, Phosphorylated MLKL; NLRP12, NLR family pyrin domain containing 12; NLRP3, NLR family pyrin domain containing 3; PAMP, Pathogen‐associated molecular pattern; RIPK1/RIPK3, Receptor‐interacting serine/threonine‐protein kinase 1/3; TGF‐β, transforming growth factor beta; TNFα, tumor necrosis factor alpha; Treg, Regulatory T cell; ZBP1, Z‐DNA binding protein 1.

1. Introduction

For organisms that regulate homeostasis, development, and tissue remodeling, cell death is an essential process. Programmed cell death (PCD), mediated by specialized molecular machinery, includes apoptosis, necroptosis, and pyroptosis [1]. Conditions, including oxidative stress, DNA damage, death receptor‐ligand binding, viral infection, or calcium ion dysregulation, can all cause apoptosis. Under these circumstances, the CASP family is activated, initiating subsequent cascades that lead to membrane blebbing and nuclear condensation [2]. Necroptosis is regulated by microbial nucleic acids or the interaction of death receptors and ligands. It mainly depends on activated Receptor‐Interacting Protein Kinase 1 (RIPK1) and RIPK3, which form the necrosome and cause Mixed Lineage Kinase Domain‐Like Protein (MLKL) to oligomerize, leading to cell membrane rupture [3]. Biomarkers with validated molecular mechanisms, such as TREM1‐mediated pyroptosis, PKM2‐mediated metabolic dysregulation, and SOX11‐mediated immune infiltration and ferroptosis, are used to evaluate treatment efficacy and adverse reactions [4]. An integrated cell death program called PANoptosis has attracted broad interest in recent years [5, 6, 7, 8]. Intracellular sensors that identify pathogen‐associated molecular patterns (PAMPs) or damage‐associated molecular patterns (DAMPs), such as absent in melanoma 2 (AIM2) and Z‐DNA binding protein 1 (ZBP1), trigger the assembly of the PANoptosome [5, 9]. This multiprotein complex simultaneously drives the crucial factors of necroptosis, apoptosis, and pyroptosis via activating CASP 3/7, cleaving GSDMD, and phosphorylating MLKL [10]. As a convergent cell death mechanism, PANoptosis triggers cytotoxicity in the tumor microenvironment (TME). Consequently, inducing PANoptosis has also been recognized as a prospective approach for reducing tumor burden and overcoming therapy resistance [11, 12]. While conventional modalities like chemotherapy, immunotherapy, and emerging regenerative medicines are widely established, combining immunotherapies with other treatments has yielded encouraging results in malignancy management [13]. The elicitation of an immune response is the central mechanism of these strategies. However, immunotherapies still encounter two significant challenges in solid tumors: immune evasion and drug resistance [14, 15, 16]. Innovative therapies targeting the mechanisms of PANoptosis could lead to new therapeutic discoveries and help overcome the existing limitations in therapies.

2. Molecular Mechanism of PANoptosis

The primary mechanism controlling PANoptosis is the formation of the PANoptosome [17]. Single‐cell analysis indicated that different pathological circumstances, including bacterial and viral infections, cytokine storms, and neoplastic alterations, can cause the intracellular assembly of the PANoptosome [18]. Although the PANoptosome assembly is reliant on specific stimuli, it typically comprises three core components: sensor proteins with sensing domains, adapter proteins with recruitment domains, and effector proteins with catalytic domains [19]. Within this framework, four different forms of PANoptosome complexes have been reported: NLRP12‐PANoptosome, ZBP1‐PANoptosome, AIM2‐PANoptosome, and RIPK1‐PANoptosome [6, 9, 20, 21, 22] (Figure 1). According to earlier research, in response to heme plus PAMPs or tumor necrosis factor‐alpha (TNF‐α), the cytosolic innate immune sensor NLRP12 triggers PANoptosome activation, inflammation, and cell death [21]. The AIM2 protein identifies PAMPs during exogenous infections, forming a complex with ZBP1 to initiate PANoptosome assembly [9]. The apical sensors of PANoptosis, ZBP1 and RIPK1, have also been associated with bacterial and fungal infections, respectively [23, 24]. Through domain‐specific mechanisms, adaptor proteins and catalytic effectors combine to form PANoptosome, which in turn initiates the PCD process. Fas‐associated death domain (FADD) recruits pro‐CASP‐8 to form the death‐inducing signaling complex (DISC). Pro‐CASP‐8 undergoes autocleavage to generate enzymatically active CASP‐8. The activated CASP‐8 then cleaves and activates downstream effector CASPs, such as CASP‐3 and CASP‐7 [25]. These effector CASPs then act on different cellular substrates, finally leading to apoptosis [26]. Simultaneously, the inflammasome complex is formed when pattern recognition receptors (PRRs) and Pro‐CASP‐1 are recruited by the adaptor protein C‐terminal CASP recruitment domain (ASC). Activated CASP‐1 cleaves GSDMD to generate its N‐terminal active fragment (GSDMD‐N), creates pores in the cell membrane, and causes cellular osmotic imbalance and the release of cellular contents [27]. RIPK3 and RIPK1 can form the necroptosis complex (necrosome). Mixed lineage kinase domain‐like protein (MLKL) is phosphorylated and activated by RIPK3 inside the necroptosis complex. The activated MLKL oligomerizes and translocates to the cell membrane, resulting in the loss of membrane integrity, disruption of ion homeostasis, and release of cellular contents [28].

