EXPLORING THE DYNAMICS OF OSTEOSARCOMA COMUNICATION BY EXOSSOMES

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Its progression is strongly influenced by interactions between tumor cells and the bone microenvironment, including osteoclasts, which play a central role in bone remodelling. Small extracellular vesicles (sEVs) are important mediators of intercellular communication and may contribute to tumor progression. This study investigated the proteomic profiles of sEVs derived from osteosarcoma cell lines and evaluated their effects on osteoclast differentiation and function. Small extracellular vesicles were isolated from three human osteosarcoma cell lines (MG63, U2OS and SAOS-2) and a non-tumoral human osteoblast cell line (HOB). Proteomic characterization was performed by mass spectrometry-based analysis, followed by protein–protein interaction (PPI) network construction using STRING and Cytoscape and module identification through Markov clustering (MCL). To investigate the biological effects of sEVs, osteoclast differentiation was assessed by TRAP staining, and bone-resorbing activity by resorption assays. Cathepsin K expression was evaluated by Western blotting. Proteomic analyses revealed distinct molecular signatures among the different cell lines. HOB-derived sEVs were enriched in proteins associated with extracellular matrix organization, collagen biosynthesis, growth factor signalling and tissue homeostasis. MG63-derived sEVs exhibited highly interconnected networks related to ribosome biogenesis, rRNA processing and translational activity, consistent with a proliferative phenotype. U2-derived sEVs displayed smaller modules associated with p53, TGF-β, PPAR and NF-κB signalling pathways, suggesting adaptive regulatory responses. SAOS-2-derived sEVs were enriched in extracellular matrix remodelling proteins, including collagens, integrins and proteoglycans, indicative of an aggressive and invasive phenotype. Functional assays also demonstrated distinct effects of tumor-derived sEVs on osteoclast biology. MG63-derived sEVs increased bone resorption activity, whereas U2-derived sEVs inhibited osteoclast differentiation. These findings were accompanied by alterations in cathepsin K expression, indicating modulation of osteoclast maturation and resorptive function. Osteosarcoma-derived sEVs reflect the intrinsic molecular programs of distinct tumor phenotypes, highlighting the complexity of tumor–bone communication and suggests that sEV-mediated signaling may contribute to the heterogeneous remodeling of the bone microenvironment during osteosarcoma progression.