TLRs are Toll-like receptors that play a crucial role in immune-related diseases. They are present on the cell surface (TLR1, TLR2, TLR4, TLR5, and TLR6) or endosomes (TLR3, TLR4, TLR7, TLR8, and TLR9) and act as sensors for various immune cells such as macrophages, monocytes, neutrophils, host cells, eosinophils, dendritic cells (DCs), and T cells (23). TLRs mediate homotypic interactions with the TIR domains of all TLRs except TLR3, by directing the recruitment of MyD88, which triggers the assembly of the “Myddosome”. The interaction is mediated by a homotypic interaction between the death domain (DD) of MyD88 and a DD-DD interaction between MyD88 and IL-1R-associated kinases (IRAK), such as IRAK1, IRAK2, and IRAK4 (25). This recruitment activates TGFβ-activating kinase 1 (TAK1), leading to the activation of mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB, and IFN regulatory factor 5 (IRF5), which secrete pro-inflammatory cytokines (26). The incidence of cancer is increased by MyD88 deficiency, which has a dual effect of reducing the ability to heal ulcers and repair DNA damage (27). TLR3 signaling occurs exclusively through TIR-domain-containing adapters that induce interferon-inducible interferon-β (TRIF) (28). TRIF interacts with TRAF3 to initiate the TANK-binding kinase 1 (TBK1)–IκB kinase ϵ (IKKϵ) axis for IRF3 activation, or with TRAF6 and receptor-interacting protein kinase 1 (RIPK1) to activate NF-κB and MAPK (29). In plasmacytoid dendritic cells (pDC), IRF6 is activated exclusively via the MyD7–IRAK88/1–TRAF4 axis in response to TLR7 stimulation (30).

According to research, CD14/TLR4-MyD88-IRAK-1 signal transduction inhibits TLR8 dependent sensing of E. coli (31). However, Pseudomonas aeruginosa can sense through TLR2, TLR4 and TLR5, and the cofactor CD14 of TLR2 and TLR4 plays a more significant role in combined sensing of most Escherichia coli (32). Tumors release DNA, which causes the accumulation, antigen uptake, and maturation of dendritic cells (DCs) in tumors in a TLR9 dependent manner. These DCs then migrate to the draining lymph nodes and major tumor specific CTLs (33). The activation of innate immune responses through TLR and NLR signaling pathways is a connection between chronic inflammation and cancer (34–36). Numerous studies have established a significant correlation between tumors and TLR sequence polymorphisms, specifically TLR4, TLR1, TLR6, and TLR10 (37). It has been observed that TLR4 inhibits tumor cell proliferation and invasion, while also inducing tumor cell apoptosis (38). Similarly, TLR3 signaling has been found to inhibit tumor cell proliferation and promote cell apoptosis (39). Additionally, TLR9 activation has been shown to sensitize tumor cells to apoptosis, resulting in tumor growth arrest (40). Therefore, TLR plays a crucial role in initiating adaptive immune response by acting as a sensor in innate immune response.

Retinoic acid induced gene I (RIG-I) is a crucial cytoplasmic pattern recognition receptor (PRR) responsible for RNA virus sensing, interferon production, and tumor suppression. Upon viral infection, RIG-I mediates RNA sensing and induces IFN production. Additionally, RIG-I promotes STAT1 activation, which enhances IFN α generation and amplifies the IFN-JAK-STAT signal. Downregulation of RIG-I expression in hepatocellular carcinoma (HCC) tissue is associated with poor patient prognosis and weakened treatment response to IFN α (41). However, RIG-I may amplify its anti-tumor effect by activating signal transduction and transcription 1(STAT1) activators in the IFN-JAK-STAT pathway through autocrine and paracrine pathways (42). The role of TBK1 in Kras-mediated tumor development is significant as it inhibits cell apoptosis, particularly in human lung cancer cell lines, and is dependent on the expression of carcinogenic KRAS. Negative regulators of type I IFN signaling, A20 and CYLD, have been linked to cancer (43). Hence, RIG-I plays a crucial role in tumor recognition, IFN production, and tumor regulation.

