Unveiling the role of the extracellular matrix in the osteosarcoma tumor microenvironment through integrated transcriptomics and experimental validation.

Osteosarcoma (OS), the most common malignant bone tumor, is characterized by heterogeneous tumor cells and abundant microenvironmental components. The extracellular matrix (ECM)-a complex and dynamic network surrounding tumor cells-plays a pivotal role in OS malignancy (e.g., cell proliferation, metastasis), making insights into ECM involvement critical for advancing OS prognosis. This study conducted bioinformatic analyses on bulk RNA-sequencing and single-cell RNA sequencing data from public databases, initially identifying collagen type V alpha 2 (COL5A2) as a key gene in OS progression. It further validated biological functions and underlying mechanisms of COL5A2 via in vitro experiments, and constructed and validated prognostic models based on ECM signature cell clusters. Results identified osteoblastic cells (OCs) and endothelial cells (ECs) as core cellular components of OS. COL5A2 was highly expressed in OCs, and high COL5A2 expression correlated with significantly reduced overall survival in OS patients. Western blot, CCK-8, and colony formation assays demonstrated that COL5A2 promoted OS cell proliferation by activating the focal adhesion pathway and inducing phosphorylation of the FAK/Paxillin/Akt signaling axis. The prognostic model highlighted the C0 OCs cluster as clinically significant. CellChat analysis uncovered significant activation of the IGFBP pathway in both C0 OCs and C1 ECs, and identified the IGFBP3-TMEM219 axis as the key ligand-receptor pair mediating their crosstalk. This study establishes COL5A2 and the C0 OCs cluster as pivotal ECM-related signatures in OS, confirming COL5A2 drives OS proliferation through focal adhesion signaling and IGFBP3-TMEM219-mediated crosstalk-both representing promising therapeutic targets. Further investigation into ECM components is warranted to refine OS treatment strategies and improve clinical outcomes.