Fig. 3

GC-derived exosomes promote angiogenesis via M2-like macrophages to facilitate liver metastasis. A. The supernatants from macrophages pre-incubated with GC cell-derived exosomes or PBS were examined to determine the secretion of TGF-β, VEGFA, and VEGFD by ELISA. B, C. Transwell assays evaluated the migration ability of HUVECs cocultured with macrophages treated with PBS, MKN45 exosomes or MKN45-HL exosomes. Representative images of migratory cells were shown. Scale bar = 200 μm. D. Tube formation of HUVECs cocultured with conditioned macrophages were determined. Scale bar = 200 μm. E, F. Aortic rings were cultured in condition media, and vascular outgrowth was quantified by counting all sprouts from one ring. Three culture environments were used: (Naked) exosomes were added directly to the culture medium of the vascular ring; (Exo) supernatant of PMA-treated THP-1 cells incubated with exosomes; (Exo + Pan) Panobinostat (0.0034 μM) was added while THP-1 was incubated with exosomes, and the supernatant was then extracted for culture of the vascular rings. Scale bar = 500 μm. G. Mice were administered Panobinostat (20 mg/kg every 2 days) intraperitoneally while receiving exosome education, and angiogenesis in the liver was observed two weeks later by immunohistochemistry with CD31. Scale bar = 100 μm. H. Mice educated with exosomes were simultaneously treated with KRN-633 (50 mg/kg, gastric lavage every 2 days) or DMSO followed by portal vein injection with luciferase-labeled MKN45 cells. The fluorescence signal in the metastases was detected and quantified using an in vivo imaging system (IVIS). I, J. The mice were then sacrificed, and their livers were photographed, weighed, and immunostained for CD31. Data are shown as mean ± standard deviation of 3 independent experiments, and statistical significance was determined using one-way ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001)