Emerging research highlights the cGAS/STING pathway as a key immunomodulatory signal in various cancers. cGAS/STING activation occurs when damage-associated molecular patterns (DAMPs) are detected in the tumour microenvironment (TME), often induced by chemotherapy or radiotherapy. This activation, though not the therapy’s direct target, results from therapy-induced immunogenic cell death, which releases tumour DNA and mitochondrial DNA, triggering the STING pathway in surrounding immune cells. In genomically unstable cancers (i.e. cancers with a high degree of chromosomal instability), dsDNA released via exosomes can also activate cGAS/STING in immune and tumour cells ( Figure 2 ) (35). This signalling plays a vital role in tumour cell apoptosis, immune cell recruitment, and enhanced antigen presentation. Type 1 interferons, such as IFN-α, further amplify this response by promoting CD8+ T-lymphocyte activity and suppressing regulatory T cells, thereby reducing immunosuppressive cytokines like IL-10 and TGF-β (20, 25, 36).

Additionally, cGAS/STING signalling influences the polarization of tumour-associated macrophages (TAMs), shifting them from an immunosuppressive M2-like phenotype to a pro-inflammatory M1-like phenotype. This shift enhances tumour cell elimination by promoting phagocytosis, tumour antigen presentation, and the production of pro-inflammatory cytokines and chemokines, such as TNF-α, CXCL9, CXCL10, and IL-1 (37–41).

Beyond its impact on immune cell function, cGAS/STING signalling also affects the vasculature of the TME, influencing neo-angiogenesis, vascular integrity, metastasis, and immune cell infiltration, primarily mediated by Interferon Beta (IFN-β) signalling (25, 42, 43). In a murine melanoma model, stromal cells were identified as an integral source of type 1 interferons, especially IFN-β (42). Briefly, IFN-β displayed anti-angiogenic activity in this model and intratumoural administration of a STING agonist resulted in type 1 interferon-dependent upregulation of various genes in surrounding endothelial cells. Notably, the cell adhesion molecules Melanoma Cell Adhesion Molecule (MCAM) and cadherin-5 were upregulated, both of which were associated with CD8+ T-lymphocyte infiltration of the TME (42). Data also indicates that IFN-β may downregulate VEGF expression, thus suppressing neo-angiogenesis. An association has also been drawn between cGAS/STING signalling and restoration of vascular integrity, contingent on type 1 interferon signalling. Vascular normalisation in this context potentiated chemotaxis and transendothelial migration of immune cells with a simultaneous mitigation of tumour cell metastasis (43).

While the cGAS/STING pathway shows promise as a complementary therapeutic target in cancer treatment, it plays a dual role in both tumour elimination and tumorigenesis. This paradox may be due to sustained low-level activation of cGAS/STING, which maintains chronic pro-tumour inflammation (19, 44). For instance high chromosomal instability has been linked to prolonged cGAS/STING activation (25, 45, 46). While persistent cGAS/STING activation promotes chronic inflammation, a hallmark of cancer (19, 47). Chronic inflammation in the TME is also associated with increased invasion, epithelial-to-mesenchymal transition (EMT), and metastatic potential (48). Furthermore, this type of cGAS/STING activation is linked to chronic NF-κB activation, driven by chromosomal instability-induced tumour cell-intrinsic micronuclei containing dsDNA (46).

The cGAS/STING pathway also contributes to tumorigenesis by altering the metabolic profile of the TME. A Lewis lung carcinoma (LLC) study found that STING signalling induced the expression of the immunosuppressive molecule IDO, leading to tryptophan depletion, which supports regulatory T-cells and causes effector T-cell exhaustion. Interestingly, STING ablation enhanced CD8+ T-lymphocyte infiltration, increased anti-tumour activity, reduced myeloid-derived suppressor cell infiltration, and decreased IL-10 production (49). Similarly, in tongue squamous cell carcinoma, STING activation increased IL-10, IDO, and CCL22 levels, promoting regulatory T-cell recruitment and an immunosuppressive TME (50) ( Figure 3 ).