FIGURE 1.

The characteristics of the different cell death mechanisms. Schematic diagram of the characteristics of four types of cell death: Apoptosis, necroptosis, pyroptosis, and PANoptosis. The cellular morphology of apoptosis, necroptosis, and pyroptosis highlights a shared pattern of organelle dysfunction, such as mitochondrial failure and ER dilation, that culminates in PANoptosis. At the molecular level, the execution of PANoptosis relies on the synchronized assembly of the PANoptosome, a master molecular complex containing core components of both the inflammasome and the necrosome. Furthermore, the evaluation of these PCDs is heavily reliant on key functional indicators, specifically plasma membrane integrity, assessed via Annexin V/PI staining, and mitochondrial functions, measured by mROS generation and membrane potential loss. AIM2, Absent in Melanoma 2; ASC, Apoptosis‐associated speck‐like protein containing a CARD; CASP (CASP1/3/6/8/9), Caspase; DAMP/PAMP, Damage/Pathogen‐Associated Molecular Pattern; DCFH‐DA, Dichlorodihydrofluorescein diacetate; DISC, Death‐inducing signaling complex; ER, Endoplasmic Reticulum; FADD, Fas‐associated protein with death domain; FITC, Fluorescein Isothiocyanate; GSDMD/GSDMD‐N, Gasdermin D/N‐terminal of Gasdermin D; IRF1, Interferon Regulatory Factor 1; MAP3K7, Mitogen‐Activated Protein Kinase Kinase Kinase 7; MLKL, Mixed Lineage Kinase Domain‐like protein; mROS, Mitochondrial Reactive Oxygen Species; NLRP3/NLRP12, NOD‐like Receptor Pyrin Domain Containing 3/12; PANoptosis, Pyroptosis, Apoptosis, and Necroptosis; PCDs, Programmed Cell Deaths; PI, Propidium Iodide; Pro‐Casp1/Pro‐Casp8, Pro‐Caspase 1/8; RIPK1/RIPK3, Receptor‐interacting serine/threonine‐protein kinase 1/3; TRADD, TNFR1‐associated death domain protein; TREM1, Triggering Receptor Expressed on Myeloid cells 1; ZBP1, Z‐DNA Binding Protein 1.

Intriguingly, expression of PANoptosis‐regulatory genes demonstrates obvious heterogeneity across diverse malignant neoplasms. Previous research has found that CASP‐8 expression is significantly elevated in glioblastoma, pancreatic carcinoma, and hepatocellular carcinoma; however, it is comparatively decreased in small cell lung carcinoma, prostate adenocarcinoma, and renal cell carcinoma [29, 30, 31]. Given its superior capacity for real‐time monitoring of cell death pathways in vivo, CASP‐8 reflects inflammatory syndromes with the help of CASP‐8‐activated bioluminescence probe Ac‐Ile‐Glu‐Thr‐Asp‐(D)‐Aminoluciferin (Ac‐IETD‐Amluc) [32]. Expression patterns of other PANoptosis genes also exhibit significant heterogeneity throughout distinct stages of tumorigenesis and progression. Researchers stratified prostate cancer specimens into distinct stages of tumor progression and revealed elevated RIPK3 expression in early‐stage prostate carcinoma, which significantly decreased in advanced stages [33]. Furthermore, APO‐positive epithelial cells in hepatocellular carcinoma (HCC) are associated with significant clinical outcomes, including clinical grades, prognosis and immunotherapy response [34]. This observation suggests that necroptosis may be initiated during tumorigenesis, while cancer cells progressively develop resistance as the disease advances. PANoptosis has been detected to have several therapeutic benefits, including immunological activation, reversal of drug resistance, and synergistic cytotoxicity in cancer [35, 36, 37].

