mtDNA is typically sequestered within the mitochondrial matrix and enclosed by mitochondrial nucleoids (12). Under stress conditions, however, it can be released from damaged mitochondria into the cytoplasm and extracellular space, serving as a key immunostimulatory signal. This release is triggered by various stressors, including oxidative stress, viral infection, and drug toxicity (9), that activate distinct but often overlapping mechanisms. These mechanisms can be classified into four major pathways based on the underlying cellular process: (i) Membrane permeabilization pathways represent primary routes for mtDNA release. Mitochondrial outer membrane permeabilization (MOMP), mediated by BAX/BAK-dependent membrane depolarization (13, 14) and promoted by PGAM5-mediated Bax translocation (15), is prominently induced by severe oxidative stress, viral infection, and apoptosis-inducing drugs. Alternatively, mitochondrial inner membrane permeability transition (MIMP) can independently mediate mtDNA release through progressive widening of outer membrane pores coupled with increased inner membrane permeability, a process particularly associated with calcium overload and metabolic toxicity (13, 16). (ii) Vesicular transport provides a membrane-preserving mechanism for mtDNA export. Mitochondrial-derived vesicles (MDVs) selectively transport mtDNA to the cytoplasm, serving as an alternative release pathway activated by metabolic stress conditions (e.g., fumarate accumulation) and during viral infection (16). (iii) Quality control failure leads to catastrophic mtDNA release. Defective mitophagy causes accumulation of damaged mitochondria, resulting in spontaneous mtDNA release through organelle rupture under conditions of prolonged stress, aging, and drug toxicity (17). (iv) Direct molecular destabilization of the mtDNA-nucleoid complex facilitates leakage. Downregulation of mitochondrial transcription factor A (TFAM), triggered by nutrient stress and viral infection, impairs mtDNA stability and promotes its release (7, 18). Overexpression of Drp1 induces mitochondrial dysfunction and stress-induced leakage, particularly under bioenergetic stress conditions (19). Additionally, excessive oxygen species (ROS) production, a hallmark of oxidative stress, oxidizes mtDNA and directly facilitates its translocation into the cytoplasm and extracellular environment (20, 21).

The cGAS- STING pathway is a core component of the innate immune system that specifically recognizes cytoplasmic dsDNA to initiate host immune responses (22). This pathway plays a crucial role in tumor immunity, antiviral responses, and autoimmune diseases (23). mtDNA in the cytosol is specifically recognized by cGAS, an enzyme that possesses unique structural features as a dsDNA sensor (24). cGAS forms a complex with negatively charged mtDNA via its positively charged DNA-binding domain; this binding induces a conformational change in cGAS and exposes its catalytic pocket (25). Experimental evidence has shown that either digestion of mtDNA by DNase I or reduction of cytosolic mtDNA using ethidium bromide (EtBr) can significantly inhibit cGAS activation (7, 26, 27). Notably, oxidatively modified mtDNA exhibits stronger cGAS-binding capacity, suggesting that oxidative stress may amplify this signaling pathway (21, 28).

Upon activation, cGAS produces the second messenger cyclic GMP-AMP (cGAMP), which binds to endoplasmic reticulum (ER)-localized STING protein and induces its conformational change (29). Activated STING translocates to the Golgi apparatus region, where it recruits and phosphorylates TANK-binding kinase 1 (TBK1) (25). Subsequently, TBK1 phosphorylates the transcription factor IRF3, promoting the dimerization of IRF3 and its translocation to the nucleus (30, 31). This cascade reaction can be affected by multiple regulatory mechanisms: epigenetic silencing of STING blocks signal transduction (5), while overexpression of mitochondrial TFAM reduces STING activation by stabilizing mtDNA (32, 33). Super-resolution imaging technology has confirmed a clear spatiotemporal correlation between mtDNA release and STING activation (13).

