Exosomes derived from Piwil2-induced cancer stem cells transform fibroblasts into cancer-associated fibroblasts

Recently, several studies have demonstrated that cancer cell-derived exosomes can facilitate tumor development and metastasis formation. However, the detailed function of exosomes released by cancer stem cells (CSCs) requires further investigation. The aim of the present study was to investigate the role of CSC-derived exosomes in tumor development. For this purpose, Piwil2-induced cancer stem cells (Piwil2-iCSCs) were used as exosome-generating cells, while fibroblasts (FBs) served as recipient cells. Exosomes were isolated by the ultracentrifugation of Piwil2-iCSC-conditioned medium and identified by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. To evaluate the effects of the exosomes on cell proliferation, migration and invasion, cell counting assay (CCK-8), a wound healing assay and a Transwell assay were performed. Protein expression [matrix metalloproteinase (MMP)2, MMP9, alpha-smooth muscle actin (alpha-SMA) and vimentin and fibroblast-activating protein (FAP)] was examined in FBs by western blot analysis. It was found that the Piwil2-iCSC-derived exosomes (Piwil2-iCSC-Exo) were oval or spherical, membrane-coated vesicles with a uniform size (30-100 nm in diameter). They are characterized by the surface expression of CD9, CD63, Hsp70 and Piwil2 proteins. Additional results from functional analyses revealed that Piwil2-iCSC-Exo enhanced the proliferative, migratory and invasive abilities of FBs, accompanied by the upregulated expression of MMP2 and MMP9. In addition, the increased expression of alpha-SMA (P<0.05), vimentin (P<0.01 vs. control group, P<0.05 vs. PBS group) and FAP (P<0.001 vs. control group, P<0.01 vs. PBS group) following exposure to Piwil2-iCSC-Exo suggested that the exosomes induced FB transformation into cancer-associated fibroblasts (CAFs). On the whole, the findings of this study demonstrate that Piwil2-iCSC-Exo induce the cancer-associated phenotype in fibroblasts in vitro, suggesting that CSCs can promote tumor development through the modulation of the tumor microenvironment.