Osteocytes constitute an essential element of the bone microenvironment, which encompasses three principal categories of bone cells: OBs, osteoclasts and osteocytes. These cells interact with stromal and immune cells by secreting a variety of cytokines and chemokines that facilitate tumor growth, invasion and metastasis. OBs originate from multipotent MSCs, and their development can be augmented by particular cytokines, including interferon (IFN)-γ, interleukin (IL)-12 and IL-13 (8). Conversely, their function may be inhibited by factors including tumor necrosis factor (TNF)-α, TNF-β, IL-1, IFN-α, IL-4 and IL-7 (10). OBs also have a notable role in influencing the formation, differentiation or apoptosis of osteoclasts through various signaling pathways, including the osteoprotegerin/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK), RANKL/leucine-rich repeats containing G protein-coupled receptor 4/RANK, Ephrin 2/ephB4 and Fas/FasL pathways (11). Osteocytes can secrete a variety of soluble factors, including growth differentiation factor 15, TGF-β, chemokines CXC-motif chemokine ligand (CXCL) 1 and CXCL2, as well as vascular endothelial growth factor (VEGF). They additionally synthesize RANKL, colony-stimulating factor 1 (CSF-1), high-mobility group box 1 (HMGB1) and IL-11, all of which promote osteoclastogenesis and the process of bone resorption (12). In OS, osteocytes activate the CXCL12/CXCR4 signaling axis by producing CXCL12, which is linked to tumor metastasis. Osteoclasts are integral to the initiation and metastasis of OS (13). Among the cytokines involved, CSF-1 and soluble RANKL are crucial for the differentiation and activation of OBs). CSF-1 is responsible for the regulation of both the proliferation and survival of pre-osteoclasts (14). By contrast, RANKL, which is synthesized by OBs and various other bone cells (15), interacts with its receptor RANK located on the surface of osteoclast precursors (16). This interaction regulates the differentiation and maturation of osteoclast precursors via paracrine signaling mechanisms. In the context of OS, heightened osteoclast activity plays a significant role in the proliferation of OS cells and the degradation of bone tissue. This mechanism leads to the liberation of pro-tumorigenic factors, such as insulin-like growth factor 1 (IGF-1) and from the bone matrix, thereby promoting tumor proliferation and metastasis (17).

Within the microenvironment of OS, monocytes and macrophages constitute 70–80% of the total BM cell population (18). Various cell types, including tumor-associated macrophages (TAMs), T cells, B cells, natural killer (NK) cells and BM MSCs, present in the tumor immune microenvironment significantly influence tumor development, invasion and metastasis. Recent investigations into TAMs have emerged as a focal point of research.

TAMs constitute the most prevalent immune cell population within the OS microenvironment, and account for >50% of all infiltrating immune cells, alongside other cell types including dendritic cells (DCs), lymphocytes and myeloid-derived cells. Collectively, these cells form the primary components of the OS immune microenvironment (19). Research indicates that TAMs are prevalent in most high-grade OS biopsy specimens, and an increase in TAMs is associated with reduced metastasis and extended survival (20); however, the underlying mechanisms remain unclear. Immune cells within the TME can either promote or inhibit OS, contingent upon the immune environment and the specific cell types involved (21). For instance, certain immune cells, including T cells, B cells and NK cells, can exert antitumor effects, while others, such as myeloid-derived suppressor cells (MDSCs), M2 macrophages and regulatory T cells (Tregs), may facilitate tumor growth and metastasis. Consequently, elucidating the intricate mechanisms of interaction between OS and immune cells is essential for advancing research into novel immunotherapies (17).

TAMs originate from peripheral monocytes and are recruited to the tumor site by various chemokines and cytokines, including C-C motif ligand 2, CSF-1 and VEGF. Upon entering the tumor, TAMs can exhibit two primary phenotypes: The pro-inflammatory M1 phenotype and the immunosuppressive M2 phenotype. The M1 phenotype is associated with protective responses and is typically linked to a favorable prognosis in patients with OS (22). Conversely, the M2 phenotype can be induced by various stimuli within the immune microenvironment, particularly through cytokines IL-4 and IL-13 that signal via the signal transducer and activator of the transcription 6 (STAT6) pathway, as well as IL-10 and glucocorticoids. M2-type TAMs secrete a range of cytokines that promote the migration and invasion of OS cells while inhibiting the immune function of T lymphocytes (23). M1 and M2 TAMs can undergo interconversion under the influence of key cytokines and signaling pathways, such as the Th2 cytokines (IL-4 and IL-13) via the STAT6 pathway, as well as IL-10 and TGF-β. This macrophage polarization is dynamic. Research has demonstrated that polarized M2-type TAMs play a significant role in the proliferation and metastasis of OS. For example, M2-type TAMs can facilitate the migration and invasion of OS cells by triggering an autocrine signaling loop that activates HMGB1 expression within the tumor cells. Furthermore, HMGB1 has been shown to encourage the polarization of M1-type TAMs into M2-type TAMs, thereby establishing a positive feedback mechanism that exacerbates the development and progression of OS (24).