3. Detection Methods for PANoptosis

Functional indicators, molecular markers, and cellular morphology are among the detection approaches that address the multi‐pathway crosstalk features of PANoptosis [38] (Table 1). PANoptotic cells combine the distinctive morphological characteristics of necrosis, pyroptosis, and apoptosis [39]. Under high‐resolution micrographs, apoptosis displays early‐stage nuclear condensation and chromatin margination. To distinguish this from necroptosis and pyroptosis, plasma membrane integrity assays should be performed with constructive analysis [40]. While phosphatidylserine externalization at the plasma membrane indicates apoptotic signaling, the rapid, progressive loss of membrane integrity, measured by real‐time dye‐exclusion assays, serves as a functional readout of gasdermin‐mediated pyroptosis pore formation and MLKL‐driven necroptosis membrane rupture [41].

TABLE 1.

Detection indicator and methods for PANoptosis.

Programmed cell death	Molecular markers		Cellular Functional indicators	Cellular morphology
Apoptosis	ZBP1, NLRP3, ASC, FADD, NLRP12, AIM2	CASP3/8/9, IRF1, TRADD, MAP3K7, Bcl‐2 family, cytochrome c	Annexin V‐FITC staining (+); JC‐1/TMRM/TMRE for mitochondrial membranes potential↓; Spectrophotometric assay (+).	Cell shrinkage; nuclear condensation; chromatin marginalization; apoptotic bodies.
Necroptosis		RIPK1/3, Phosphorylated MLKL	Annexin V‐FITC/PI staining (+); DCFH‐DA/MitoSOX for mitochondrial functions (+); Spectrophotometric assay (+).	Cell swelling; plasma membrane rupture; organelle swelling; severe cytoplasmic vacuolization.
Pyroptosis		CASP1/6/8, GSDMD, TREM1, AIM2	Annexin V‐FITC/PI staining (+); JC‐1/TMRM/TMRE ↓; DCFH‐DA/MitoSOX(+).	Cell swelling/expansion; large plasma membrane pores; intact nucleus with minimal condensation
PANoptosis		PANoptosome complex, including AIM2, RIPK1, RIPK3, CASP1, CASP8, etc.	Annexin V‐FITC/PI staining (+); JC‐1/TMRM/TMRE ↓; DCFH‐DA/MitoSOX(+) Spectrophotometric assay (+); Explosive cytokine release	Mixed/hybrid phenotype: concurrent apoptotic blebbing and lytic membrane perforation

Synthesizing these diverse cellular changes remains a primary challenge, as organelle‐level characteristics of PANoptosis include premature opening of mitochondrial permeability transition pores (mPTP) and rapid loss of mitochondrial membrane integrity [42]. Concurrently, PANoptosis cells also exhibit hallmarks of pyroptotic and necroptotic lytic cascades, such as cellular expansion and the formation of plasma membrane holes, which drive the release of cellular cytokines and the subsequent inflammatory responses. According to electron microscopic results, apoptosis is characterized by cell atrophy, an increased nucleo‐cytoplasmic ratio, and chromatin agglutination. In contrast, pyroptosis and necroptosis exert a framework marked by total cellular and organelle swelling, loss of ribosomes, and severe cytoplasmic vacuolization. At the ultrastructural level, PANoptotic cells exhibit morphological traits at the ultrastructural level, simultaneously presenting features of three pathways [43]. The morphological features of PANoptosis vary dynamically according to stimulus specificity and cell type, making morphological identification challenging. Therefore, an integrated approach combining ultrastructural observations with rigorous molecular marker and functional pathway validation is necessary for a thorough evaluation of PANoptosis.

Detecting molecular biomarkers of PANoptosis requires careful analysis of key proteins implicated in all three PCD pathways. One to three biomarkers from each pathway must be selected, and markers from all three pathways should be included [44]. In the apoptotic process, CASP activation is a crucial detection criterion, particularly for CASP‐3, CASP‐8, and CASP‐9, which can be quantified by Western blotting, flow cytometry, or immunofluorescence [45]. At the same time, indicators of mitochondrial function, including Bcl‐2 family expression and cytochrome c release, are also crucial detection signs [46]. Within the pyroptotic pathway, the cleavage and oligomerization of gasdermin family proteins, especially GSDMD, and inflammasome assembly by crucial biomarkers, including NLRP3, ASC, and CASP‐1, are critical processes detected by immunoblotting of cleaved fragments [47, 48]. For the necroptotic cascade, analysis focuses on the phosphorylation and activation status of the necrosome core, including p‐/t‐RIPK1, p‐/t‐RIPK3, and p‐/t‐MLKL, which can be examined using phosphorylation‐specific antibodies to track their oligomeric assembly [49].

