Integrative Single-Cell and Bulk Transcriptomics Identifies a Proliferative Malignant State and Supports a Functional...

Osteosarcoma contains heterogeneous malignant-cell states whose clinical relevance is difficult to infer from small single-cell cohorts.

We reanalyzed six treatment-naive osteosarcomas from GSE162454, identified CNV-supported malignant osteoblastic cells, and characterized a recurrent proliferative/mitotic state using patient-paired pseudobulk analysis and program-selection sensitivity tests. A 50-gene module was evaluated for association with metastatic occurrence in 53 pretreatment tumors from GSE21257. Candidate ranking was audited across transparent weighting scenarios, and TROAP was depleted with two independent siRNAs in HOS and U2OS cells.

Of 52,489 input barcodes, 39,063 singlets were retained, including 10,456 CNV-supported malignant osteoblastic cells. M6 comprised 725 cells and was detected in all six tumors. It ranked first in six of eight program-selection scenarios but fell to fifth when G2M checkpoint and E2F targets were jointly excluded, supporting its interpretation as a proliferative/mitotic state. Its weighted module was higher in metastatic tumors (p = 0.0147; apparent AUC = 0.704), but the association was attenuated after macrophage adjustment (OR = 1.646, 95% CI 0.649-4.170; p = 0.2937), whereas the macrophage score remained inversely associated with metastasis (OR = 0.251, 95% CI 0.081-0.773; p = 0.0161). TROAP was strongly enriched in M6 and supported by paired pseudobulk analysis, but it was not the top-ranked candidate under every weighting scenario. TROAP depletion reduced colony formation, overall wound closure, and Transwell migration and invasion in both cell lines.

A recurrent proliferative/mitotic malignant state is detectable across the six osteosarcomas and yields a bulk expression program associated with metastatic occurrence, although this association is sensitive to macrophage composition. TROAP is supported as a functional contributor to osteosarcoma cell phenotypes rather than an established driver of the state or an independently validated clinical marker.