Finally, the cGAS/STING pathway also plays a role in promoting tumorigenesis through non-canonical STING signalling that primarily activates NF-κB rather than IRF3. Briefly, following nuclear DNA damage, STING can be activated independently of cGAS by an ATM/IFI16 complex, leading to NF-κB activation (51). NF-κB drives tumorigenic pathways such as cell survival, proliferation, and chronic inflammation, and is linked to osteosarcoma pathogenesis (52, 53). Inhibiting NF-κB reduces the proliferative capacity of osteosarcoma cells (53) and suppresses tumour growth and angiogenesis in mouse models (54). NF-κB signalling also contributes to chemoresistance, anti-apoptotic protein expression, and metastasis in osteosarcoma (19, 55–58). Given the variable expression of cGAS/STING components and high chromosomal instability in osteosarcoma, some tumour subpopulations may activate NF-κB through non-canonical STING pathways, driving malignant progression even after cytotoxic treatment. Thus, understanding which signalling pathway is activated is crucial when targeting the STING pathway.

However, despite its potential for tumorigenesis, the cGAS/STING pathway is being explored as a novel immunological target for osteosarcoma due to its vital anti-tumour inflammatory effects. A previous study found impaired STING expression in osteosarcoma cell lines U20S and Saos-2 (22). These cells, along with an immortalized human lung fibroblast cell line (HEL), were infected with a mutant HSV-1 virus lacking the ICP0 protein. While HEL cells showed STING activation, U20S and Saos-2 did not, even when treated with the STING ligand 2’3’-cGAMP. Western blot and PCR analyses confirmed low STING levels in osteosarcoma cells but normal levels in HEL cells. Restoring STING in osteosarcoma cells reduced viral replication and increased ISG15 and IL-6 expression, indicating intrinsic STING deficiency. Together, the data indicate an intrinsic deficiency of STING expression in these human osteosarcoma cell lines at both the protein and gene expression levels.

Withers et al., 2023 further investigated STING pathway components in osteosarcoma cell lines, confirming significant STING downregulation in U2OS and Saos-2 cells compared to human osteoblasts (23). Saos-LM6 and MG63 cells had similar STING levels to osteoblasts, but MG63 cells showed reduced cGAS. Following X-ray irradiation, STING-deficient Saos-2 cells exhibited poor chemokine expression (CCL5 and CXCL10), whereas Saos-LM6 and MG63 cells expressed CCL5, and U2OS and MG63 cells produced CXCL10. siRNA-mediated STING knockdown in MG63 cells reduced chemokine expression, mirroring Saos-2 results. This study highlights the need for STING signalling in chemokine expression following DNA damage and reveals variations in cGAS/STING components among osteosarcoma cell lines, which may impact patient responses to radiotherapy. Most research on STING expression in osteosarcoma is based on in vitro cell line studies. While these studies provide valuable insights, it is important to acknowledge their limited translatability. Recently, an analysis of approximately 18,000 patient tumour samples from 139 different types of tumours was conducted using tissue microarrays. Among the 29 OS samples analysed, only 48.2% showed positive STING expression. Of this 48.2%, more than half (27.6%) exhibited weak STING expression, with only 10.3% of samples having strong STING expression (59). This data suggests that STING expression in OS is relatively low and often weak; however, similar to cell line data, there is variability in STING expression between patients. Unfortunately, there is still very little data available regarding the expression of cGAS/STING pathway components in patient samples, and further analysis is vital to truly understand the role the STING pathway plays in OS development.

Although the cGAS/STING pathway plays an essential role in inducing an anti-tumour inflammatory response, the current approach does not favour relying on cGAS/STING as a standalone immunotherapeutic target. Instead, cGAS/STING activation is being used as a complement to other therapeutic approaches, specifically chemotherapy and radiotherapy. Sodium-glucose cotransporter 2 (SGLT2) was found to be overexpressed in osteosarcoma patient derived samples at the protein level, as confirmed by western blot analysis and immunohistochemical staining, though not at the mRNA level (validated by RT-qPCR) (60). Administration of SGLT2 inhibitors as monotherapies suppressed tumour cell growth while simultaneously promoting CD4+ and CD8+ T-lymphocyte infiltration. Importantly, SGLT2 inhibitor administration induced STING upregulation, at both the protein and mRNA levels in a dose- and time-dependent fashion. This upregulation is believed to occur through SGLT2 inhibitor-mediated suppression of PI3K/Akt signalling. Beyond STING upregulation, SGLT2 inhibitors also enhanced the phosphorylation of key pathway components, including STING, TBK1, and IRF3. SGLT2 inhibitors also increased the expression of IFN-β, which is central to the activation of the infiltrating immune cells. Silencing of STING suppressed all such effects, thereby confirming STING as the primary mediator of these effects. Critically, co-administration of 2’3’-cGAMP alongside SGLT2 inhibitors enhanced all aforementioned effects in vitro. Lastly, using a syngeneic murine model, SGLT2 inhibitors in concert with 2’3’-cGAMP constrained osteosarcoma tumour growth and potentiated CD45+ CD4+/CD45+ CD8+ T-lymphocyte infiltration at tumour sites.