NLRs are proteins that generate immune responses to microorganisms and danger signals (44). They mediate dNTPase activity and oligomerization of NLR proteins. NOD2, also known as NLRC2, recognizes viral genomic ssRNA during RSV infection and mediates IRF3-dependent production of type I IFN by MAVS in hematopoietic and non-hematopoietic cells (45). NLRs act as receptors for PAMPs and induce NF-κB signaling, mediating NLRP3 as a linker to form different types of inflammasomes to regulate IL-1β and IL-18 secretion (46). NLRs also sense viral dsRNA in complex with specific DEAH-box RNA helicases, triggering type I IFN and/or inflammasome-dependent antiviral responses. Some NLR members regulate other RNA-sensing pathways, particularly the RLR-MAVS pathway. NLRP6 is expressed highly in intestinal epithelial cells and serves as an antimicrobial defense mechanism. It also plays a crucial role in antiviral immunity mediated by type I and type III IFNs. Recent mass spectrometry analysis of NLRP6 has identified the RNA helicase DHX15 as the NLRP6-DHX15 complex that interacts with long viral dsRNA in EMCV-infected cells (47). This complex is recruited to MAVS to induce transcription of type I and type III IFNs and ISGs, ultimately limiting virus propagation. However, NLRs can also act as negative regulators of innate immune responses, such as inflammation, antiviral immunity, and autophagy (48). NLRP3-dependent IL-18 production is known to have a protective role in colorectal tumorigenesis by inducing IFN-γ production and STAT1 signaling (49). The deficiency of NLRP6 inflammasome has been linked to abnormal inflammation in the colon and colitis-induced tumorigenesis (50). Additionally, it has been found to regulate epithelial cell repair upon injury and maintain a healthy gut microbiota (51). On the other hand, deletion of the NLRC4 inflammasome has been associated with enhanced epithelial cell proliferation and reduced apoptosis (52). As a result, the NLRP6 and NLRC4 inflammasomes have been identified as potential therapeutic targets for inflammation-induced cancers. TLRs also play a crucial role in the perception of tumor occurrence and regulating the damage repair of epithelial cells.

GMP-AMP synthase (cGAS) is the primary cytoplasmic sensor for detecting long and short dsDNAs, along with binding proteins, at both low and high cGAS concentrations (53). Cytoplasmic IFI16 specifically recognizes dsDNA from herpes simplex virus type I (HSV-1), cowpox virus (VV), and ssDNA from human immunodeficiency virus type 1 (HIV-1)-infected CD4⁺ T cells, leading to the production of IFN-I. Following the detection of dsDNA from HSV-1, sarcoma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV), nuclear IFI16 oligomer and cGAS translocate into the cytoplasm. This translocation is facilitated by the generation of CDN messengers, which enable long-distance communication with the effector platform, STING. This, in turn, triggers STING-mediated IFN-I production and/or inflammasome-mediated IL-1β production. Additionally, DEAD-box deconjugase 41 (DDX41) has been identified as a dsDNA sensor during HSV-1 infection and B-type DNA sensor transfection (54). Recognition of cytosolic DNA activates the interferon gene-stimulating factor (STING)-mediated IFN-I production. This process occurs through two signaling pathways: IKKα/β-NF-κB and TBK1-IRF3/7 (55, 56). Type I IFNs then signal through the Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathways. This signaling leads to the induction of interferon stimulated genes (ISGs), triggering an immunostimulatory response (57). As a result, pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-1), interleukin-1β (IL-44β), and the type I IFNs themselves (IFN-α, IFN-β, and IFN-γ), are secreted.

The role of cGAS/STING agonists in cancer immunity has been widely acknowledged (58, 59). cGAS is an enzyme that can be activated by the DNA of invading pathogens, resulting in the synthesis of 2′-3′-cyclic GMP-AMP (cGAMP) from GTP and ATP (60). The cGAMP molecule then binds to STING, which subsequently activates TANK-binding kinase 1 (TBK1) and κB kinase inhibitors (IKKs) (61, 62). TBK1 and IKK, in turn, activate the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), respectively. IRF3 and NF-κB induce the production of type I interferon (IFN) and other inflammatory cytokines. Additionally, STING triggers autophagy through a mechanism that is independent of TBK1 and IKK (63). While initially identified as a pattern recognition receptor for invading microbial DNA, cGAS can also be activated by its own DNA in certain cases, leading to autoimmune diseases (64–66). In the context of tumors, DNA from cancer cells passes through the cytoplasm of antigen-presenting cells, activating the cGAS-STING pathway. This activation results in the induction of type I interferon and other immunostimulatory molecules, which promote anti-tumor immunity by activating T cells and natural killer (NK) cells (67, 68).

