Formononetin suppresses osteosarcoma by targeting MYO1B and remodeling the tumor immune microenvironment

Resistance to and associated toxic side effects of neoadjuvant chemotherapy remain major obstacles to improving the prognosis of osteosarcoma patients. Consequently, there is an urgent need to discover effective therapeutic agents with lower toxicity. In this study, the patient-derived xenograft (PDX) model was established and single-cell multi-omics sequencing was performed to comprehensively analyze changes in cellular heterogeneity and gene expression patterns of under formononetin treatment. We found that formononetin can significantly inhibit tumor growth in the osteosarcoma PDX model, on which the single-cell sequencing identified MYO1B as a key target mediating the anti-osteosarcoma effects of formononetin. <i>In vitro</i> experiments demonstrated that MYO1B overexpression enhanced the proliferation, invasion, and migration of osteosarcoma cells, while MYO1B silencing exhibited the opposite effects. Further investigation revealed that formononetin treatment markedly downregulated MYO1B expression, effectively suppressing the proliferative, invasive, and migratory phenotypes of osteosarcoma cells. Moreover, single-cell transcriptomic analysis of murine-derived cells showed that formononetin enhanced the cytotoxic activity of NK cells, promoted M1 macrophage polarization and inhibited M2 polarization, and reduced the proportion of senescent neutrophils, thereby alleviating the immunosuppressive state of the tumor microenvironment. Overall, our findings provide a comprehensive single-cell-level elucidation of the molecular mechanisms underlying the anti-osteosarcoma effects of formononetin, primarily involving downregulating the expression of MYO1B and remodeling the tumor immune microenvironment.