Another combinatorial approach targeted the DNA damage checkpoint protein ataxia telangiectasia mutated and Ras3 related (ATR), a major regulator of the G2/M cell cycle checkpoint (61). Detection of DNA damage by ATR is one means of ATR activation therefore leading to cell cycle arrest, thus preventing cells with DNA lesions from replicating. This investigation centred on the combination of ATR inhibitors (VE822/AZD6738) with radiotherapy in vitro as a means of activating the cGAS/STING pathway the elicit an IFN-based inflammatory response. Herein, the levels of phosphor-STAT1 (pSTAT1) served as a measure of cGAS/STING activation and type 1 interferon activation. STAT1 phosphorylation is a known effect of autocrine and paracrine type 1 interferon signalling through interferon receptors. Application of low level (5Gy) Ionizing radiation (IR) alone to U2OS cells yielded increased pSTAT1 6-days post treatment. The combination of IR with ATR inhibitors expedited STAT1 phosphorylation in U2OS, despite the low levels of STING expression reported in these cells. Interestingly, STING levels increased following combination treatment, concurrent with increased generation of micronuclei. The localisation of cGAS to generated micronuclei is believed to be the precipitating factor. Indeed, cGAS co-localisation with micronuclei was detected in distinct intracellular foci. Lastly, siRNA-mediated depletion of STING abolished type 1 interferon production and STAT1 phosphorylation following combination treatment. Taken together, this data presents DNA damage checkpoint inhibitors as enticing anti-cancer agents when used in concert with radiotherapy. The suppression of DNA repair and activation of replication stalling, in conjunction with the generation of DNA lesions, supports the production of micronuclei due to cancer cell replicative stress. Micronuclei are known potent activators of the cGAS/STING pathway, which drives type 1 interferon production, providing a rationale for leveraging existing DNA checkpoint inhibitors as a novel means of eliciting an immunological anti-tumour response in combination with low-dose radiotherapy.

The efficient delivery of the immunotherapeutic target continues to pose a significant challenge due to poor serum stability and cellular internalisation. Recently, innovative approaches using combinatorial strategies facilitated by nanotechnological drug delivery platforms, have been employed to activate cGAS/STING signal transduction. Nanotechnological delivery systems (NTS) are a popular contemporary research avenue for the treatment of various cancers. An NTS refers to a method for delivering drugs, genes, or other therapeutic agents to specific cells or tissues in the body using various nanoscale technologies. These systems include, but are not limited to, nanoparticles, nanosheets, nanofibers, nanocapsules. nanoframeworks, and nanocomposites. NTS’s allow the simultaneous employment of different therapeutic approaches simultaneously to achieve maximal therapeutic benefit. Depending on the formulation of the nanomaterials, small molecule drugs can be delivered alongside atypical therapeutic strategies such as photothermal therapy and photosensitization. The most common NTS are nanoparticle delivery systems (NDS). NDS have key advantages over typical treatment approaches which facilitate more innovative therapeutic approaches. Firstly, many chemotherapeutic drugs have broadscale toxicity with a range of off-target effects due to their lack of specificity. NDS can help reduce off-target toxicity by providing tissue- or site-specific delivery. This ensures that cytotoxic payloads are more precisely released at the site of action rather than systemically. Due to reduced toxicity, NDS also facilitate the administration of combination therapies that might otherwise have too high a toxicity profile. Lastly, NDS allow the contemporaneous delivery of different types of molecules, such as nucleic acids and adjuvants molecules like a STING agonist, along with chemotherapeutic agents to bolster anti-tumour effects of chemotherapy ( Figure 4 ). A summary of these studies can be found in Table 1 .

