Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles

Colorectal cancer (CRC) with mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by accumulation of cancer-associated fibroblasts in the stroma. We investigated whether epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knock down, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF ric