First, using sequential ultracentrifugation method [27] we isolated extracellular vesicles (EVs) from conditioned media (CM) of ER-α positive MCF-7 breast cancer cells grown (Lep-EVs) or not (C-EVs) in the presence of leptin (500 ng/mL) for 48 h as previously described [12]. Here, LEP is linked to breast cancer.