FOXC2 regulates macrophage polarization and osteosarcoma progression via the CXCL12 / CXCR4 signaling axis

Forkhead box protein C2 (FOXC2) is frequently overexpressed in osteosarcoma (OS) and has been implicated in tumor proliferation and metastasis; however, its role in regulating the tumor immune microenvironment remains poorly understood. In this study, integrative bioinformatics analyses of the TCGA-SARC (The Cancer Genome Atlas sarcoma cohort) dataset were performed to evaluate the clinical significance of FOXC2 in OS. High FOXC2 expression was significantly associated with poorer overall survival in patients with OS (p < 0.001). Functional experiments demonstrated that FOXC2 knockdown significantly reduced the overall viable cell/metabolic signal, migration, and invasion in U-2 OS and SaOS-2 cells (p < 0.05). Mechanistically, FOXC2 was found to regulate the CXCL12/CXCR4 signaling axis. ELISA, RT-qPCR, and western blotting analyses showed that FOXC2 knockdown decreased CXCL12 expression, whereas FOXC2 overexpression significantly increased CXCL12 levels. ChIP-qPCR analysis demonstrated significant enrichment of FOXC2 at the CXCL12 promoter and detectable enrichment at the CXCR4 promoter in SaOS-2 cells, and dual-luciferase reporter assays further showed that FOXC2 overexpression significantly increased CXCL12 promoter activity whereas FOXC2 knockdown reduced it, supporting direct transcriptional regulation of the CXCL12/CXCR4 axis by FOXC2. In macrophage co-culture experiments, FOXC2 knockdown promoted M1-like macrophage polarization and reduced M2-like macrophage markers, whereas activation of CXCR4 partially reversed these effects. Flow cytometric analysis further confirmed that FOXC2 knockdown significantly increased the M1/M2 macrophage ratio. In vivo, FOXC2 knockdown markedly reduced tumor growth and lung metastases in a xenograft mouse model, with tumor volume and lung metastatic burden reduced by approximately 50% and 43%, respectively. Tumor tissues from FOXC2-knockdown mice exhibited increased M1-like macrophage infiltration and reduced M2-like macrophage markers. Together, these findings demonstrate that FOXC2 regulates macrophage polarization and OS progression through transcriptional modulation of the CXCL12/CXCR4 signaling axis, highlighting FOXC2 as a potential regulator of the tumor immune microenvironment in OS. © 2026 The Pathological Society of Great Britain and Ireland.