After translocating into the nucleus, activated IRF3 initiates the transcription of type I interferon (IFN-α/β) genes (25). These interferons activate the JAK-STAT pathway through autocrine and paracrine effects, inducing the expression of hundreds of interferon-stimulated genes (ISGs) (5, 34, 35). Overall, upon binding cytosolic mtDNA, cGAS synthesizes the second messenger 2’3’-cGAMP, activating STING on the ER membrane. STING then recruits TBK1 and IRF3, triggering IRF3 phosphorylation and nuclear translocation to drive IFN-I transcription. IFN-I is a pleiotropic cytokine with antiviral, antiproliferative, and immunomodulatory functions. In tumors, IFN-I can activate anti-tumor immune responses by recruiting natural killer cells and expanding CD4+ and CD8+ T cells; conversely, mild and persistent IFN-I signaling can lead to immunosuppression by inducing mediators involved in T cell exhaustion, such as PD-L1, IDO, and IL-10 (36). Studies have shown that STING-deficient mice exhibit a significantly attenuated inflammatory response (37, 38), whereas the introduction of exogenous mtDNA can restore IFN-β production (39, 40). Notably, this pathway can also synergistically activate the NLRP3 inflammasome, facilitating the release of pro-inflammatory cytokines such as IL-1β (21, 41).

NK cells are cytotoxic lymphocytes with the ability to kill tumor cells and secrete pro-inflammatory cytokines (42). Owing to their role in tumor suppression, NK cells play a critical function in tumor immune surveillance, particularly in preventing tumor metastasis (43). The mtDNA-cGAS-STING pathway activates cytotoxic immune cells through multiple mechanisms. In NK cells, mtDNA released by tumor cells partially triggers intrinsic STING activation in NK cells via recognition by cGAS, thereby maintaining the antitumor activity of the TCF-1+ NK cell subset (a subset with long-term memory potential) (8).

T cells exhibit multiple mechanisms of action in tumor therapy and provide robust support for tumor treatment through various approaches, including directly killing tumor cells, regulating immune responses, enhancing immune memory, and participating in immune checkpoint inhibition (44, 45). In particular, CD8+ T cells can directly recognize and kill tumor cells: they identify complexes of tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) with MHC class I molecules on the surface of tumor cells via T cell receptors (TCRs). Once recognition occurs, cytotoxic T lymphocytes (CTLs) release cytotoxic granules, such as perforin and granzyme, which can induce tumor cell apoptosis (46). In CD8+ T cells, IFN-I produced after STING pathway activation can promote their proliferation and the expression of cytotoxic granules (47). Experimental evidence shows that direct activation of STING using cyclic GMP-AMP (cGAMP) significantly enhances the IFN-γ production capacity of NK cells; meanwhile, the ferroptosis-induced mtDNA-releasing can effectively recruit CD8+ T cells infiltration into tumors by activating the STING pathway (48). Additionally, the cGAS-STING axis activated by cytoplasmic mtDNA can promote the occurrence of pyroptosis, which in turn activates CD8+ T cells in a paracrine manner (49).

DCs play a crucial role as a link between innate and adaptive immunity in the immune system and serve as important hubs for immune responses. As a bridge connecting innate and adaptive immunity, DCs have attracted significant attention due to their excellent antigen-presenting ability (50). In the process of antitumor immunity, DCs perform an indispensable and vital function; their roles are closely associated with the cancer-immune cycle, ultimately facilitating the elimination of tumor cells by effector T cells (51). Studies have revealed that DCs are key effector cells for the activation of the cGAS-STING pathway (52, 53). Acute STING activation promotes DCs function through two mechanisms: first, it directly induces the upregulated expression of DCs maturation markers (e.g., CD80, CD86); second, it enhances DCs’ antigen-presenting ability, which mainly depends on the autocrine loop of IFN-I triggered by mtDNA (47, 54, 55). Immunogenic cell death (ICD) induced by photothermal therapy can synergize with STING pathway activation to promote the uptake and processing of tumor antigens by DCs, forming a positive feedback loop (55).

