Abstract: Objective To explore the effect of M2 macrophage-derived exosomes on stemness of 4T1 cells. Method M2 macrophage-derived exosomes were isolated by ultracentrifugation. And transmission electron microscopy, nanoparticle tracking technology and Western blot were performed to identified the exosomes. Then, the M2 macrophage-derived exosomes were labeled with Dil which makes it available for us to observe their transfer to breast cancer cell line 4T1 by immunofluorescence. After that, flow cytometry, co-culture system and transwell chamber were used to investigate the tumor phenotype and stemness of 4T1 under the influence of M2 macrophage-derived exosomes. Results First, the M2 macrophages were successfully induced in vitro. In addition, the M2 macrophage-derived exosomes were successfully isolated and verified. M2 macrophage-derived exosomes labeled with Dil could be transferred into 4T1 cells. After co-incubation with M2 macrophage-derived exosomes, the migration (P<0.001) and sphere formation (P<0.05) ability of 4T1 cells were significantly enhanced. While after using exosome inhibitor GW4869, the number and volume of spheres formed by 4T1 stem cells were reduced (P<0.05). The proportion of CD44highCD24low cells in 4T1 cells (P<0.001) was significantly increased after incubating with M2 macrophage-derived exosomes. Conclusions M2 macrophage-derived exosomes can increase the migration and sphere formation ability of 4T1 tumor cells, so is the proportion of tumor stem cells in 4T1 cells.

Key words: M2 macrophage, exosome, migration, sphere formation, tumor stem cell