CAFs can arise from multiple cellular sources, including tissue-resident fibroblasts, pericytes, bone marrow–derived mesenchymal stem cells (MSCs), and endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) (13).

In the bone metastatic microenvironment, TGF-β reshapes the niche by modulating osteoclasts, osteoblasts, and fibroblast-like stromal cells, thereby promoting tumor cell colonization. Its recruitment and activation of stromal fibroblasts contribute to establishing a pro-metastatic and immunosuppressive milieu (5, 14).

Activated CAFs exhibit a secretory and contractile phenotype with enhanced production of ECM and secretion of immunomodulatory and survival factors. These characteristics are generally maintained through persistent paracrine signaling and epigenetic remodeling (10).

CAFs cells do not share one common marker. They are often identified by α-smooth muscle actin (α-SMA), FAP, and platelet-derived growth factor receptors (PDGFRα/β) (15). Immunohistochemical examination identified multiple FAP⁺ and α-SMA⁺ fibroblasts in primary osteosarcoma lesions and lung metastatic nodules (7, 16, 17). Single-cell transcriptomics defined that CAFs are not a homogeneous population. At least two dominant subtypes are invariably observed: (1) myofibroblastic CAFs (myCAFs): with strong α-SMA expression, with ECM remodeling gene patterns and contractility; (2) inflammatory CAFs (iCAFs): with enrichment for cytokines and chemokines IL-6, CXCL12, and CCL2 (18–20). These types could both be found within tumors and possibly alternate each other according to signals by cancer and immune cells, leading to their functional plasticity.

In osteosarcoma, CAFs secrete CXCL12, which can bind to CXCR4 on the surface of T cells, hindering their infiltration into the tumor core area (21). This effect forms an “immune shell” around the tumor, physically excluding T cells from contact with tumor cells. Secondly, TGF - β produced by CAFs significantly weakens T cell function, inhibits its proliferation, cytotoxicity, and cytokine production, while promoting the differentiation of initial CD4 ⁺ T cells into immunosuppressive regulatory T cells (Tregs). These processes collectively establish an immunosuppressive tumor microenvironment, promoting cancer progression and enhancing resistance to treatment (10, 22).

In addition to directly inhibiting effector T cells, CAFs also recruit multiple immune suppressive cell populations by secreting chemokines such as CCL2, IL-6, and CXCL8 (5). These factors attract bone marrow-derived suppressor cells (MDSCs), M2 polarized macrophages, and Tregs, synergistically creating an inhibitory immune microenvironment (23). For example, IL-6 mediated activation of the JAK/STAT3 pathway has been shown to be closely associated with enhanced immune suppression and accelerated tumor development in osteosarcoma (24).

Cancer associated fibroblasts promote immune escape by reshaping the ECM, which increases matrix density and stiffness, forming a physical barrier that hinders the infiltration and migration of cytotoxic T lymphocytes (10). In the microenvironment of osteosarcoma, ECM components such as type I collagen and fibronectin are significantly elevated, while the expression of matrix metalloproteinases (MMPs) is also upregulated (25). These changes not only hinder the entry of immune cells into the tumor area, but are also associated with poor response to immune checkpoint inhibitors, further highlighting the role of ECM remodeling in immune suppression of osteosarcoma (26).

The latest research shows that CAFs alter the metabolic characteristics of the TME by secreting lactate and acidifying the extracellular environment (27). This metabolic change can inhibit T cell effector function and promote immune tolerance (28). In addition, extracellular vesicles derived from CAFs may carry immunosuppressive miRNAs and proteins that can regulate the phenotypes of dendritic cells and macrophages (29). Similar phenomena have also been observed in osteosarcoma: fibroblasts exhibit high glycolytic properties, while lactate acts as a “metabolic signal” that promotes epithelial mesenchymal transition (EMT) of tumor cells and activates immunosuppressive pathways (30).

