Figure 4
NSCLC-derived exosomes at early and advanced stages affect cell proliferation and induce angiogenesis and cell transformation. (A) Representative fluorescence image of NSCLC-derived exosome (exo-NSCLC) uptake by human bronchial epithelial cells (BEAS-2B). BEAS-2B cells, cultured in exosome-depleted serum, were incubated with acridine orange (AO)-labelled exo-NSCLC (20 µg/ml), and their internalization was visualised using a fluorescent microscope (Zeiss, Axiocam MRc5; magnification 400–600 x). Scale bar for all images equals 100 μm. (B) Cell proliferation (left panel) and colony-forming assay (right-panel) of BEAS-2B cells incubated with healthy subject-derived exosomes (CTRL), and NSCLC-derived exosome at early (exo-NSCLC Stage I/II) and advanced (exo-NSCLC Stage III/IV) stages. Representative images at 3 months after treatment are shown (magnification 100 x). (C) EAhy926 cells and their anti-miR-transfected counterparts were seeded in 96-well plates with 50 μl of Matrigel per well so that suspension of 200 μl of complete medium with 2 × 104 cells were added to each well. Control cultures as well as those incubated with exosomes from healthy subjects (CTRL) and NSCLC-derived exosome (20 μg/ml) at early (exo-NSCLC Stage I/II) and advanced (exo-NSCLC Stage III/IV) stages were evaluated by counting the number of complete tubes connecting points of individual polygons of the capillary network in a light microscope at 24 h (lower panel). The data and images are relative of three independent experiments. Comparisons among groups were determined by one-way ANOVA with Tukey post-hoc analysis. The symbol “*” indicates significant differences compared with control, and the symbol “°” indicates significant between CTRL and anti-miR, with p < 0.05.
