Images showed that the exosomes were between 30-100 nm in diameter and had a round with cup-like concave morphology (Figure ?(Figure2A),2A), consistent with known exosome morphology 6. in some patients. Our results suggest that while exosomes derived from macrophages normally inhibit proliferation and metastasis of MCF-7 or MDA-MB-231 cells, exposure of macrophages to breast cancer cells that have experienced chemotherapy are modified them to promote these processes. Exosomes from macrophages exposed to apoptotic cancer cells have increased amounts of IL-6 that increases the phosphorylation VU661013 of STAT3, which likely explains the increased transcription of STAT3 target genes such as CyclinD1, MMP2 and MMP9. These observations suggest that the inhibition of exosome secretion and STAT3 signaling pathway activation might suppress the growth and metastasis of malignant tumors, and provide new targets for therapeutic treatment of malignant tumors after chemotherapy. apoptotic cancer cell model. Results from flow cytometry showed that H2O2 induced apoptosis in the MCF-7 cells, with 0.3 mM H2O2 treatment for 24 h generating nearly 100% apoptosis in these cells (Figures ?(Figures1B-C),1B-C), with results from the cisplatin induced-apoptotic MCF-7 cells shown in Figures S1A-B. All further experiments used 0.3 mM H2O2 or 25 M cisplatin to generate apoptotic breast cancer cells. Open in a separate window Figure 1 Verification of THP-1-derived macrophages and apoptosis of MCF-7 cells in vitro. (A) Cells were fixed and immunolabeled for the detection of CD163, CD68, CD204 and CD206 using specific antibodies (labeled green or red). Nuclei were stained with DAPI VU661013 (blue). Scale bar is 50 m. (B) Flow cytometry analysis of Annexin V-FITC/PI co-stained untreated (left panel) and apoptotic (right panel) MCF-7 cells. (C) Quantification of the flow cytometry results. Results are typical of three independent experiments. Data represent means S.E. ( s) (n=3). Rabbit polyclonal to PLD4 *** p<0.001 indicates statistical VU661013 significance of the apoptosis group to the untreated group. Characterization of exosomes by TEM and Western blotting We characterized exosomes that had been purified by ultracentrifugation by TEM. Images showed that the exosomes were between 30-100 nm in diameter and had a round with cup-like concave morphology (Figure ?(Figure2A),2A), consistent with known exosome morphology 6. In addition, Western blotting showed that the isolated exosomes were enriched with HSP70, TSG101 and CD9 (Figure ?(Figure2B),2B), which is consistent with previous reports 30, thus we concluded that both macrophages and macrophages exposed to apoptotic MCF-7 cells produce exosomes. Open in a separate window Figure 2 Characterization of exosomes and the co-culture of macrophage-derived exosomes increased the proliferation of MCF-7 cells. (A) Exosomes isolated from macrophages (Mac-exo) and co-cultured macrophages (Co-exo) imaged by TEM are approximately 100 nm in size. Scale bar = 100 nm. (B) Levels of exosome markers HSP70, TSG101 and CD9 in Mac-exo (left lane) and Co-exo (right lane) groups were determined by Western blotting. (C) Confocal microscopy visualization of exosomes taken up by MCF-7 cells. Exosomes were stained with DiD dye, and then co-cultured with MCF-7 cells for 24 h, 48 h and 72 h. Nuclei were stained with DAPI. Scale bar = 25 m. (D) Proliferation of MCF-7 cells in control (CON), MCF-7Mac-exo and MCF-7Co-exo groups was measured over 72 h by MTS assay. Results are typical of three independent experiments. Data represent means S.E. ( s) (n=3). * p<0.05, ** p<0.01 and *** p<0.001 indicate statistical significance in comparisons of the MCF-7Mac-exo and MCF-7Co-exo groups to the CON group. ### p<0.001 indicates statistical significance in comparisons of the MCF-7Co-exo group with the MCF-7Mac-exo group. Co-exo increases proliferation ability of breast tumor cells We next identified whether Mac-exo and Co-exo exosomes were taken up by MCF-7 cells. MCF-7 cells were incubated with exosomes labeled with DiD dye for 72 h. DiD staining was observed by confocal microscopy in the MCF-7 cells exposed to both Mac-exo (top panels) and Co-exo (lower panels) exosomes (Number ?(Figure2C)2C) at 24 h, 48 h and 72 h. Over time, the DiD dye appeared in the MCF-7 cells, having a maximum at 48 h and lower slightly lower levels at 72 h. These data demonstrate that MCF-7 cells efficiently internalize these two groups of exosomes. Next, to further investigate the effects of these two groups of exosomes within the proliferation of MCF-7 cells, we co-cultured MCF-7 cells, in DMEM medium, with 10% exosome-depleted FBS, with 100 g/ml of added exosomes for 72 h..