Beyond the isolated molecular targets, organelle dysfunction is another early indicator of PANoptosis [37]. The instability of mitochondrial membrane potential can be quantitatively detected using fluorescent probes such as JC‐1, TMRM, or TMRE [50]. Additionally, activation of mPTP can be quantitatively assessed by calcein‐cobalt quenching tests or cytochrome c release assays [39]. Mitochondrial formation of reactive oxygen species (ROS) can be detected through fluorescent probes such as DCFH‐DA or MitoSOX [51]. At the endoplasmic reticulum (ER) level, induction of the unfolded protein response (UPR), manifesting with the activation of the PERK, IRE1α, and ATF6 signaling axis, serves as a significant indicator of ER stress [52]. Furthermore, lysosome‐selective dyes can monitor alterations in lysosomal function, whereas lysosomal enzyme release, particularly cathepsin B and L, is inferred from enzymatic activity using fluorogenic substrates or immunostaining techniques. Finally, changes in cellular membrane permeability can be detected using membrane‐impermeant dyes such as Annexin‐V, propidium iodide (PI), or Oxazole Yellow‐Pro‐1 (YO‐PRO‐1) [53, 54].

RNA sequencing has provided new perspectives for the research of PANoptosis due to whole‐transcriptome coverage, high‐throughput capabilities, and high sensitivity. Single‐cell RNA sequencing (scRNA‐seq) offered significant advantages to identify vital PANoptosis regulatory genes and analyze PCD modes among different cellular subpopulations within the microenvironment. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) activates PANoptosis, leading to podocyte damage in diabetic kidney disease (DKD) [55]. Proteomics enables the simultaneous detection of expression and post‐translational modification changes in thousands of intracellular proteins; it can be integrated with other omics data (such as transcriptomics and metabolomics), providing a comprehensive perspective for elucidating the regulatory network of PANoptosis. Proteomic analysis revealed that Y‐box binding protein 1 (YBX1) elevated in neurons following spinal cord injury (SCI), mechanistically promotes ZBP1 expression by stabilizing its mRNA, thereby exacerbating ZBP1‐mediated PANoptosis [56].

4. PANoptosis in Cancer: From Immunosuppression to Immunostimulation

4.1. PANoptosis as an Immunogenic Cell Death

PANoptosis pattern in tumor cells can stimulate innate immune pathways and predict anti‐tumor immunotherapy response. Before being identified as a structural factor in PANoptosis, ZBP1 was recognized as part of the DNA‐dependent activator of type‐I interferon (IFN), promoting the release of pro‐inflammatory cytokines, CD8+ T cell infiltration, and effector T cell response [57]. In CRC, PANoptosis was a highly immunogenic form of cell death, driven by mitochondrial dysfunction. In colon tumor cells, calcium overload, mitochondrial membrane dissipation, and mPTP opening trigger mitochondrial oxidative stress and energy metabolic disorders [58]. The cGAS/STING pathway is a key facilitator of innate immune signaling, mediating DNA recognition, ZBP‐1 PANoptosome formation, and inflammatory cell death. As an immunogenic cell death, PANoptosis via the cGAS/STING pathway contributes to immune activation against primary tumor development and malignancy. Specifically, the STING pathway activated by SAMHD1 deficiency mediates MLKL phosphorylation, caspase‐3 cleavage, GSDME cleavage, and PANoptosis via the CASP8/RIPK3/ASC complex [59].

4.2. PANoptosis Pattern as an Immunosuppression Signature

As a host strategy to induce spontaneous inflammatory cell death, PANoptosis affects immune and tumor cells separately, characterizing TME, which serves as an immunosuppressive breeding ground for tumor metastasis and proliferation. PANoptosis‐related signatures, such as AIM2, NLRP3, and CASP5, play a dual role in immunogenicity and immunosupression in tumor microenvironment. The immunosuppressive phenotype in renal clear cell carcinoma could be identified in the PANs model, mediated by downregulated CD4 + T cells and natural killer cells, but increased M2 macrophages and regulatory T cells [60]. As prognostic predictors in LUAD, PANoptosis scores can independently reflect the suppressed TIME, limited lymphocyte infiltration, and resistance to chemotherapy and immunotherapy [61]. This is consistent with the molecular mechanism. In gastric cancer, the immune‐inflammatory microenvironment was defined by the overexpression of PANoptosis‐related genes. Lower PANs scores indicated higher sensitivity to immunotherapy in clinical models [62].

