Butyrate derived from intratumoral microbiome facilitates pulmonary metastasis in osteosarcoma

Osteosarcoma, the predominant primary malignant bone tumor, has a dismal prognosis due to pulmonary metastasis, necessitating innovative strategies for early prediction and targeted therapy. While microbial metabolites such as butyrate are increasingly recognized as cancer modulators, their mechanistic involvement in osteosarcoma metastasis remains undefined. Here, we integrated multiomics integration of 16S rRNA sequencing and circulating microbial DNA (cmDNA) profiling to investigate intratumoral microbiome dynamics in osteosarcoma pulmonary metastasis. Metastatic tumors presented significantly reduced microbial diversity, as assessed via the observed Shannon and Simpson indices. Roseburia, Fusobacteriales, and Clostridiales were enriched in the metastatic cohort, whereas Bifidobacteriales and Pseudomonadales predominated in the nonmetastatic cohort. Both intratumoral microbiome signatures and cmDNA levels have emerged as novel prognostic biomarkers for pulmonary metastasis. RNA sequencing and single-cell analysis revealed butyrate metabolism remodeling as a hallmark of metastatic progression. Butyrate supplementation significantly enhanced the migratory capacities of U2OS, 143B and MG-63 cells through activation of the Wnt/β-catenin pathway. In vivo models further confirmed butyrate-induced acceleration of metastasis. Our findings mechanistically correlated intratumoral microbiome-derived butyrate with Wnt/β-catenin-driven osteosarcoma metastasis, suggesting that cmDNA-based liquid biopsy and microbial modulation are precision medicine approaches.