As the main matrix component in the microenvironment of osteosarcoma, CAFs secrete a large amount of soluble factors such as IL-6, TGF-β, and CXCL12 (also known as SDF-1). Recent studies demonstrate that cytokines secreted by CAFs—such as IL−6, IL−11, TGF−β, and growth differentiation factor—can activate several oncogenic survival pathways in tumor cells. Moreover, emerging evidence highlights that CAFs can also suppress ferroptosis, a novel form of regulated cell death central to anticancer therapy. In osteosarcoma, FSP1 has been identified as a key determinant of cellular susceptibility to ferroptotic death, where high FSP1 expression confers resistance, while its inhibition markedly sensitizes tumor cells to ferroptosis-inducing agents (32). Similarly, in pancreatic ductal adenocarcinoma, CAFs secrete cysteine via a TGF−β/SMAD3/ATF4–dependent transsulfuration pathway, enhancing glutathione (GSH) synthesis in tumor cells and thereby blocking lipid peroxidation–driven ferroptosis (33). Mechanistically, hyperactivation of the PI3K/AKT axis in cancer cells is also known to inhibit ferroptosis by modulating downstream regulators such as SLC7A11, GPX4, NRF2, and iron metabolism components (34). Taken together, these observations support the hypothesis that CAFs−mediated metabolic rewiring and signaling crosstalk may contribute to ferroptosis resistance and therapy failure in osteosarcoma.

ABC transporters are a large class of transporters that rely on ATP (hydrolysis) for energy to transport various substrates across the membrane. In osteosarcoma, a key mechanism by which CAFs mediate therapeutic resistance is through induction of ABCB1/P−glycoprotein in tumor cells, leading to active efflux of chemotherapy agents (35, 36). Also, a recent study demonstrated that the glycosyltransferase C1GALT1, which can be upregulated in the tumor stroma, plays a critical role in promoting doxorubicin resistance by inducing ABCC1 expression in osteosarcoma cells (37). Furthermore, targeted disruption of ABCB1 using CRISPR/Cas9 has been shown to restore doxorubicin sensitivity in resistant osteosarcoma cell lines (e.g., KHOSR2, U-2OSR2), further confirming the role of CAFs-induced signaling in drug resistance (35).

Recent studies suggest that tumor-derived exosomes not only remodel the pre-metastatic niche but also modulate stromal cells to support tumor progression and drug resistance. For instance, osteosarcoma-secreted exosomal linc00881 can be internalized by lung fibroblasts, inducing their transformation into CAFs-like phenotypes (38). And once activated, CAFs can further contribute to tumor progression and therapy resistance by releasing their own exosomes enriched with oncogenic non-coding RNAs and proteins. These CAFs-derived exosomes can be internalized by osteosarcoma cells and reprogram gene expression to enhance survival pathways—such as PI3K/AKT—and suppress apoptotic responses (39, 40). For instance, exosomal miR-1228 derived from cancer-associated fibroblasts has been shown to promote osteosarcoma cell migration and invasion by directly targeting the tumor suppressor SCAI. This exosome-mediated intercellular communication reinforces a pro-tumorigenic microenvironment and may contribute to the development of aggressive and potentially chemoresistant phenotypes in osteosarcoma (41).

One main strategy for targeting CAFs is to block its signaling pathway activated in osteosarcoma cells. CAFs can secrete various soluble cytokines and growth factors, including IL-6, TGF - β, and CXCL12, which bind to receptors on tumor cells and activate oncogenic signaling pathways such as JAK/STAT3, PI3K/AKT, and MAPK. Among them, the IL-6/STAT3 axis is particularly crucial in promoting resistance to amphotericin B and Cisplatin, by enhancing survival signaling and anti-apoptotic ability (4, 42). The treatment methods for blocking this axis include IL-6 receptor antibodies (such as tocilizumab), STAT3 small molecule inhibitors, or JAK kinase inhibitors, which are expected to reverse chemotherapy resistance induced by CAFs. Similarly, TGF - β signaling plays an important role in EMT, tumor stemness, and matrix deposition. Small molecule inhibitors such as Galunisterib (LY2157299) target TGF - β type I receptors and have been studied in various solid tumors, particularly in the CAFs rich microenvironment (43–45).