PANoptosis molecules also present an immunosuppressive mechanism in cancers. Within the PANoptosis‐related gene risk model, the high‐risk group exhibited elevated immune checkpoint scores, highlighting its robust capacity for immune evasion in ccRCC. CASP5 is upregulated in ccRCC tissue, contributing to the model as the predominant molecular mechanism [63]. CASP2 also remodels the immune microenvironment as a PANoptosis‐related modulator during HCC progression [64]. In NSCLC, HMGB1‐positive tumor‐associated neutrophil subgroups are prone to PANoptosis and tumor lesion, contributing to immune evasion and serving as a pro‐tumoral signature via the GATA2/HMGB1/TIM3 axis [65]. Finally, at the transcriptional level, PANoptosis‐related LncRNAs, including AC133552.2 and AC034229.4, also play an immunosuppressive role in the tumor progression model [66].

4.3. Mechanisms of Immune Cell‐Mediated PANoptosis in Cancer Cells

As a microscopic insight into the tumor microenvironment, the collaborative innate‐adaptive immune response to PANoptosis is worth exploring. The T cells‐mediated anti‐tumor immunity is stimulated by the recognition of tumor‐derived antigenic peptides‐MHC complexby TCR [67]. Antigen presentation activates T cells to differentiate into cytotoxic T cells (CTLs) and helper T cells (Th cells) [68]. The Th cells combat tumor through cytokine secretions. CTLs eliminate tumor cells through releasing perforin and granzymes, or express death receptor pathways, such as Fas‐Fas ligand (FasL) and TRAIL‐death receptor signalings, which trigger PCD of tumor cells such as PANoptosis [69].

A pan‐cancer analysis revealed that the PANoptosis gene expression pattern correlates with the infiltration of various immune cells, especially NK cells, which can release inflammatory cytokines, such as IFNγ and TNFα [70]. It has been demonstrated that local intratumoral TNFα and IFNγ suppress tumor growth and restore sensitivity to PD‐1 blockade. Mechanistically, we assemble inflammatory cytokines that contribute to PANoptosome assembly and PANoptosis activation. Additionally, oncolytic viruses can enhance the expression of IFN‐stimulated genes, subsequently activating PANoptosis [71]. Another IFN‐stimulated signaling pathway, including IRF1, IRF3, and STING, can also stimulate inflammation in tumors, mediating PANoptosis in tumor cells and subsequently enhancing the efficacy of immunotherapy [72]. Previous studies revealed that immunotherapy resistance has been connected to abnormalities in TNF signaling [73, 74]. Notably, dysregulation of TNF signaling, such as blocking TBK1, also restores sensitivity to immune checkpoint inhibitors (ICIs), inducing tumor cell death that is dependent on both RIPK1 and CASP [75].

4.4. PANoptosis as Tumor Immune Evasion Mechanism

Emerging evidence indicates that PANoptosis can promote immune evasion, even in microenvironments characterized by active immune cell infiltration. As a pivotal component of PANoptosis machinery, CASP5 functions as an oncogenic driver in cutaneous clear cell squamous cell carcinoma (ccSCC), promoting tumor cell growth, migration, and invasion while facilitating their immune evasion. Instead of undergoing silent, noninflammatory apoptosis, CASP5‐overexpressing tumor cells leverage PANoptosis to sustain a pro‐tumorigenic inflammatory microenvironment [64]. Tumor DNA–RNA hybrids or dsRNA can activate PANoptosome assembly to establish a chronic inflammatory loop. Tumor‐associated macrophages can activate the local inflammasome; this interaction not only enhances the invasive capacity of malignant cells, but also drives progressive cell membrane perforation and rupture. The resulting lytic event culminates in the massive release of IL‐1β, IL‐18, and DAMPs [76]. AIM2 inflammasome and pro‐inflammatory cytokines inherently promote tumor growth and may be a tumorigenic pathway in the tumor microenvironment, contributing to immunosuppression [65]. Additionally, as a key regulator of MHC‐I antigen presentation, NLRC5 co‐occurs with T cell exhaustion, enhanced immune checkpoint upregulation, and poor prognosis in ESCC. The enrichment of PANoptosis‐related transcriptional programs in NLRC5‐high tumors indicates immunosuppressive mechanisms in TME [77].

4.5. PANoptosis: A Potential Role in Immune Remodeling

Accumulating reports indicate that PANoptosis remodels the tumor microenvironment towards either immunogenicity or immune evasion in a context‐dependent manner. Specifically, as an immunostimulatory remodeling mechanism, Platycodon D‐induced, ROS‐mediated PANoptosis promotes DC maturation, triggering the release of ATP and HMGB1, and suppresses tumor growth in lung cancer. Conversely, PANoptosis can act as a pro‐tumoral, immunosuppressive signature [78]. In nonsmall cell lung cancer (NSCLC), tumor‐associated neutrophils promote PANoptosis coupled with HMGB1 overexpression, contributing to immune evasion and serving as a pro‐tumoral signature via the GATA2/HMGB1/TIM3 axis [65]. Inflammasome formation regulates dual efficacy of PANoptosis: anti‐tumoral and pro‐tumoral. As an inflammasome, ZBP1 enhances the anti‐tumor efficacy of PANoptosis, whereas ADAR1 restricts PANoptosis by interacting with the Zα2 domain of ZBP1, thereby promoting tumorigenesis [79].

