miRNAs regulate cell proliferation, differentiation, apoptosis, and development by binding to the 3’ untranslated region (3’-UTR) of target mRNAs and are able to degrade or induce translational silencing in OS cells (21). miR-223-3p has been shown in studies to limit cadherin-6 expression by directly binding to the 3’-UTR of cadherin-6 and to inhibit the invasion, migration, growth, and proliferation of OS cells (22). The expression of miR-18b-5p, which is mediated by HIF-1α, is substantially increased in OS and is associated with a poor prognosis (23). In addition, miR-18b-5p promotes the incidence and development of OS by inhibiting the expression of the tumour suppressor gene PHF2 (23). miRNA-98-5p is under-expressed in OS and inhibits cell cycle progression and migration potential by down-regulating CDC25A, thereby inducing OS apoptosis (24). Overexpression of miRNA-1236-3p in HOS cells reduces proliferation, stops the cell cycle in the G0/G1 phase, and promotes apoptosis (13). A differential analysis of miRNA expression in OS ( Figure 1A ) shows that the expression of let-7A-2 and miR-323 is decreased, whereas the expression of miR-182 is increased, suggesting that miR-182 could be a possible therapeutic target in OS. The detailed information of differentially expressed ncRNAs in A-C is presented in Table S1 .

The expression of lncRNA MELTF-AS1 is significantly increased in OS and promotes OS metastasis by upregulating the expression of MMP14 (25). lncRNA ODRUL can act as a competitive endogenous RNA (ceRNA) sponge of miR-3182 and promotes the proliferation, migration, invasion, and tumour growth of OS by upregulating the expression of matrix metalloproteinase (MMP) II (26). The oncogenic effects of LncRNA CBR3-AS1 are executed by regulating the network of the miR-140-5p/DDX54-NucKS1-mTOR signalling pathway, which encourages stemness and epithelial–mesenchymal transition (EMT) of OS (27). The overexpression of lncRNA EBLN3P promotes the progression of OS cells, which is indicative of the stimulating effects of EBLN3P (28). In OS cells, the expression of the lncRNAs ENSG00000233086.8 and ENSG00000269821 is much higher ( Figure 1B ). By examining the molecular pathways and regulatory mechanisms further, one may be able to control the development of OS.

circECE1 is highly expressed in OS tissues and cells, and its association with c-Myc promotes tumour proliferation and metastasis by boosting glucose metabolism in OS cells to prevent speckle-type POZ-mediated ubiquitination and degradation of c-Myc (29). C-Myc-targeting checkpoint inhibitors have been demonstrated to impede OS development via modulating the production of ncRNAs (30). Studies have shown that knockdown of circRNA circ_001422 significantly inhibits the proliferation and metastasis of OS cells and promotes apoptosis. Regulating the miR-195-5p/FGF2/PI3K/AKT axis produces the opposite impact of overexpression (31). circMYO10 has been confirmed as a promoter of OS progression by regulating the miR-370-3p/RUVBL1 axis and chromatin remodelling, consequently boosting the transcriptional activity of the β-catenin/LEF1 complex (32). The number of circRNAs with decreased expression was much greater than those with enhanced expression ( Figure 1C ), a finding that could be leveraged to design targeted therapies once the regulatory mechanisms of these circRNAs have been elucidated.

Recent research has increasingly focused on the impacts and mechanisms of microRNAs, whereas research into lncRNAs and circRNAs is still in its infancy ( Table 1 ) (102). More research points to the importance of noncoding RNAs in OS, both in terms of diagnosis and treatment (9). An alternative mechanism for OS chemotherapeutic resistance has been proposed through the construction of ceRNA networks, in which noncoding RNAs bind to mRNAs (103). Differential expression of noncoding RNAs and the formation of ceRNA networks may lead to the development of more effective treatment techniques and the ability to overcome drug resistance in OS ( Figure S1 ).