T cells are integral to both cellular and humoral immunity and comprise a wide array of functionally specialized subsets, including T helper cells (Th1, Th2, Th9, Th17, Th22 and follicular helper T cells), cytotoxic T lymphocytes (CTLs) and Tregs. This diversity is crucial for orchestrating the antitumor immune response in OS, with the composition and balance of these infiltrating T cell subsets determining the immunological outcome. In OS, tumor-infiltrating lymphocytes are predominantly located in areas expressing human leukocyte antigen class I, while CD4⁺ and CD8⁺ T cells primarily aggregate at the interface between lung metastases and normal tissue (25). Studies have indicated that the density of T cells in metastatic lesions is significantly greater than that in primary and recurrent lesions, suggesting that T cells may also serve as potential prognostic indicators (26,27). Analysis of biopsy tissues and peripheral blood from patients with primary OS has revealed that T cell levels in biopsy specimens exceed those in peripheral blood, suggesting that the immune microenvironment within tumor lesions is suppressive, potentially inhibiting T cell immune activity through TAMs, further suggesting that T cells may serve as an auxiliary biomarker for clinical diagnosis (28). Furthermore, the depletion of CD163⁺ macrophages, a hallmark immunosuppressive subset in the OS TME, has been shown to enhance T cell growth and pro-inflammatory factor production in vitro. Therefore, targeting these cells represents a promising approach to reprogram the immunosuppressive OS TME and potentiate antitumor immunity (28). The prognostic value of T cells is well-established across various cancer types, with studies confirming that specific T-cell signatures, such as the CD8 T-cell signature, are robust predictors of patient survival (29,30). Furthermore, prognostic models based on T-cell-related genes have been successfully constructed for cancer types such as hepatocellular carcinoma (29,30).

B cells can be classified into three categories: Naive B cells, memory B cells and effector B cells/plasma cells, with the latter being the primary source of antibodies. Regulatory B cells, a subset of B cells, exert immunosuppressive effects by inhibiting CD4⁺ T cells, CTLs, macrophages and DCs through the secretion of inhibitory cytokines such as IL-10, TGF-β and IL-35, as well as the expression of membrane surface regulatory molecules such as FasL and CD1d (31). Regulatory B cells also promote the conversion of T cells into T lymphocytes, thereby diminishing the antitumor immune response (32)]. Current research on B cells in OS remains limited; however, a recent pan-cancer immune-infiltration analysis showed that patients with high B-cell abundance exhibit a significantly improved overall survival, and the proportion of activated B cells (CD19⁺CD27⁺) correlates positively with metastasis-free survival in OS (33). Thus, the presence of effector B cells may serve as a favorable prognostic indicator. Antibodies play a crucial role in the regulation of tumor growth and metastasis through various mechanisms, including antibody-dependent cell-mediated cytotoxicity, regulatory effects, complement activation, tumor cell receptor blockade and alterations in tumor cell adhesion (34,35). Nonetheless, some studies have also indicated that certain antibodies may bind to antigens on the surface of tumor cells, thereby obstructing their cytotoxic effects (32,36).

NK cells are a type of innate lymphocyte characterized by the expression of the intracellular transcription factor E4 BP 4+ and are identified as CD3⁻ CD19⁻ CD56⁺ CD16⁺. NK cells have been shown to not only directly eliminate tumor cells but also regulate tumor progression and metastasis (37,38). They can induce tumor cell death in the TME by releasing perforin, granzyme and TNF-α, as well as expressing FasL (39). The programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis plays a role in modulating the antitumor effects of NK cells. Research has demonstrated that blocking the PD-1/PD-L1 axis with PD-L1 antibodies enhances the cytotoxic activity of NK cells against human OS cells by inhibiting NK cell toxicity through the secretion of granzyme B (40). Comprehensive analyses of immune infiltration in the OS microenvironment have revealed that male patients exhibit a higher presence of NK cells compared with female patients (41). Additionally, it has been observed that NK cells are suppressed in the OS microenvironment, with increased expression of TGF-β (42). This suppression may involve the inhibition of the activating receptor natural killer group 2 member D and a reduction in perforin release by NK cells, thereby promoting angiogenesis, bone remodeling and cellular migration.

