Functional microRNA is shuttled from macrophages to breast cancer cells. (A) Schematic illustration of a miRNA transfer model. Macrophages were preloaded with Cy3 fluorescently-labeled miR-223 or the non-mammalian miRNA lin-4. A transwell system with a 0.4- μm pore size film, which allows small size materials (e.g., miRNAs), but not cells, to pass through, was used to separate SKBR3 cells from macrophages. (B) and (C) SKBR3 cells were cultured alone (blank) or co-cultured with macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. The Cy3 fluorescence signal in SKBR3 cells was determined by fluorescence microscopy (B), and the percentage of Cy3-positive cells in the images was calculated in (C). Arrowheads indicate Cy3-positive SKBR3 cells, and images are shown at 200×. (D) SKBR3 cells were cultured as shown in (B), and Cy3-positive cells (%) were quantified by flow cytometry. (E) A lin-4-targeting luciferase reporter gene carrying a lin-4 complementary sequence in the 3'-UTR was transfected into SKBR3 cells. SKBR3 cells were then co-cultured in a transwell with macrophages pre-transfected with miR-NC or lin-4. SKBR3 cells that were directly transfected with lin-4 were used as a control. Luciferase activities in SKBR3 cells were measured, and relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).