Extracellular vesicles of cancer cells induce FOXP3+ fibroblasts and facilitate tumor invasion via the Wnt3-β-catenin pathway

Forkhead-box-protein P3 (FOXP3) is a key transcription factor in T regulatory cells (Tregs). However, its expression and significance in non-immune stromal cells in the tumor microenvironment remain unclear. Here, we demonstrated FOXP3 expression in stromal fibroblasts of mouse and human gastrointestinal tumors. Immunohistological examination revealed FOXP3 expression in αSMA⁺ collagen I⁺ myofibroblasts. In the mouse omentum inoculated with gastric cancer cells, cytokeratin⁽⁻⁾/CD45⁽⁻⁾/FoxP3⁽⁺⁾ stromal cells were identified via flow-cytometry, and high FOXP3 expression was noted in fibroblasts surrounding the tumor glands, where CD8⁺ T cells were exclusively infiltrated. Extracellular vesicles (EVs) from mouse gastric cancer cells upregulated Foxp3 transcription in fibroblasts, which partly depends on increase of transcription factors including NFAT1 and c-Rel, and activation of TGF-β and STAT5 pathways. In FOXP3⁽⁺⁾ fibroblasts, immunosuppressive cytokines including IL-10 and CCL2 were upregulated. FOXP3 overexpression in NIH/3T3 fibroblasts enhanced Wnt3a-induced β-catenin responses, accompanied by cell growth and tumor invasion in mice stomach. As the mechanism, FOXP3 induced CDH11 expression in fibroblasts, which augmented the Wnt3/β-catenin pathway, and blocking of CDH11 suppressed tumor invasion mediated by FOXP3⁽⁺⁾ fibroblasts. Our results suggest that cancer cell-derived EVs regulate FOXP3 expression in stromal fibroblasts, attenuating antitumor immunity, and facilitating tumor invasion.