Single-Cell and Spatial Profiling of Tumor Microenvironment Heterogeneity in Human Osteosarcomas

Osteosarcoma, a highly heterogeneous malignant bone tumor, exhibits substantial interpatient and intratumoral heterogeneity. Utilizing integrated single-cell RNA sequencing and spatial transcriptomics, we uncovered distinct tumor microenvironment (TME) transcriptional profiles across patients, highlighting profound interpatient heterogeneity. Intratumorally, malignant cells primarily followed two differentiation trajectories, converging into osteoblastic and chondroblastic functional states, and hypoxia may be the influencer; notably, within individual patients, tumor cells exhibited greater transcriptional similarity driven by specific transcription factors, despite these divergent states. Spatial analysis revealed patient-specific TME cellular co-localization patterns, alongside conserved spatial relationships: vascular components (endothelial cells, pericytes) demonstrated strong co-localization, while immune cells (T cells, myeloid cells) clustered within shared regions. Crucially, these functional states occupied distinct microniches: osteoblastic regions were enriched with osteoclasts, vascular components, and immune cells, whereas chondroblastic regions displayed the opposite composition and were preferentially located in hypoxic, vascular-poor niches, exhibiting significant enrichment of hypoxia-related signaling pathways. Furthermore, our data suggest osteosarcoma cells may activate fibroblasts via the SPP1 signaling pathway, indicating fibroblasts act as key intermediaries in tumor-directed TME modulation. This study comprehensively delineates the intricate landscape of osteosarcoma heterogeneity, defining distinct functional cellular states, their spatially organized TME niches, and a potential SPP1-mediated tumor-fibroblast regulatory axis. This comprehensive analysis elucidates the intricate interpatient and intratumoral heterogeneity in osteosarcoma, revealing functional and spatial organization within the tumor and its microenvironment.