Single-Cell Fixed RNA Profiling of Osteosarcoma Evolution Reveals a Coordinated Multi-cellular Network Driving Metastasis

Osteosarcoma remains a profound clinical challenge, particularly for patients with metastatic or recurrent disease where survival rates have stagnated for decades. In this study, we present the first systematic application of Single-cell Fixed RNA Profiling (FLEX) to the osteosarcoma clinical spectrum. We profiled over 40,000 cells across treatment-naïve, post-chemotherapy, recurrent, and lung metastatic stages. By identifying the ADAMTS12/RNASE1/S100A4/PLVAP multi-cellular network, we move beyond simple cell-type identification toward an integrated understanding of how malignant cells and their microenvironment co-evolve. These findings offer a translational framework for improved risk stratification and the development of next-generation therapies designed to disrupt the interactive networks supporting metastatic osteosarcoma.