Neutrophils exposed to a cholesterol metabolite secrete extracellular vesicles that promote epithelial-mesenchymal transition and stemness in breast cancer cells [smRNA-seq]

Small extracellular vesicles (sEVs) are emerging as critical mediators of intercellular communication in the tumor microenvironment. Here, we investigate the mechanisms by which sEVs derived from neutrophils treated with the cholesterol metabolite, 27-hydroxycholesterol (27HC), influence breast cancer progression. sEVs released from 27HC treated neutrophils enhance epithelial-mesenchymal transition (EMT) and stem-like properties in breast cancer cells, resulting in loss of adherence and increased migratory capacity. Decreased miRs within the sEVs resulted in activation of the WNT/ β-catenin signaling pathway in recipient cells and suggest that this may be a predominant pathway for stem-like phenotype and EMT. Our findings underscore a novel mechanism by which 27HC-modulated neutrophils contribute to breast cancer pathophysiology through EV-mediated intercellular communication, suggesting potential therapeutic targets in cancer treatment. Total RNA isolated form sEVs was used for small-RNA-Seq. sEVs were isolated, using comercial SBI kit, from primary neutrophils (Ly6G+ cells; from WT BALB/c female mice) treated with DMSO or 10uM 27HC for 48h. Total RNA from extracellular vesicles was isolated using miRNeasy Micro kit (Qiagen) according to the manufacturer’s protocol with supported modification for small RNA quantities. The quantity and quality of isolated RNA were determined with Qubit Fluorometer (Thermo Fisher Scientific), Bioanalyzer 2100 and Fragment Analyzer (Agilent).