Immature bone marrow cells (IMCs) differentiate into mature macrophages, dendritic cells, and granulocytes under physiological conditions and transform into immunosuppressive MDSCs when regulated by chemokines in the TME (104). MDSCs generate pro-inflammatory substances such as NO, IL-1, and IL-6, which expose OS cells to a persistently inflammatory environment and dramatically enhance the risk of DNA damage and tumour cell proliferation, which may contribute to the progression of OS (105, 106). Through the activation of the activator for transcription 3, miR-21 and IL-6 can synergistically enhance the development of MDSCs and influence treatment resistance (107). Reactive oxygen species (ROS) produced by oxidative stress can activate the NF-κB and Nrf2 pathways, allowing tumour cells to survive (108). MDSCs generate excessive ROS via NOX2 and suppress the antitumor effects of T cells and natural killer (NK) cells, hence mediating OS immune escape while maintaining oxidative balance via glycolysis upregulation (109, 110).

The TME alters the lipid metabolism of MDSCs to enhance the uptake of fatty acids and the activation of fatty acid oxidation (FAO), thereby improving the immunosuppressive activity of MDSCs and promoting tumour growth (110). In addition to LXR agonists, liver-X nuclear receptors (LXRs) regulate cholesterol and lipid metabolism via the transcription target Apolipoprotein E (111). LXR agonists have been demonstrated to play a role in MDSC depletion, which could be related to FAO inhibition in MDSCs (112). By increasing the activities of arginase-1, MDSCs compete with T cells for the consumption of arginine, which leads to T cell dysfunction (113). L-arginine supplementation may improve the anticancer impact of cyclophosphamide (CP) and minimize T cell dysfunction caused by increased MDSCs generated by CP (114).

TAMs are the primary immune cells in the TME, which are usually produced from bone marrow monocytes, and the presence of TAMs is indicative of a poor prognosis in OS patients (115, 116). TAMs, via stimulating the COX-2/STAT3 axis and causing epithelial– mesenchymal transition, can increase OS pulmonary metastasis (117). C–C motif chemokine ligand 18 secreted by TAMs has been shown to promote the proliferation and metastasis of OS cells via the EP300/UCA1/Wnt/β-catenin pathway, which significantly reduces the survival rate of OS patients (118). Studies have demonstrated that miR-363 inhibitors can promote the migration of TAMs after transfection of OS cells (119).

TAMs can be divided into classically activated macrophages (M1), with antitumor activity, and selectively activated macrophages (M2), with tumour-promoting activity, both of which can coexist in the TME (120). It has been found that M2 can promote the deterioration of OS cells through the SOCS3/JAK2/STAT3 axis, and OS cells can enhance the M2 polarisation of TAMs (121). LncRNA RP11-361F15.2 enhances M2 polarisation mediated by cytoplasmic polyadenylate element binding protein 4 through miR-30c-5p and further promotes the occurrence of OS (122).

TAMs substitute glycolysis with FAO as a source of energy by expressing a high amount of the scavenger receptor CD36, which enhances lipid accumulation and reprograms TAMs into M2 types (123). S100A4 has been reported in mice to upregulate FAO and mediate TAM polarization to M2, as well as to have carcinogenic activity (124).

Extensive Treg cell infiltration into tumour tissues is often associated with a poor prognosis, whereas their removal enhances antitumor immune responses (125). FOXP3+ expression in Treg cells has been shown to predict the prognosis of osteosarcoma in vivo and in vitro and could potentially be used as a diagnostic marker in clinical practice (126–128).

Glycolysis and oxidative phosphorylation, which are essential for Treg cell metabolism, require FAO (129). Treg cells in tumours, in contrast to normal tissues, have considerably decreased glucose uptake and are dysfunctional in a high-glucose environment (130). P13K inhibitors can reduce the immunosuppressive effects of Treg cells by upregulating glycolysis and reducing FOPX3 expression (131). miR-34a targets the 3’ UTR to inhibit the expression of FOXP3, which is controlled by the NF-κB pathway and downregulated by IL-6 and TNF-α (132). It has been demonstrated that the transcriptional regulator c-Myc influences oxidative phosphorylation in Tregs via regulating mitochondrial activity, hence limiting accumulation and functional activation (133). Targeting c-Myc and associated signalling pathways as a means of treating OS has drawn a lot of interest (29, 134).