The balance between positive and negative regulation of cGAS-STING-triggered innate immune responses is influenced by post-translational modifications (PTMs). These modifications, including phosphorylation, ubiquitination, SUMOylation, acetylation, methylation, and glutamylation, can significantly impact the activity and function of cGAS-STING-associated proteins. They play a crucial role in dynamically regulating immune homeostasis (69). As a result, various targeting modes of regulation have been identified, such as C-176, C178, and H-151. These strategies aim to antagonize STING regulation by covalently binding to STING at Cys91 (70). These findings provide a foundation for the development of potential drugs.

Autophagy plays a crucial role in the activation and regulation of both innate and adaptive immune responses (71). p62 belongs to a family of autophagy receptors that are involved in linking ubiquitin and autophagy. It contains a ubiquitin-binding structural domain and an LC3 interaction region. The protein p62/SQSTM1, which is responsible for selective autophagy, is vital for the degradation of STING stimulated by DNA and cGAMP. STING is ubiquitinated through the K63 chain and recruited to the p62-positive compartment. In cells lacking p62, STING is not degraded, resulting in the production of high levels of IFN and IFN-stimulated genes (ISGs). Therefore, p62 is essential for the degradation of STING through autophagosomes following stimulation of the cGAS-STING pathway (72). STING is ubiquitinated and packaged into autophagosomes with the assistance of p62. These autophagosomes are eventually sorted into lysosomes (73). The digestion of cGAS or STING occurs in autophagic lysosomes immediately after the activation of downstream signaling transients (74). ER-Golgi body activation leads to the binding of STING molecules on the intermediate compartment to microtubule-associated protein 1 light chain 3 (MAP1LC3; also known as LC3) on the autophagic membrane, resulting in the degradation of STING and termination of activation signaling. This degradation process also contributes to the destruction of cellular DNA derived from the host or microbes in autolysosomes through enzymatic destruction (75). Autophagy induced by cGAS-STING has been observed after radiotherapy or Mycobacterium tuberculosis infection (76, 77). Interactions between bacterial DNA and STING stimulate resistance to infection by triggering a T helper 17 cell immune response (78). Autophagy acts as a negative feedback loop, ensuring transient cGAS-STING signaling and preventing sustained overactivation of the pathway.

In certain cell types, strong STING activation can induce apoptosis. This pro-apoptotic process is driven by the activation of the mitochondrial B-cell lymphoma 2 homologous structural domain 3 (BH3 only) protein and is observed in T cells, but not in macrophages or dendritic cells (79). Additionally, the BH3-only protein PUMA can also lead to increased necrotic apoptosis, which requires the activation of STING following mtDNA release (80). Furthermore, certain viruses, such as murine gamma herpesvirus, can induce necrotic apoptosis in a TNF- and STING-dependent manner (81).

Necrotic apoptosis is a process that occurs downstream of receptor-interacting protein kinase (RIPK)1 and 3. The pseudokinase mixed-spectrum kinase-like structural domains (MLKL) activate and disrupt the plasma membrane (82, 83). IFN plays a crucial role in regulating the host immune response by binding to receptors and activating the STAT1/2 transcription factors. This activation occurs through various gene families of ISGs (84). DNA from DNA damage repair or mitochondrial stress activates the cGAS-STING pathway, which leads to the production of constitutive IFNs. These IFNs then provide feedback to the cell and help maintain the expression of many interferon-stimulated genes (ISGs). One such ISG is MLKL, which needs to be fully expressed in order to promote oligomerization and cell death (85). Furthermore, the cGAS-STING pathway triggers necrotic apoptosis in primary macrophages when cysteine asparaginase is inhibited upon detection of DNA (86). This cell death response requires STING-dependent production of IFN and TNF, which induce necrotic apoptosis via STING activation. The signaling of these two pathways is reciprocal and synergistic.