Oxidative stress signals released by mitochondrial damage and mtRNA are the most potent activators of intracellular pattern recognition receptors, directly inducing the assembly of the PANoptosome and orchestrating a profound tumor immune remodeling. AIM2‐inflammasome‐related PANoptosis can be activated by glycosylation‐related protein NDUFA4L2 inhibition, which remodels the immunosuppressive microenvironment and enhances anti‐tumor efficacy and chemotherapy sensitivity in HNSCC [80]. Overexpression of USP13, a deubiquitinase, reflects CD8+ T cell infiltration blockade and poor prognosis. USP13‐ACLY inhibition, mediated by Spautin‐1, in DLBCL cells promotes PANoptosis by inducing mitochondrial damage, presenting enhanced anti‐tumor efficacy in an immune‐competent model [81].

5. Therapeutic Implications of PANoptosis: Modulating Tumor Responses to Immunotherapy and Combination Therapies

Tumors might readily exploit alternative cellular mechanisms to evade adoptive immune clearance due to the inherent vulnerability of depending on a single cell death pathway. For example, tumor cells can bypass T cell recognition by downregulating specific antigens targeted by T cells or shedding the MHC‐I complex [82]. A prime sample of single‐pathway vulnerability selectively targets T‐cell‐mediated autophagy cargo receptor NBR1, which selectively mediates the lysosomal degradation of MHC‐I molecules in pancreatic ductal adenocarcinoma (PDAC), thus enabling immune evasion [69]. Furthermore, with the emergence of either inherent or acquired resistance mechanisms, the clinical effectiveness of current therapeutic strategies remains limited in patients [82]. Immune‐induced PANoptosis provides a promising strategy to overcome immune evasion. The development of PANoptosis has been reported to advance in cancer therapies, involving chemotherapy, immunotherapy, and the emerging field of regenerative medicine [83].

5.1. Induction of PANoptosis by Conventional Therapies

Targeting upstream regulatory molecules of PANoptosis significantly enhances chemosensitivity. Emerging evidence indicates that the efficacy of chemotherapy by DNA‐damaging agents is regulated by PANoptosis, as DNA lesion molecules upstream signal to trigger PANoptosome assembly. Specifically, CASP8, as a PANoptosis signature, can trigger resistance to commonly used standard chemotherapies, including ionizing radiation and Temozolomide, but increases sensitivity to ionizing radiation via interacting with PARP inhibition in glioblastoma (GBM) [84]. The bioactive small molecule, bavachinin (BVC), also activates DNA damage accompanied by ROS production, mitochondrial membrane potential collapse as a component of PANoptosis, exerting anti‐tumor effects in endothelial cancer [85]. Pyridostatin derivatives and anthraquinone derivatives share PANoptosis‐related mechanisms with classic cisplatin to ameliorate tumor progression [86].

Recently, an ultrasmall Bi2Sn2O7 nanozyme with ultrasound‐magnified multienzyme‐mimicking properties was designed for the inhibition of hepatocellular carcinoma therapy and lung metastasis. The engineered BSO nanozyme regulates mitochondrial function in tumor cells and enhances accumulation of toxic ROS, therefore triggering the activation of PANoptosis [87]. The potential of oncolytic viruses (OVs) to induce PANoptosis in tumor cells and enhance the effectiveness of T‐cell‐based immunotherapies has been explored by recent studies [71]. Fusobacterium nucleatum outer membrane vesicles (Fn‐OMVs) that promote the expression of PANoptosis effect proteins were discovered by Wang et al. [88]. Through activation of the ZBP1 sensor and release of tumor antigens or inflammatory factors, including HMGB1 and IL‐1β, Fn‐OMVs cause PANoptosis and facilitate CTL infiltration. Nanoparticles have also garnered significant attention in recent years as a strategy for inducing PANoptosis [89]. Nano/genetically engineered extracellular vesicles that can cause highly immunogenic PANoptosis in tumor cells were developed recently [90]. Through the repeated release of DAMP, this process triggers the cancer‐immunity cycle and sets off a variety of antigen‐specific T cell responses [8].