NETs are mesh-like structures released by neutrophils, mainly composed of DNA, histones, and related proteases. The release of NETs is usually triggered by reactive ROS, which subsequently activates peptidylarginine deiminase 4 (PAD4), leading to the citrullination of histone 3 (H3) and further causing DNA unwinding and nuclear membrane rupture (56). Immediately after, gasdermin D (GSDMD)-mediated cell perforation further results in the release of DNA, histones, and related proteases, a process termed NETosis (57). During the release of NETs, mtDNA leakage often occurs, and the interaction between mtDNA and the cGAS-STING signaling pathway can sometimes promote the release of NETs (24, 58). In addition, various cell types can capture NETs through phagocytosis by recipient cells; intracellular cGAS recognizes NETs-derived DNA, thereby activating the cGAS-STING signaling pathway and increasing the expression levels of interleukin-6 (IL-6) and IFN-I (59). Most myeloid cells, such as macrophages and DCs, have been reported to recognize NETs-DNA intracellularly (60). Other epithelial-derived cells and tumor cells can also take up NETs and recognize them via intracellular cGAS (61). For example, hepatic tumor cells possess the ability to take up NETs, thereby activating the cGAS-STING pathway and inhibiting tumor migration (62). In summary, the release of NETs can amplify inflammation mediated by the mtDNA-cGAS-STING pathway, while activation of the mtDNA-cGAS-STING pathway can in turn promote the release of NETs—yet the specific mechanisms remain unclear.

Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME. Their plasticity allows them to differentiate into antitumor M1-like phenotypes or protumor M2-like phenotypes. Classically activated M1 macrophages possess potent tumor-killing, phagocytic, and antigen-presenting capacities; however, TAMs in the TME are typically educated into M2-like phenotypes, which assist tumor progression by supporting angiogenesis, promoting tumor proliferation, and establishing an immunosuppressive network (63–65). Therefore, targeting and reprogramming the activation status of TAMs has emerged as a crucial strategy to enhance antitumor efficacy. Recent studies have shown that cytoplasmic mtDNA induces inflammatory cascades by activating the cGAS-STING-IRF3 signaling axis, effectively reshaping macrophage function—this not only inhibits their protumor activities but also significantly enhances their antigen-presenting capacity (66, 67). For instance, when exogenous mtDNA is taken up by TAMs, it can directly drive their conversion from a protumor to an antitumor phenotype, providing a novel intervention target for cancer immunotherapy (68).

The mtDNA-cGAS-STING axis exerts dual roles in regulating the polarization of TAMs. In the hepatocellular carcinoma model, mtDNA induces the polarization of TAMs toward the M2 subtype through the TLR9- nuclear factor κB (NF-κB) signaling, forming an immunosuppressive microenvironment (69, 70); however, whether the cGAS-STING signaling is involved remains to be further studied. There is evidence that cGAS-STING activation induced by mtDNA can activate JAK–STAT3 signaling (71), and this signaling plays an important role in the differentiation of TAMs into the M2 subtype (72–74). Interestingly, oxidatively modified mtDNA can escape from tumor cells and act as an immunogenic damage-associated molecular pattern to induce the polarization of TAMs toward the M1 subtype, thereby reactivating the immune response of macrophages against cancer cells (75, 76). These findings reveal the complex role of mtDNA-cGAS-STING axis in regulating macrophage polarization in shaping the TME.

The mtDNA-cGAS-STING axis critically regulates tumor immune evasion through coordinated interaction with the PD-1/PD-L1 pathway. Activation of this axis promotes IFN-I secretion, which upregulates PD-L1/PD-1 expression and drives T cell exhaustion (77–79). Vesicle-mediated mechanisms further integrate these pathways. Necrotic tumor cells release extracellular vesicles (EVs) enriched in mtDNA and PD-L1 that induce macrophage IFN/IL-6 production to weaken T cell responses while directly triggering T cell apoptosis (80, 81). Similarly, IL-6-induced EVs promote mtDNA leakage in endometrial cancer, an effect reversible by anti-PD-L1 therapy (80). Although the precise role of cGAS-STING activation in these vesicle-mediated processes requires further investigation, these findings establish a strong rationale for combination therapies targeting both the mtDNA-cGAS-STING pathway and immune checkpoints.

The core function of MDSCs is to construct an immunosuppressive microenvironment in tumors, which inhibits anti-tumor immune responses through various mechanisms, while directly or indirectly promoting tumor growth, metastasis, and drug resistance (82). mtDNA released by senescent cells can be packaged in extracellular vesicles, which are selectively transferred to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in TEM. This process enhances the immunosuppressive activity of PMN-MDSCs via the cGAS-STING-NF-κB signaling (83). Additionally, exogenous mtDNA can also activate the STING pathway, which in turn creates an immunosuppressive microenvironment in MDSCs. This ultimately provides favorable conditions for the survival and proliferation of tumor cells (83, 84).