Another strategy is to physically or functionally clear CAFs by targeting surface markers or lineage specific proteins. FAP is selectively overexpressed in CAFs and osteosarcoma cells, but shows minimal expression in normal fibroblasts (46, 47). In osteosarcoma, FAP has been shown to promote tumor progression by enhancing angiogenesis through activation of the AKT and ERK signaling pathways, and by facilitating cell proliferation, migration, and invasion (48). These findings not only support its role as a prognostic marker associated with poor outcomes (49). Although direct in vivo evidence in osteosarcoma is limited, the presence of FAP ⁺ CAFs is associated with poorer prognosis and stronger drug resistance. Therefore, the use of anti FAP strategies in combination with cytotoxic drugs may improve therapeutic efficacy (50). Therefore, integrating FAP-targeted strategies with conventional chemotherapy may offer a synergistic approach to overcome resistance and improve clinical outcomes in osteosarcoma (47, 48).

In addition to direct clearance, recent studies have also attempted to reprogram activated CAFs into a resting state or anti-tumor phenotype. In osteosarcoma models, recent studies have begun to explore reprogramming activated CAFs into a quiescent or tumor-suppressive phenotype. Vitamin D (via VDR activation) was shown to inhibit EMT and ROS (reactive oxygen species) signaling—key drivers of metastasis and tumor survival—and thus may indirectly modulate stromal fibroblasts toward a less tumor-supportive state in osteosarcoma xenograft models (51). Although direct data on ATRA(All-Trans Retinoic Acid) in osteosarcoma CAFs is still lacking, preclinical evidence from pancreatic and other cancers supports the concept that vitamin A derivatives can normalize CAFs and suppress their pro−tumor secretome (52). And the use of HDAC (histone deacetylase) inhibitors or BET(Bromodomain and Extra-Terminal domain) inhibitors for epigenetic remodeling is also expected to transform CAFs into a low tumorigenic state (4). However, studies on the application of ATRA and HDAC inhibitors in osteosarcoma remain limited, so further research is needed to evaluate their therapeutic potential in this context.

CAFs also plays a role in remote regulation by releasing extracellular vesicles (EVs), which contain miRNA, lncRNA, and proteins. In osteosarcoma, CAFs have been shown to release EVs enriched in non−coding RNAs—particularly miRNAs and lncRNAs—that modulate tumor cell behavior. For instance, CAFs−derived exosomal miR−21−5p promotes OS cell proliferation and chemoresistance by targeting PIK3R1, thereby activating the PI3K/Akt/mTOR pathway (53, 54). Similarly, exosomal lncRNA−SNHG17 transported from CAFs acts as a competing endogenous RNA (ceRNA) to sponge miR−2861, leading to MMP2 upregulation, enhanced migration and invasion of osteosarcoma cells (54). Additionally, lncRNA PVT1—abundant in exosomes from bone marrow–derived mesenchymal stromal cells—has been shown to promote metastasis via the miR−183−5p/ERG axis, and may likewise be relevant in CAFs−EV cargos (53). These non-coding RNAs often dysregulate key signaling nodes such as PTEN/PI3K/AKT under chemotherapy pressure, fostering apoptosis resistance and aggressive phenotypes. Although direct CAFs-specific PVT1 data in OS is still emerging, the functional parallels with SNHG17 and miR-21 support a convergence on PI3K/Akt signaling modulation (53, 55). And potential intervention strategies targeting these EVs include: (1) using drugs such as GW4869 or sphingomyelinase inhibitors to suppress their generation or secretion; (2) blocking osteosarcoma cell uptake of EVs; (3) selectively targeting pathogenic RNA cargo, for example, using antisense oligonucleotides to interfere with SNHG17 or miR-21, to disrupt cancer-promoting EV signaling while maintaining normal stromal cell communication.

The latest research has found that CAFs can inhibit ferroptosis by regulating iron metabolism, promoting glutathione synthesis, and upregulating SLC7A11 (xCT) expression in tumor cells. CAFs helps tumor cells resist oxidative stress induced by chemotherapy by maintaining redox balance (33). Although direct experimental validation in osteosarcoma is currently insufficient, targeting xCT mediated cysteine uptake with inhibitors such as sulfasalazine can make tumor cells more sensitive to ferroptosis inducers. This strategy is expected to overcome drug tolerance dominated by CAFs by disrupting redox homeostasis and metabolic support, especially in tumors with high CAFs activity and strong antioxidant stress resistance (56).