5.2. Sensitizing Tumors to Immunotherapy and Combination Therapy

The development of immunotherapies in clinical practice can be combined with a PANoptosis activator, resulting in improved tumor response [91]. Recent studies demonstrated that the STING agonist DMXAA, functioning as a potent immune agonist, efficiently induces PANoptosis in diffuse large B‐cell lymphoma (DLBCL) by promoting the assembly of the CASP8/RIPK3/ASC complex [92]. IFN‐γ and TNF‐αproduced by immune cells have been demonstrated to induce PANoptosis in animal models, and the capacity of PANoptosis induction in tumor cells is a key intrinsic factor determining their response to immunotherapy [93]. Studies in DNA mismatch repair proficient (pMMR) tumors revealed that combinatorial treatment with IFN‐γ and TNF‐α induces PANoptosis in DNA mismatch repair deficient (dMMR) tumor cells, while DNA mismatch repair proficient (pMMR) tumor cells exhibit defective PANoptosis pathways and resistance to the combinatorial IFN‐γ and TNF‐α therapy [73]. Conversely, through functionally deactivating the cGAS‐STING‐TBK1‐IRF3 signaling axis, mutant p53 (mtp53) can disrupt the cytoplasmic DNA sensing and shift cytokine production patterns, thereby inducing immune evasion [91].

A nanoformulation containing FeS and GSDMD has been developed to induce PANoptosis and ferroptosis for cancer immunotherapy activation [94]. SAMHD1 triggered DNA damage and activated STING‐dependent PANoptosis to regulate innate immune cells and promote the efficacy of PD‐1 inhibitors in Diffuse Large B‐cell Lymphoma (DLBCL) [95]. Cinobufagin, identified as a PANoptosis activator, increases immune cell infiltration and sensitivity to immunotherapy, thereby transforming tumor‐associated macrophage (TAM) polarization towards an M1‐like phenotype and the infiltration of effective T cells in GBM [37]. Consequently, PANoptosis provides a mechanism to counteract tumor immune evasion and establishes positive feedback for immune activation in TME, suggesting a promising therapeutic strategy worthy of further exploration.

Furthermore, chemotherapy‐induced DNA damage can reshape immune cell responses by triggering PANoptosis within the tumor microenvironment. The potent anticancer agent CBL0137 exerts its cytotoxic effects by modifying DNA structure, thereby inducing ZBP1‐dependent PANoptosis in macrophages. This effect is observed both when CBL0137 is used as a monotherapy and in combination with LPS [66]. DNASE1L3 facilitates the generation of dsDNA breaks during sorafenib‐induced HCC, subsequently inducing PANoptosis [49]. As a rate‐limiting enzyme in iron–sulfur (Fe‐S) cluster biogenesis, high expression of NFS1 was found to prevent PANoptosis in tumor tissues and was correlated with poor survival and hyposensitivity to chemotherapy in patients with CRC [95]. As a subunit of the respiratory chain complex I, NDUFA4L2 also inhibits PANoptosis associated with the poor progression and low sensitivity to chemotherapy in HNSCC patients [83]. The blockade of cell death signals rendered by βIII‐tubulin silencing can increase chemotherapy sensitivity in PDAC cells [96].

The interaction between PANoptosis and ferroptosis revolves around reactive oxygen species (ROS). The ROS‐induced PANoptosis is influenced by ferroptosis and cuproptosis, offering a potential co‐regulation therapy [44]. Photodynamic therapeutic strategies, including the ROS‐producing photosensitizers and newly developed long‐acting liposomal photosensitizers, drive immunogenic ferroptosis and PANoptosis in cancer cells via targeting mitochondria [97]. ROS generation efficiency is also promoted with ultrasound excitation. Ultrasonic excitation also enhances the efficiency of ROS generation using BiF₃@BiOI@Pt‐PVP nanocomposites [98]. Nanoformulation containing metal sulfide of FeS and GSDMD plasmid (NPFeS/GD) also induces immunogenicity and rescues immunosuppression through dual activation of ferroptosis and PANoptosis [94]. Under hypoxia, DHODH also plays a dual role in the ferroptosis and PANoptosis pathways, maintaining the mitochondrial quantity and promoting the selection of mitochondria favorable to cell survival [99]. In lung, pancreatic, and other cancers, DHODH has been found to regulate radioresistance and other forms of resistance [100].

6. Future Perspectives

While triggering PANoptosis in immunotherapy and combination therapy has demonstrated remarkable anti‐tumor performance, severe immune evasion and the highly immunosuppressive TME remain major barriers to their clinical translation. The immunoregulatory features of PANoptosis heavily influence both inflammatory tumor cells and active immune cells, thereby driving immunogenic cell death and robust immune response. Intriguingly, metabolic rewiring, especially glycolysis, stands out as an underlying determinant of PANoptosis sensitivity in malignancy. Consequently, a pivotal challenge in current therapeutic strategies lies in preferentially activating PANoptosis rather than other alternative cell death pathways. To achieve this, a critical next step is to delineate the precise upstream targets governing PANoptosome assembly. Exploiting key components of PANoptosome, such as ZBP1, AIM2, and RIPK1, offers a promising avenue for overcoming immune tolerance and enhancing therapeutic outcomes.

