Single-Cell Transcriptomics Uncovers β-Elemene-Induced Macrophage Reprogramming as a Mechanism to Suppress Osteosarcoma Stemness via the CCL2-Akt Axis.

BACKGROUND: β-elemene (ELE), a natural sesquiterpene with antitumor properties, exerts immunomodulatory effects on osteosarcoma (OS), but its impact on the tumor microenvironment and cancer stemness remains unclear. This study investigated how ELE reprograms tumor-associated macrophages (TAMs) to suppress OS progression. METHODS: We combined single-cell RNA sequencing (scRNA-seq) of a murine OS model with functional validations, including macrophage polarization assays, co-culture systems, western blot, RT-PCR, ELISA, tumorsphere formation, and rescue experiments (CCL2 overexpression, Akt activation). RESULTS: ELE treatment significantly inhibited OS tumor growth and promoted apoptosis. scRNA-seq revealed that ELE skewed TAMs from immunosuppressive M2 to antitumor M1 phenotypes, concurrently reducing the osteosarcoma stem-like cell (OSC) population. Cell communication analysis showed enhanced M1-OSC pro-inflammatory signaling (e.g. TNF, IL-1) and suppressed M2-derived CCL2 signaling. Mechanistically, ELE downregulated CCL2 secretion from M2 macrophages, inhibiting Akt phosphorylation and subsequent β-catenin activation, which led to reduced expression of stemness markers (SOX2, NANOG, CD133) and impaired OSC self-renewal and invasion. These effects were reversed by CCL2 overexpression or Akt activation. CONCLUSION: ELE attenuates OS stemness by polarizing TAMs toward an M1 phenotype and disrupting the CCL2/Akt/β-catenin axis, revealing a novel immunometabolic mechanism for OS therapy.