Epstein-Barr virus (EBV), human high-risk tumor virus (HPV), Kaposi’s sarcoma-associated herpesvirus (KSHV), hepatitis B virus (HBV), hepatitis C virus (HCV), Merkel cellular polyomavirus (MCPV), and human T-cell lymphotrophic virus type 1 (HTLV) have been identified as the primary pathogens responsible for cancer (87). These viral oncoproteins enable cells to evade immune destruction, maintain proliferation and immortalization, cause mutations and genetic changes, enhance chronic inflammation, and promote metastasis and angiogenesis. Additionally, viral oncoproteins disrupt cellular energy balance (88). Oncolytic viruses can influence cellular gene expression through various mechanisms such as modifying host DNA methylation, triggering chromatin reorganization, expressing virally produced non-coding RNAs, and impacting cellular non-coding RNAomics (89).

Tumor progression is influenced by the local microenvironment, with macrophages being the most abundant component. Both clinical data and preclinical studies in various mouse models of cancer indicate that tumor-associated macrophages (TAMs) play a significant role in promoting cancer. Within primary tumors, TAMs facilitate tumor cell invasion and infiltration, enhance the viability of tumor stem cells, and stimulate angiogenesis. At metastatic sites, macrophages associated with metastasis contribute to the extravasation of tumor cells, support their survival and growth, and in certain cases, maintain tumor cell dormancy. Moreover, TAMs hinder the function of cytotoxic T and natural killer cells, which possess the ability to eliminate tumors. These findings strongly suggest that targeting TAMs could be a crucial approach for therapeutic intervention (90). pDCs have been shown to have anti-tumor effects by secreting more type I-IFN upon Toll-like receptor (TLR) stimulation. Proper activation of pDCs has been found to initiate an effective immune response of T cells against established tumors in vivo (91). In mouse models, pDCs display negative immunomodulatory properties within the tumor microenvironment. This regulatory phenotype is acquired due to the presence of immunosuppressive mediators, such as the expression of the transcription factor Forkhead box O3 (Foxo3) within the tumor. Foxo3 expression leads to defective type I-IFN production, reduced expression of co-stimulatory molecules, and up-regulation of IDO and PD-L1 expression (92, 93). Silencing of Foxo3 in pDCs partially restores their stimulatory function in a mouse tumor model (94, 95). These findings suggest that the tumor microenvironment promotes the expression of Foxo3 in pDCs, leading to the acquisition of a tolerogenic phenotype.

The protein cGAS targets dsDNA derived from viruses and activates downstream interferon signaling. It can also recognize DNA intermediates produced during reverse transcription of the HIV genome, which in turn stimulates downstream STING-TBK1 signaling (96). In cancer, endogenous retroviral elements are often not inhibited, and epigenetic modifications can worsen this situation (97) ( Figure 2 ).

The role of cGAS as an indirect immunosensor against dsRNA. The presence of dsRNA, such as dengue RNA virus, triggers cGAS-STING dependent IFN signaling, leading to activation of the immune system (98). In cancer, chronic STING activation is sensitive to dsRNA response, and tumors expressing ISG upregulate RIG-I (99). However, some oncoviruses, such as HPV18 and human adenovirus 5, have evolved to inhibit the DNA cGAS-STING-IFN perception pathway through their oncoproteins E7 and E1A, respectively. This allows the virus to promote malignant tumors while inhibiting innate immune signaling (100). KSHV and hepatitis B, which are also associated with cancer, have been found to interfere with the cGAS-STING pathway by expressing interferon regulatory factor 1, envelope protein ORF52, and viral polymerase binding, according to studies (101).