For example, Liu et al., 2022 developed PEGylated titanium carbide MXene nanosheets to which an ovalbumin-Mn2+ complex was bound (collectively referred to as TPOM nanocomplexes) (62). Ovalbumin served as an adjuvant antigen to support strong activation of dendritic cell (DC) maturation while Mn2+ ions were incorporated due to their notable enhancement of cGAS sensitivity for dsDNA, and the increased affinity of STING for cGAMP. The cell cytotoxicity of the TPOM nanocomplexes was first assessed by incubating TPOM nanosheets with LM8 murine osteosarcoma cells. In the absence of 808nm near-infrared (NIR) laser application, TPOM nanocomplexes were deemed biocompatible as no detectable death of LM8 cells was observed. In contrast, 808nm NIR laser administration to cells incubated with the TPOM complexes induced robust cell death in a time-dependent manner. Following confirmation of biosafety, 808nm NIR irradiation was shown to induce mt-DNA release within LM8 cells in vitro. Furthermore, using a transwell assay, 808nm NIR irradiation in combination with TPOM nanocomplex administration led to significant maturation of BMDCs due to the induction of tumour cell immunogenic cell death. Alongside mt-DNA and dsDNA release due to tumour cell irradiation-induced cell death, TPOM nanocomplex irradiation led to the liberation of ovalbumin and Mn2+ in vitro. Consequentially, the levels of BMDCs STING, phospho-IRF3, and IFN-β were elevated, revealing that TPOM nanocomplex irradiation is a biocompatible and potent means of activating BMDC STING signalling through the release of tumour antigens and co-stimulatory adjuvant molecules (ovalbumin and Mn2+). Lastly, in vivo murine models bearing subcutaneously implanted LM8 cell tumours were treated with a combination of NIR irradiation + TPOM and then compared to either NIR irradiation alone or no treatment. Ultimately, NIR irradiation of the TPOM nanocomplex led to efficient release of Mn2+ ions, ovalbumin, and mt-DNA synergistically activated dendritic cell cGAS/STING, culminating in improved dendritic cell maturation, enhanced tumour antigen presentation, increased inflammatory cytokine release, and increased CD8+ cytotoxic T-lymphocyte recruitment and activation at both local and metastatic tumour sites. These findings present this nanoplatform as a viable avenue for the synchronous activation of the innate and adaptive anti-tumour immune response through a multi-faceted stimulation of the cGAS/STING cascade. Importantly, a limitation to this study would be the subcutaneous injection of the LM8 tumour cells for the development of tumours. NIR irradiation does not possess the necessary penetration depth for use in an orthotopic model and therefore a degree of translatability is lost due to the non-native locations of primary tumours.

Another study synthesised a therapeutic composite nanoparticle containing both a platinum IV prodrug molecule (Pt(IV)-C12) and an indolamine-2,3-dioxygenase inhibitor (NGL919) (63). A critical feature of these nanoparticles was the presence of reactive oxygen species-sensitive amphiphilic thiol-ketal bonds. These polymers were used to encapsulate both molecules within the nanoparticles. The nanoparticles were observed to efficiently penetrate cancer cells after which the amphiphilic polymers would undergo reactive oxygen species-induced hydrolysis leading to intracellular release of Pt(IV)-C12 and NGL919. Pt(IV)-C12 induced DNA damage, subsequently activating the cGAS/STING pathway. NGL919 simultaneously inhibited indolamine-2,3-dioxgenase thus inhibiting tryptophan depletion in the TME. In response to nanoparticle administration, tumour cell apoptosis coincided with increased CD8+ T-lymphocyte recruitment and activation in vivo validated using a K7M2 (murine osteosarcoma cell line) orthotopic mouse model.