The impact of PANoptosis on the TME is bidirectional, capable of both stimulating anti‐tumor immunity and facilitating tumor progression. This paradoxical outcome is largely orchestrated by Caspase‐8 (CASP8) [31]. As a core structural and functional component of PANoptosome assembly, CASP‐8 exerts a context‐dependent, dual role in cancer, acting as a canonical tumor suppressor and a noncanonical tumor promoter. On the one hand, activated CASP8 inhibits tumor cell viability and growth through extrinsic apoptosis. To limit the progression of DLBCL, CASP8 is activated by STING overexpression, promoting the formation of the RIPK3‐ASC complex, contributing to PANoptosis therapy and inhibiting tumor growth [101]. The CASP8‐targeting protein c‐FLIPs in the caspase‐8/c‐FLIP(L) heterodimer has been reported to induce apoptosis, especially enhancing tumor cell death in pancreatic cancer [102]. Pro‐tumor inflammation caused by CASP‐8 depends on STING‐activated necroptosis. Conditional deletion of CASP8 in SCLC leads to tumor cell necroptosis rather than apoptosis, thereby inducing pro‐tumor inflammation and immunosuppression. Whereas CASP8‐mediated apoptosis is the major mechanism of cell regulation in cancer, CASP8 is exploited by cancer cells to override the p53‐dependent G2/M cell‐cycle checkpoint in some tumors, shifting the balance from apoptosis to proliferation/cell cycle progression [103]. In various tumors, including glioblastoma (GBM), CASP8 is generated from pro‐CASP8, which is upregulated and localized to the nucleus, correlated with treatment resistance and poor clinical outcome. Src‐dependent phosphorylation of CASP8 on Tyrosine blocks pro‐CASP8 maturation, therefore inhibiting apoptosis in tumor cells while simultaneously promoting energy metabolism [30]. In several tumors, phosphorylation events strengthen the interaction of p62 and KEAP1 and activate MTORC1, sustaining NRF2 signaling and modulating cancer cell metabolism in glioblastoma [104]. Together, the phosphorylation and metabolic reprogramming strictly determine the dual fate of CASP‐8.

Multiple programmed cell death pathways can be intricately interconnected within TME. Future PANoptosis‐ferroptosis combinatorial strategies should amplify ROS‐driven TME remodeling via nanoplatforms [105]. The goal of ROS‐targeted strategies is mainly the disruption of mitochondrial hemostasis, simultaneously fueling PANoptosome assembly and lipid peroxidation in TME. The delivery systems carrying specific plasmids can activate PANoptosis and focus solubility on tumor cell ferroptosis, dismantling the lipid‐driven immunosuppressive barriers. The immunosuppressive TME can be reversed by PANoptosis and the ferroptosis stress mechanism. Furthermore, PANoptosis‐ferroptosis combinatorial strategies identify the mitochondrial enzyme DHODH as a critical therapeutic target to prevent cancer cells from selecting survival‐favorable mitochondria.

Recent research mainly focuses on identifying PANoptosis‐related phenotypes and predicting cancer prognosis and treatment response through bioinformatics analysis. Given the immune remodeling character in PANoptosis, future studies must shift towards observation in large tumor populations and robust validation through in vivo and in vitro studies [106, 107]. Furthermore, Nanotechnology presents an innovative frontier for PANoptosis therapy strategies [65]. Leveraging the inherent characteristics of the TME, such as hypoxia, low pH, and altered redox reactions, nanoplatforms can be engineered to precisely deliver PANoptosis inducers into tumor cells and remodel the TME, thus reactivating potent anti‐tumor immune responses [108, 109].

7. Conclusions

As a newly recognized form of PCD, PANoptosis has emerged as a potential therapeutic target in oncology. Advancements in novel technologies are facilitating its translation of PANoptosis‐based mechanisms into strategies that may enhance current anti‐tumor therapies. Further exploration of multi‐targeted cell death programs holds great potential to overcome current therapy resistance and provide precise, personalized cancer treatment.

Author Contributions

All authors contributed to various aspects of this study. F.L. performed the literature review, prepared the figures, and drafted the manuscript. Z.L. and X.Z. assisted with literature collection and preparation of the initial draft. J.L. and Q.T. designed the review topic and framework. Q.T. supervised the review and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from Capital's Funds for Health Improvement and Research (2024‐1‐4065).

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

No data were used for this review.