BM MSCs are classified as multipotent stem cells that significantly contribute to the development of OS tumors through the modulation of immune responses and the facilitation of cell fusion and differentiation (43). MSCs and OBs are regarded as potential precursors to OS cells (33). Research suggests that MSCs play a crucial role in mediating the bidirectional crosstalk between OS tumor cells and the TME through the secretion of a variety of cytokines, chemokines, ILs and other signaling molecules (44). MSCs are actively involved in the paracrine signaling mechanisms of OS tumor cells, influencing multiple facets of tumor behavior, such as angiogenesis, proliferation, invasion, metastasis, immune modulation and resistance to chemotherapy (45). Furthermore, MSCs facilitate the growth, metastasis, and angiogenesis of OS by releasing an array of chemokines, including CCL5, stromal-derived factor 1, CXCL12, IL-6 and VEGF (45).

MSC-derived extracellular vesicles have been shown to enhance the proliferation, invasion and migration of OS cells via the metastasis-associated lung adenocarcinoma transcript 1/microRNA (miR)-143/Neurensin-2/Wnt/β-catenin signaling pathway, as well as through miR-655-mediated β-catenin signaling (46). Research indicates that TGF-β is significantly upregulated in patients with OS (47). Furthermore, OS cells enhance the secretion of extracellular vesicles that contain TGF-β. This process subsequently stimulates the release of IL-6, which is activated by signal transducer and activator of transcription 3 (STAT3) from MSCs, thereby facilitating lung metastasis (48). Additionally, single-cell RNA sequencing has uncovered a considerable heterogeneity among MSCs associated with OS (18).

MSCs facilitate the proliferation and metastasis of OS cells through two primary non-immune mechanisms. First, the interaction between OS cells and MSCs is mediated by IL-8 and aquaporin 1. Second, aberrant gene expression, including retinoblastoma, c-Myc, TP53, KRas and Indian Hedgehog, contributes to the transformation of MSCs into OS cells (49). Furthermore, studies have demonstrated that when MSCs are located within the microenvironment of OS cells, they have the capacity to differentiate into CAFs (50,51). This differentiation notably contributes to the increased proliferation, migration and invasion of OS cells. Under the influence of MSCs, OS cells are capable of inducing the migration and invasion of endothelial cells, which in turn promotes angiogenesis (52). From an immunological perspective, MSCs secrete anti-inflammatory factors and inhibit pro-inflammatory substances, thereby assisting OS cells in evading immune surveillance, particularly through autocrine or paracrine exosomal mechanisms. Lagerwei et al (53) demonstrated that MSCs suppress T cell proliferation and immune responses by releasing exosomal vesicles (EVs) containing miRNA/RNA and proteins. Additionally, Zhang et al (54) reported that MSC-derived EVs express TGF-β and TGF-β1. Moreover, MSC-derived exosomes can enhance OS tumorigenesis and metastasis through the induction of autophagy, as supported by evidence from other studies (55,56).

OS cells are present not only within the tumor tissue but also in the bloodstream, where they are identified as CTCs (57). CTCs demonstrate the capacity to circumvent localized therapeutic approaches, including surgical resection and radiation therapy, and can remain present in small numbers during systemic treatments. This persistence ultimately facilitates the metastasis and recurrence of OS (57,58). Current research suggests that CTCs play a unique role within the immune microenvironment associated with OS. For instance, Zhang et al (59) demonstrated that the inhibition of IL-6 can suppress the proliferation of OS cells and decrease the presence of CTCs. In vitro studies revealed that IL-6 activates the Janus kinase (JAK)/STAT3 and mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. While both pathways facilitate the proliferation of OS cells, only the JAK/STAT3 pathway is implicated in promoting cell migration (59–61). A clinical study has shown that the level of IL-6 in OS samples is significantly upregulated compared with normal bone tissue, indicating that IL-6 plays an important role in the progression of OS (62). Furthermore, MSCs have been shown to enhance OS proliferation and metastasis through the secretion of IL-6 (63,64). Liu et al (65) further identified that IL-8 also contributes to the progression of OS. Their research involved isolating and culturing CTCs from patients, revealing that IL-8 promotes tumor growth and lung metastasis in both in vitro and in vivo models, indicating that targeting IL-8 may yield antitumor effects. Although the precise mechanisms governing the interaction between CTCs and tumor immunity remain unclear, CTCs may represent promising targets for therapeutic intervention and serve as biomarkers for predicting clinical outcomes.

