Heterogeneity in Osteosarcoma Revealed by scRNA Sequencing: CLU+ Endothelial Cells as Key Players in Tumor Progression

Osteosarcoma is a common malignant bone tumor in children and adolescents, characterized by high heterogeneity and poor prognosis. The role of intratumoral heterogeneity (ITH) in osteosarcoma progression remains poorly understood. To explore this, we collected paired tumor tissue samples from the central region (CR) and peripheral region (PR) of six osteosarcoma patients and performed single-cell RNA sequencing. Our findings reveal significant microenvironmental differences between these regions. The CR harbors a higher proportion of tumor cells, while the PR contains a higher proportion of endothelial cells, particularly the CLU+ subcluster. Functionally, the CR hosts a higher proportion of immune-activated myeloid cells and tumor-infiltrating lymphocytes (TILs), whereas the tumor cells in the PR show increased activation of hypoxia-related pathways. In the PR, CLU+ endothelial cells (CLU+_ECs) promote tumor metastasis by interacting with tumor cells through various ligands, including collagen family members, via ITGB1. Furthermore, CLU+_ECs induce CD8+ T cell exhaustion via the Nectin2-TIGIT pathway, suppressing the anti-tumor immune response. Overall, our study highlights the substantial spatial heterogeneity in osteosarcoma and identifies CLU+_ECs in the peripheral region as promising therapeutic targets.