The STING pathway is activated by the cyclic dinucleotide ring GMP-AMP (cGAMP) synthesized by cGAS, which senses both self and foreign cytoplasmic DNA (102). STING, an endoplasmic reticulum linker, is activated and translocates to the Golgi apparatus, where it triggers the transcription of IRF-3 and NF-κB, leading to the expression and release of type I interferon. This promotes the recruitment of immune cells, DC maturation, and antigen-specific immune activation (103). The cGAS-STING pathway is involved in detecting cytoplasmic DNA associated with viral infection and tumorigenesis. STING, which is expressed by various immune and non-immune cells, has the ability to detect tumor-derived DNA, making it useful for cancer treatment purposes. In a mouse tumor model, it was discovered that STING-dependent cytoplasmic DNA sensing by tumor resident DC can induce the production of type I-IFN, which is necessary for CD8⁺T cell infiltration and immunogenic tumor rejection (104). The STINGVAX vaccine is made up of a CDN ligand prepared with GM-CSF and has strong anti-tumor activity as a single therapy for various mouse models. It has been proven to upregulate PD-L1 on TME and lead to the combination regression of anti PD-1 monotherapy for drug-resistant tumors (105). STING agonists have been shown to enhance anti-tumor immune response when combined with chemotherapy and radiotherapy in both preclinical and clinical settings (106). However, it is important to consider the increase in solute DNA levels in cancer cells with high chromosomal instability (CIN) which can lead to endogenous cGAS/STING activation. This activation can promote tumor occurrence, immune evasion, and metastasis (107). Therefore, the presence of this phenotype should be taken into account when developing a strategy for incorporating STING agonists. Additionally, the mechanisms of intermittent and continuous STING pathway activation in generating persistent anti-tumor responses require further exploration.

The cGAS-STING pathway plays a crucial role in the anti-tumor T cell response and radiation-induced anti-tumor response (108). Researchers have proposed studying the mechanism of STING signal transduction in DC triggered by DNA derived from tumor cells by examining CD8α DC phagocytosis of apoptotic or necrotic tumor cells (109). This leads to the secretion of type I IFN and subsequent activation of the secretory and paracrine pathways, which drive CD8α/CD103 DC to effectively process antigens and present antigenic peptides on class I molecules of the major histocompatibility complex (MHC) to cytotoxic CD8⁺T cells. This promotes antigenic cross-presentation and T cell initiation and is not affected by TLR or RIG-I/MAVS pathways (110). According to research, insufficient type I-IFN production in glioblastoma hinders T cell immunity (111). Indirect STING activation can be achieved in cancer therapy through cGAS or STING-activated molecules. Anticancer drugs can activate endogenous DNA accumulation in cytoplasm and transfer to DC through exosomes or GAP connections, leading to downstream cGAS-STING signal transduction activation (112, 113). Administration of DMXAA or other STING agonists has shown significant efficacy in treating glioblastoma when given within tumors or throughout the body, alone or in combination (111). Additionally, DNA exonuclease Trex1 was induced at a dose of 12-18Gy to degrade and stimulate the cytoplasmic DNA required for effective Sting-dependent type I-IFN response (114). The cGAS-STING pathway has been found to have a dual effect on tumors, both promoting and inhibiting their growth. This pathway is crucial in adjuvant anti-tumor therapy and the initiation of adaptive immune response.

According to a study, animals that lack STING or IRF3 exhibit T cell initiation defects and are unable to reject immunogenic tumors (115). The study also found that tumor-derived DNA was present in the cytoplasm of tumor infiltrating DC during in vitro analysis. This was associated with the translocation of IRF3 to the nucleus and the expression of IFN-β. Therefore, the study suggests that the host STING pathway is the main innate immune sensing pathway for tumor detection in vivo. The activation of this pathway in the APC in the tumor microenvironment drives subsequent T cell activation against tumor-associated antigens (116). Additionally, the expression of OVA peptides (B16.OVA and EL4.OVA respectively) generate adaptive immune responses to tumor-associated antigens after cryoablation in an IfNar-dependent manner (117). According to the study, CD11c subpopulation is a significant source of type I-IFN after sensing the DNA released by dying cells. The activation of the STING/TBK1/IRF3 pathway controls this mechanism. The innate immunity controlled by STING plays a crucial role in controlling tumorigenesis. In this model, STING gets activated by DNA leakage from the carcinogen damaged nucleus in the dermis, leading to cytokine production, recruitment of infiltrating phagocytes, and driving inflammatory processes that promote tumor development. The ablation of tumors in STING deficient mice indicates that the activation of this pathway is a necessary component of inflammation-induced carcinogenesis (118).