Exosomes are a key subtype of EVs. EVs are membrane-bound structures that are released into the ECM following the fusion of intracellular multivesicular bodies with the cell membrane. These vesicles encapsulate a diverse array of biomolecules, including long non-coding RNAs, miRNAs, proteins, lipids and metabolites. Notably, cancer cells tend to produce a greater quantity of exosomes compared with normal cells, and these exosomes are implicated in various aspects of tumor biology, including tumor initiation, progression, immune evasion and drug resistance (66). Evidence suggests that EVs play a critical role in the onset, advancement and metastasis of OS (15). Specifically, exosomes can facilitate tumor growth by influencing endothelial cells to enhance angiogenesis, while also mediating intercellular interactions and participating in the regulation of cellular functions and the transmission of biological information, which in turn affects the expression and secretion of specific cytokines (67,68). Furthermore, exosomes can modify signaling pathways in recipient cells, thereby promoting the metastasis of cancer cells (69). Concurrently, exosomes can activate multiple signaling pathways that lead to the development of drug resistance in previously drug-sensitive cells or assist cancer cells in expelling cytotoxic agents (70). Research has also indicated that exosomes play a role in modulating the immune response, aiding tumor cells in evading immune surveillance and facilitating cancer metastasis. For instance, exosomes have been shown to promote lung metastasis in OS by releasing PD-L1 (9). Recent studies have advanced our understanding of the role of exosomal miRNAs in OS (71,72). For instance, in the context of OS, it has been demonstrated that exosomal miRNAs, such as miR-148a-3p, can stimulate the release of pro-angiogenic factors and induce angiogenesis by influencing the activity of osteoclasts and endothelial cells (73). Additionally, exosomes can enhance the invasiveness of OS through immune modulation; the surface of exosomes is adorned with tumor-associated antigens that interact with antigen-presenting cells, thereby inducing tumor-specific cytotoxic T cell immune responses (74). Moreover, it has been observed that exosomes secreted by metastatic OS cells can induce M2 polarization via TGF-β2, further promoting tumor invasion and metastasis (75). In a study, Shimbo et al (76) encapsulated synthetic therapeutic miRNA-143 within exosomes and administered it into the OS microenvironment, resulting in a significant reduction in OS cell migration. The investigation of exosomes has emerged as a prominent area of research and ongoing advancements in this field have the potential to yield novel breakthroughs in the clinical management of OS.

The ECM is a complex, mesh-like structure comprised of collagen, proteoglycans, glycoproteins and glycosaminoglycans, including hyaluronic acid. This matrix not only supplies essential nutrients and structural support to tumor cells but also plays a critical role in the construction and remodeling of the ECM, thereby influencing the physical and chemical characteristics of the TME (77). Cytokines and the ECM serve as pivotal mediators within this microenvironment, capable of modulating various biological behaviors of tumor cells, including proliferation, apoptosis, invasion and metastasis (78). OS is known to produce a large amount of ECM, which significantly impacts tumor invasiveness and the response to therapeutic interventions (66). Specific ECM components, such as collagen, fibronectin and laminin, are implicated in aberrant signaling pathways and structural irregularities that facilitate sarcoma growth and metastasis through diverse mechanisms, including integrin-mediated signaling activation, the promotion of EMT and the enhancement of cell migration and invasion (79). Furthermore, the ECM has the capacity to regulate the IGF axis, which is instrumental in modulating OS growth and conferring resistance to conventional chemotherapy agents (66). Targeting the ECM in the treatment of OS presents promising potential; for instance, ECM-like hydrogels can be utilized for the delivery of therapeutic agents, thereby offering a novel platform for OS treatment and bone regeneration (80). Additionally, ECM-associated factors, such as neurotrophic EGF-like molecule 1, have emerged as promising candidates for novel therapeutic strategies aimed at impeding OS progression (20). Evidence suggests that the invasiveness of OS can be mitigated by targeting the underlying mechanisms of ECM degradation and angiogenesis (14,81). Therapeutic strategies include suppressing key enzymes such as matrix metalloproteinases (MMPs; including MMP-2 and MMP-9) to prevent ECM breakdown and employing monoclonal antibodies or small-molecule inhibitors against pro-angiogenic factors such as VEGF to block new blood vessel formation (14,81). The interplay between the ECM and CAFs is also known to foster the development of OS (82). This cooperative relationship drives disease progression by creating a stiffened, pro-fibrotic microenvironment that supports tumor cell survival, proliferation and invasion. The critical nature of this interplay is evidenced by studies showing that therapeutic strategies designed to destroy CAFs and disrupt the ECM can effectively suppress OS tumor growth (82). Moreover, ECM components, including EVs, hold potential as biomarkers for the diagnosis and prognosis of OS (83). Engineered AttIL 12-T cells, which are tumor-specific T cells with IL-12 anchored to their cell membrane, have demonstrated enhanced efficacy by targeting CAFs within the ECM, disrupting the tumor stroma and promoting T cell infiltration, indicating promising avenues for future research (82). This engineering approach involves uniformly tethering the IL-12 cytokine onto the surface of the adoptively transferred T cells, which allows for dose-controlled and localized delivery of IL-12 directly to the tumor site (82). In conclusion, the significant role of the ECM in OS is increasingly becoming a focal point for therapeutic strategies.