Neutrophils exposed to a cholesterol metabolite secrete extracellular vesicles that promote epithelial-mesenchymal transition and stemness in breast cancer cells [RNA-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/ ß-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. Overall design: Total RNA was used for bulk RNA-Seq from 2D culture of 4T1 cells in complete media with EVs depleted FBS (Gibco). Cells were treated with small extracellular vesicles (sEVs) for 48h and 72h. 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. Cells after 27HC_sEVs lost adherence and started to float. Two subpopulation of cells after that treatment was used: adherent and non-adherent. 5 replicates from each treatment group was performed.