Results from studies using STING agonist cancer models have demonstrated that local administration of STING agonists reduces tumor size and improves survival in mice with melanoma, prostate adenocarcinoma, and glioma (119–122). In melanoma studies, it has been observed that activation of STING in tumor cells and DCs leads to increased infiltration of NK cells in the tumor microenvironment (TME) through the secretion of CXCL10 and CCL5 cytokines. Additionally, the secretion of IL-33 has been found to inhibit tumor growth. It has been reported that minimal activation of CD8+ T cells is accompanied by the migration of NK cells (122). Combination therapies involving STING and anti-PD-1/PD-L1 have shown that STING agonists enhance T cell infiltration and increase PD-L1 expression within the TME (123). Furthermore, these therapies have the potential to promote M2 repolarization to an M1 phenotype and increase NK cell infiltration. The efficacy of this treatment approach has been evaluated in various preclinical studies with differing levels of success in non-GBM cancer models (124).

The cGAS and STING proteins are known to mediate the sensing of both DNA and RNA through RLRs. When ATRX was knocked down in HFF cells, it was found that there was impaired secretion of type I-IFN after activation of both DNA and RNA sensing pathways (125). Additionally, ATRX was found to specifically regulate the expression of certain interferon-stimulated genes (ISGs) induced by type I and type II-IFN. ATRX was found to positively regulate ISG expression by affecting IRF3-mediated production of type I-IFN and modulating type I and type II-IFN signaling. Therefore, ATRX is an important co-regulator of the initial innate immune response and plays a crucial role in both the cGAS-STING-DNA sensing pathway and the RIG-I-dependent RNA sensing pathway (126).

The innate immune defense primarily involves a pro-inflammatory response that is mediated by type I interferon (IFN) and IL-1. Type I-IFN activates stimulator genes (ISGs) that have antiviral or immunomodulatory functions through IFNα/β (IFNAR) receptors. On the other hand, IL-1β of the IL-1 cytokine family binds to homologous IL-1 receptors to induce inflammation during microbial infection. Thus, during microbial infections and autoinflammatory diseases, there is a widespread ‘crosstalk’ between type I-IFN and IL-1 responses, with reported synergistic or negative regulatory circuits (127). The production of type I-IFN and IL-1β relies on the recognition of specific patterns by receptors known as pattern recognition receptors (PRRs). These receptors are able to distinguish between foreign or abnormal molecules and host cell components in specific cellular compartments, which triggers an immune response. Host RNAs can also trigger innate immune responses during viral infections or when they are chronically upregulated, misaligned, or mishandled in disease settings such as autoimmune disorders, which can lead to chronic inflammation. Most RNA sensors that stimulate the immune system (both pathogen and host-derived RNA) are located in endosomes and cytoplasm, although RNA sensing can also occur in the nucleus and mitochondria through PRR-mediated mechanisms. RNA sensing plays a crucial role in regulating polymorphisms in genes and human disease conditions (128). It has been observed that 5S ribosomal RNA pseudogene transcripts (RNA5SP141) associated with KSHV and IAV virus infection can activate RIG-I to detect DNA viruses (129). Additionally, RIG-I activation of RNA polymerase (Pol) III transcripts during DNA virus infection can detect RNA from both viral and host sources (130). During KSHV infection, the virus down-regulates the cellular triphosphatase DUSP11 (double specific phosphatase 11), leading to the accumulation of 5’-triphosphorylated vault RNA, which then activates RIG-I. Moreover, Pol III driven small RNA transcripts from EBVs (EBER RNA) and adenoviruses (VA RNA) can induce type I-IFN responses in a RIG-I-dependent manner. The replication of DNA viruses, stranded RNA viruses, and dsRNA viruses is known to produce long dsRNA (131). These irregular dsRNA activate mRNA associated with the virus L gene and MDA5, which mediates the type I-IFN response (132).