Immunosuppressive cells can regulate the occurrence and development of OS through crosstalk with stromal cells in the TME ( Figure 1D ), which are regulated by ncRNAs in OS cells, according to the studies on the metabolic heterogeneity of immunosuppressive cells and the regulatory mechanisms of ncRNAs.

Interferon (IFN) is a cytokine that white blood cells generally secrete during infections (135). Due to its effects as an agonist of antitumor activity in adaptive and innate immune cells, it leads to the establishment of antiproliferative and antiangiogenic activity in osteosarcoma and antagonizes inhibitory immune subsets (135, 136). IFN-γ induces PKR-dependent autophagy in OS cells through signal transduction and activation of transcription 1, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase-dependent pathways (137). miR-142-5p enhances the transcription of IFN-γ by downregulating the expression of interaction protein domain 2 (138). miR-31 reduces interferon-γ production, thereby attenuating Th1 response (139). The efficiency of IFN therapy could be increased by modulating the aberrant expression of ncRNAs, which has a good synergy for drug development in the treatment of OS.

The protein EWS-FLI1, which is overexpressed in OS, has become a specific Treg antigen for vaccine development (150). EWS-FLI1 inhibits effector T cell responses and has been found circulating in or infiltrating tumours in Ewing patients, resulting in unfavourable clinical outcomes (150). Double sialic ganglioside (GD2) is extensively expressed in osteosarcoma (OS) and soft tissue sarcomas, and immunotherapies including GD2 vaccines have been utilized to treat solid tumor (151). miR-34a can target GD-2 to enhance tumour apoptosis, which is anticipated to be a novel OS target (152). Previous studies developed fusion cell vaccines by chemically fusing human γδT cells with SAOS-2 cells, eliciting cytotoxic T lymphocyte responses against two human OS cell lines that were specific to cancer antigens (153). CD103⁺cDC1 vaccines produced in vitro elicited systemic and long-lasting tumour-specific T cell-mediated cytotoxicity, thereby inhibiting the growth of primary and metastatic osteosarcoma (154).

Chimeric antigen receptor T cell therapy has been shown to be effective in leukaemia and lymphoma, and current studies have increasingly focused on CAR-T therapy for solid tumours, such as OS (155). The efficacy of B7-H3-CAR-T cell therapy in treating solid tumours was initially proven in a model of childhood cancer (156). Following that, the efficacy of B7-H3-CAR-T cells in OS and preventing lung metastasis progression was demonstrated in a dose-dependent manner in a mouse model with orthotopic OS of the tibia and lung metastases (157). Human EpHA2-directed CAR-T cells can target human OS cells in vitro, and the injection of CAR-T cells can eradicate tumour deposits in the liver and lungs of metastatic OS models in vivo (158). CD166 is selectively expressed in OS cells and can be used as a new target for CAR-T cell therapy, which has been demonstrated in mice models of OS by injection of CD166.BBζ CAR-T cells (159). Human epidermal growth factor receptor 2 (HER2)-CAR-T cells have entered phase II clinical trials, and the safety and efficacy of this therapy have been demonstrated in a study of 19 patients with HER2-positive solid tumours (160).

To treat OS, immunotherapy particularly targets immune cells and immunosuppressive cells in the TME. ncRNAs play a crucial regulatory role and have the potential to be exploited as synergistic agents for checkpoint inhibitors as well as novel targets for interferon treatments and cancer vaccines. The evidence of clinical data in interferon therapy and checkpoint inhibitors is shown in Table S2 . CAR-T cells are a new therapeutic for solid tumours that can eradicate tumour cells from primary and metastatic lesions and may provide a unique immunotherapy treatment for patients with metastatic OS.