Single-cell fixed RNA profiling of the osteosarcoma clinical Spectrum identifies ADAMTS12 and RNASE1 as key drivers of the metastatic niche.

Osteosarcoma (OS) suffers from stagnant survival rates due to high metastatic potential and genomic complexity. Here, we present a high-resolution single-cell transcriptomic atlas of the OS ecosystem using single-cell fixed RNA profiling (FLEX) of 40,401 cells across four clinical stages: treatment-naïve, post-chemotherapy, recurrent, and lung metastatic. We identified a metastasis-enriched malignant subpopulation, OB_3, characterized by aggressive transcriptional signatures and governed by ADAMTS12, which we validated as a prognostic marker for reduced overall survival. Functional in vitro experiments further demonstrated that ADAMTS12 silencing significantly reduced the migratory capacity of HOS and 143B OS cells and suppressed AKT and ERK phosphorylation, thereby supporting its role as an active driver of metastatic progression. Within the microenvironment, metastatic progression was defined by the emergence of RNASE1+ M2-like tumor-associated macrophages, an expansion of highly immunosuppressive S100A4+ regulatory T cells, and the specialization of PLVAP+ stalk endothelial cells driving tumor angiogenesis. Integrative interactome analysis revealed that these populations function as central coordinating hubs, utilizing MIF, SPP1, and Galectin signaling to orchestrate the metastatic niche. Our findings, validated across multiple external cohorts and tissue microarrays, delineate a coordinated multi-cellular network involving ADAMTS12+, RNASE1+, S100A4+, and PLVAP+ cells. This study clarifies how malignant and microenvironmental components co-evolve to facilitate systemic dissemination, providing a translational framework for precision risk stratification and the development of next-generation therapeutic strategies to counteract metastatic OS.