APCs, as the main target cell population for mRNA cancer vaccines, play a crucial role in the immunostimulatory effects of exogenous IVT mRNA. This mRNA is recognized by various cell surface, endosomal, and cytoplasmic PRRs (145). Within endosomes, the recognition of IVT mRNAs is primarily mediated by Toll-like receptors TLR-7 and TLR-8. Subsequently, these receptors activate the MyD88 pathway, leading to the activation of the type I interferon (IFN) pathway and the secretion of pro-inflammatory cytokines (146). In the cytoplasm, other PRR families such as retinoic acid-induced gene I-like (RIG-I-like) receptors, oligoadenylate synthase (OAS) receptors, and RNA-dependent protein kinases (PKR) also sense these exogenous mRNAs. These PRRs detect different types of RNAs, including dsRNA and ssRNA, resulting in the inhibition of mRNA translation. The activation of multiple PRRs and the production of type I-IFNs can have either beneficial or detrimental effects on anticancer immunotherapy. Activation of the type I-IFN pathway drives antigen-presenting cell (APC) activation and maturation, facilitating antigen presentation and triggering a robust adaptive immune response, which is beneficial. However, innate immune sensing of RNA may suppress antigen expression and inhibit immune responses (147). For instance, during in vitro transcription (IVT), phage RNA polymerase can produce unwanted double-stranded RNA (dsRNA), which can activate protein kinase R (PKR) and lead to phosphorylation of eukaryotic initiation factor (eIF)-2. This, in turn, can block mRNA translation and activate innate immunity through oligoadenylate synthetase (OAS), toll-like receptor 3 (TLR-3), and melanoma differentiation-associated protein 5 (MDA-5), a receptor similar to retinoic acid-inducible gene I (RIG-I). Simultaneously, dsRNA binding to OAS activates RNase L (148), which degrades exogenous RNA. Ultimately, dsRNA binding to MDA-5 and TLR-3 activates type I-IFN, which triggers other genes that inhibit mRNA translation (149). Apart from dsRNA impurities, improperly designed mRNA structures can also activate pattern recognition receptors (PRRs) like MDA-5 and PKR, thereby suppressing antigen expression.

The activation of type I-IFNs has paradoxical effects, not only limited to antigen expression, but also observed in CD8⁺T cell activation. The effects of type I-IF on CD8⁺T cell activation can be stimulatory or inhibitory, depending on the timing and kinetics between activation of IFNAR signaling and TCR signaling. These effects may also be influenced by the route of administration of the mRNA cancer vaccine (150). To appropriately activate innate immunity and initiate an adaptive immune response, adjustments are made to the purity of the mRNA product, modification of the mRNA sequence, delivery system, and route of administration. This is done to avoid toxic over-activation that inhibits antigenic protein expression and immune response.

mRNA is a versatile and powerful cancer vaccine platform that significantly enhances our ability to fight against cancer. During vaccination, naked or loaded mRNA vaccines effectively express tumor antigens in antigen-presenting cells (APCs), thereby promoting APC activation and stimulating innate/adaptive immune response. Compared to other conventional vaccine platforms, mRNA cancer vaccines offer advantages such as high efficacy, safe administration, rapid development potential, and cost-effective production. However, the application of mRNA vaccines is limited by issues such as instability, innate immunogenicity, and inefficient in vivo delivery (151). By adjusting the route of administration and co-delivering multiple mRNA vaccines with other immunotherapeutic agents (e.g., checkpoint inhibitors), the host’s anti-tumor immunity can be further enhanced, increasing the likelihood of tumor cell eradication. Nucleic acid (DNA or RNA) based vaccines have shown great promise as a vaccine platform. These vaccines have the advantage of delivering multiple antigens simultaneously, which can target various tumor-associated antigens (TAA) or somatic tumor mutations. This leads to the activation of both humoral and cell-mediated immune responses, increasing the chances of overcoming vaccine resistance. Nucleic acid vaccines can encode full-length tumor antigens, allowing antigen-presenting cells (APCs) to present multiple epitopes of class I and class II patient-specific human leukocyte antigens (HLAs). Consequently, these vaccines are less restricted by human HLA type and are more likely to stimulate a broader T-cell response (152). By synthesizing mRNA neoantigen vaccines in real-time from surgically resected PDAC tumors, it is possible to generate individualized vaccines with a wide range of neoantigens. These vaccines can efficiently activate antigen-presenting cells and can be combined with clinical applications to improve patient prognosis (153, 154).