Tao Li et al., 2022 developed a metallo-organic nanoparticle composed of a tantalum-zirconium framework combined with tetrakis(4-carboxyphenyl)porphyrin (TCCP) as a photosensitising ligand (64). In vitro, X-ray irradiation of nanoparticles induced extensive reactive oxygen species (ROS) generation. ROS generation, in conjunction with DNA damage caused by the X-ray itself, lead to immunogenic cell death of K7M2 and U2OS (human osteosarcoma cell line) cells that had internalised the nanoparticles. The apoptotic phenotype was deemed to be immunogenic based on the elevated levels of markers HMGB1 and calreticulin in the affected cells. Utilising murine models, intravenous injection of the TZM nanoparticles, accompanied by X-ray irradiation constrained to tumour sites, resulted in a significant reduction in tumour burden without detectable systemic toxicity. Next, TZM nanoparticles were co-administered with an anti-PD-L1 therapy followed by X-ray irradiation of tumour sites. This multifaceted approach led to profound immunogenic tumour cell death in both local and metastatic tumours. Tumour cell death was accompanied by the release of IFN-β, TNF-α, and IL-6 (attributed to cGAS/STING activation in the surrounding immune milieu following immunogenic cell death), all of which are drivers of anti-tumour inflammation. X-ray-induced DNA damage, coupled with the release of tumour cell nucleic acids following tumour cell death, efficiently activated cGAS/STING both in vitro and in vivo. Lastly, the release of tumour antigens, in conjunction with cGAS/STING activation, potentiated dendritic cell maturation and activation, thus facilitating the recruitment and activation of anti-tumour immune cells. Most recently, platinum prodrug-containing nanoparticles were synthesised via the incubation of Pt(IV) alongside L-lysine diisocyanate and mPEG (65). Following synthesis of the core nanoparticle, alendronate was conjugated to the nanoparticle exterior via electrostatic interactions. Essentially, nanoparticle composition capitalised on the high degree of glutathione overexpression in osteosarcoma tumours. Glutathione facilitated nanoparticle dissociation, thus releasing the cytotoxic phenanthridine molecule, derived from the Pt(IV) platinum prodrug. In vitro, fluorescence microscopy confirmed efficient internalisation of the nanoparticles in both murine osteosarcoma K7M2 2D cell monolayers and 3D spheroids. Intracellular concentrations of phenanthridine in these models were compared against unconjugated cisplatin as a measure of nanoparticle penetration and intracellular dissociation. Indeed, a 6-fold increase in intracellular phenanthridine was observed versus cisplatin alone, indicating efficient release and catabolism of Pt(IV) to active phenanthridine following cellular uptake. Next, researchers interrogated the capacity of their nanoparticles to induce DNA damage. Immunofluorescent staining of cytoplasmic dsDNA of K7M2 cells revealed significant DNA damage following nanoparticle uptake. Western blot analysis of cGAS/STING pathway components displayed a significant increase in STING, IRF3 and TBK1 phosphorylation following nanoparticle internalisation by K7M2 cells. The immunostimulatory effects of cGAS/STING activation in this context were assayed through co-culturing of K7M2 cells alongside immature bone marrow-derived dendritic cells (BMDCs). Importantly, BMDC maturation and secretion of type 1 interferons and IL-6 were enhanced following nanoparticle administration in vitro. Lastly, an orthotopic K7M2 murine osteosarcoma model was developed for the analysis of nanoparticle biodistribution and anti-tumour efficacy. Collectively, in vivo intracavitary administration of the alendronate coated nanoparticles displayed selective accumulation of the nanoparticles at tumour sites with minimal loss of Pt(IV) in circulation indicating excellent biosafety. Furthermore, alendronate nanoparticles yielded a 2-fold higher suppression of tumorigenesis versus cisplatin. Selective cytotoxicity of the nanoparticles was complemented by enhanced dendritic cell activation, increased CD8+ T-lymphocyte infiltration and activation, beneficial polarisation of tumour associated macrophages to an inflammatory M1-like phenotype, and elevated central memory T-cell levels in the spleens of treated mice. Researchers concluded that alendronate coating of the nanoparticles resulted in bone-specific accumulation and selective release of the active compound at tumour sites. This was coupled with efficient activation of the cGAS/STING pathway, through phenanthridine-induced DNA damage, resulting in type 1 interferon release and the genesis of an anti-tumour immune profile in the TME.