Exosomal shuttling of miR-223 from macrophages to breast cancer cells promotes breast cancer cell invasion. (A) IL-4-activated macrophages promote breast cancer cell invasion. The breast cancer cell lines SKBR3 and MDA-MB-231 were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages. Approximately 24 to 48 h after co-culture, SKBR3 and MDA-MB-231 cells were subjected to an invasion assay. Data are averages of triplicates from more than three independent experiments and are presented as the number of invading cells per field. (B) miR-223 mimics enhanced the invasion of SKBR3 and MDA-MB-231 breast cancer cells. miR-223 was transfected into SKBR3 and MDA-MB-231 cells. Cell invasion was then determined by a transwell invasion assay. Relative invasion activities are presented as fold increases in the miR-223 group. The miR-NC group was normalized to 1.0. (C) Exposure to exosomes derived from macrophages resulted in similar effects on breast cancer invasion as the actual macrophages. SKBR3 cells were incubated with exosomes derived from unactivated or IL-4-activated macrophages and then treated with miR-NC ASO or miR-223 ASO. Cell invasion assays were performed as shown in (A). Data are averages of triplicates from three independent experiments and are presented as the number of invading cells per field. * p < 0.05; ** p < 0.01. (Un-Mac-exosome, exosomes originated from unactivated macrophages; IL-4-Mac-exosome, exosomes from IL-4-activated macrophages).