The paracrine Ephrin A4/BMP4 feed-forward loop sustains a tumor–CAF vicious cycle in osteosarcoma

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Abstract Background Advanced osteosarcoma (OS) remains a clinical challenge due to its aggressive behavior and poor prognosis. Cancer-associated fibroblasts (CAFs) have been shown to contribute to tumor progression, but the tumor–CAF crosstalk in OS is still poorly defined. Elucidating these interactions could reveal novel therapeutic targets. Methods Human OS surgical samples and cell lines were implanted into immunocompromised mice to generate 10 patient-derived (PDXs) and 10 cell line-derived xenografts (CDXs), respectively. RNA-sequencing was performed on the xenografts to distinguish murine stromal from human tumor transcripts. Stromal subpopulation metagenes and matched tumor-stroma ligand-receptor pairs were analyzed. The expression and roles of the candidate ligands were validated using histological analyses, RT-qPCR, Western blot, ELISA, proliferation, migration, invasion, sphere-forming, and RNA silencing assays. Their prognostic relevance was evaluated in human OS samples using a publicly available dataset with clinical annotations and by immunohistochemical analysis. Results Stromal contribution and tumor growth varied across OS xenografts, with no significant differences between PDXs and CDXs. CAF, white blood cell, and endothelial cell metagenes correlated with stromal content, and myofibroblastic CAFs were the most prevalent subpopulation. The Ephrin A4-Epha2-Bmp4 axis was strongly associated with CAF content. Conditioned medium (CM) from OS cells, which can generate xenografts with high-CAF (HC) content, promoted normal fibroblast (NF) activation and CAF proliferation, migration, and invasion more than CM from low-CAF (LC) cells. Similar effects were observed upon direct treatment with soluble Ephrin A4, but not with CM from Ephrin A4-silenced HC OS cells. In turn, CAF-derived CM or direct treatment with soluble BMP4, but not the CM from BMP4-silenced CAFs, enhanced proliferation, migration, and invasion, and increased stem-like traits in HC OS cells. Ephrin A4 induced EphA2 expression in NFs and BMP4 production in CAFs, whereas BMP4 stimulated Ephrin A4 production in HC OS cells, establishing a tumor-stroma feed-forward loop. The co-expression of human Ephrin A4 and murine BMP4 mRNA was confirmed in HC OS xenografts. Tumor Ephrin A4 and stromal BMP4 were more highly expressed in HC human OS, characterized by higher stromal alpha-smooth muscle actin (αSMA) expression, as well as in OS patients with poor prognosis. Conclusion Tumor-derived Ephrin A4 and stromal-derived BMP4 generate a feed-forward loop in OS, sustaining tumor aggressiveness. This axis represents a promising therapeutic target that warrants further translational investigations.