Formononetin Antagonizes Osteosarcoma Metastasis by Targeting CD44: An Integrated Multi-Omics Study.

BACKGROUND: The leading cause of death in osteosarcoma (OS) is metastasis, to which chemoresistant cell subpopulations contribute. A major obstacle is the limited understanding of the metastatic mechanisms of these cells and the identification of effective drugs. PURPOSE: This research aimed to identify a chemoresistant, metastasis-prone OS subpopulation, elucidate its pro-metastatic mechanism via endothelial dysfunction, and investigate the natural compound formononetin (FMN) as a potential inhibitor. STUDY DESIGN AND METHODS: We performed an integrated analysis of bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) data from OS patients to characterize tumor heterogeneity and identify key target. In silico molecular docking and molecular dynamics simulations were utilized to predict the binding interaction between FMN and the candidate target, which was further validated via CETSA assays. We performed in vitro functional verification including Transwell assays, transendothelial migration assays and conditional co-culture experiments in two OS cell lines. Finally, in vivo animal experiments were carried out to verify the lung metastatic capacity mediated by CD44 as well as the anti-metastatic effect of FMN against lung metastasis. RESULTS: ScRNA-seq analysis revealed a chemoresistant tumor cell cluster (TC1) and TC1 cells exhibited close spatial proximity to a specific endothelial cell subtype (C5) associated with metastasis. Cellphone DB analysis identified the CD44 as a critical target of TC1-induced endothelial dysfunction. Proprietary algorithm analysis confirmed predominant CD44 expression by TC1 cells. Computational modeling demonstrated a stable binding interaction between CD44 and FMN with high affinity (-7.0 kcal/mol), and CETSA assays yielded a ΔTm50 = 3.2 °C. In vitro, CD44 overexpression enhanced OS cell migration, invasion, and endothelial barrier disruption. FMN treatment significantly inhibited these phenotypes in a concentration-dependent manner. Crucially, rescue experiments confirmed that FMN specifically attenuated the enhanced metastatic capabilities induced by CD44 overexpression. In vivo experiments showed that CD44 overexpression promoted lung metastasis, whereas FMN reversed the lung metastasis induced by CD44 overexpression. CONCLUSION: Our research uncovers a novel mechanism whereby chemoresistant OS cells facilitate metastasis via CD44-mediated endothelial dysfunction. We demonstrated that FMN is a highly promising therapeutic agent, which likely acts by directly binding to the CD44 protein, thereby inhibiting OS cell transendothelial invasion. This research provides new insights into the treatment of metastatic chemoresistant OS.