Acquired cell therapy (ACT) is a prominent form of immunotherapy that involves injecting patients with tumor-infiltrating lymphocytes or immune cells derived from peripheral blood, particularly T cells. Currently, ACT utilizes genetically engineered peripheral blood T cells that are activated in vitro and modified to express antigen receptors like T-cell receptors (TCRs) or antibody-based chimeric antigen receptors (CARs). These modified receptors, which are specific to tumor cells, undergo a brief expansion outside the body before being infused back into the patient (155). Recent advancements in CD19-targeted CAR-T cell-based ACTs have led to the approval of four CAR-T cell products by the U.S. Food and Drug Administration (FDA): Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Tecartus (brexucabtagene autoleucel), and Breyanzi (lisocabtagene maraleucel) (156, 157). Currently, the efficacy of CD4⁺ T cells and their subpopulations in ACT is not as high as that of CD8⁺T cells. Clinical data have shown that over-transplantation of CD19-targeted CAR-T cells at a 1:1 ratio of CD4⁺CAR-T cells to CD8⁺CAR-T cells can increase therapeutic efficacy in adult B-cell acute lymphoblastic leukemia (158). Further studies are needed to understand the function of persistent CD4⁺T cells in the context of over transplantation and to determine if these cells can enhance the persistence and function of CD8⁺CAR-T cells. Strategies should be explored to promote the formation of T_CM, T_SCM, and TPEX cells, which can retain the ability to memorize transformation of effector cells, or to combine with additional agents to facilitate this process. This can help prevent potential tumorigenesis and limit cytokine release syndrome (159, 160). Optimizing the in vivo generation of CAR-T cells holds promise as a less invasive and potentially more cost-effective approach.

Studies have found that CAR-T cell therapy has low clinical efficacy in treating solid tumors. The effectiveness of ACT in fighting tumors is highly dependent on the expansion of permissive metastatic cells, which is hindered by poor immunosuppression, limited persistence and sustained activity in the tumor microenvironment (TME), and dysfunctional or depleted T cell terminal differentiation and function. It is crucial to develop strategies that enhance the self-renewal potential and drive the differentiation of memory or precursor-type T-cell subsets. These strategies will improve CAR-T cell maintenance, promote long-lasting antitumor efficacy, and have a positive impact on patient prognosis. Research has been conducted to determine the applicability of these strategies to patients with different types of solid cancers (161).

In the context of infection, a subset of T cells differentiate into memory precursors that have the potential to give rise to long-term memory cells. CD8⁺T cells expand and differentiate into effector cells that mediate target cell lysis (162). Tumor-resident T cells can acquire a T_RM cells phenotype, which has been associated with increased survival in patients with various cancers, including melanoma, bladder uroepithelial cell carcinoma, non-small cell lung cancer, and breast cancer (163, 164). In humans, cellular memory T cells (T_SCM cells) express the primitive cell markers CD45RA and CXC chemokine receptor 3 (CXCR3) and possess the ability to self-renew and proliferate (165). In comparison to acute infections, CD8⁺ T cells lose their ability to produce effector cytokines like interferon-γ (IFNγ) and tumor necrosis factor (TNF) when responding to progressive cancers and chronic infections. This reduction in cytokine production is accompanied by elevated expression of inhibitory receptors or checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immunoreceptor with immunoglobulin and ITIM structural domain (TIGIT), and 2B4 (also known as CD244), as well as programmed cell death 1 (PD1), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin and mucin structural domain 3 (TIM3). These inhibitory receptors restrict T-cell activation, proliferation, and function, leading to a state of T-cell exhaustion. Studies in metastatic melanoma have demonstrated that complete response and T-cell persistence are associated with tumor-infiltrating lymphocytes that acquire a T_CM-like phenotype after overt metastasis (166). Therefore, it is advantageous to infuse T cells with a less differentiated phenotype prior to infusion, as they have a greater ability to mount an anti-tumor response.