Fig. 2
EMT-CRC exosomes increase vascular endothelial cell permeability and facilitate invasion by disrupting VE-cadherin expression. (a) Electron microscope images of exosomes isolated from the conditioned medium of cultured Nor-HCT116 and EMT-HCT116 cells (scale bar = 200 nm). (b) Nanoparticle tracking analysis of isolated exosomes. (c) Western blot analysis of exosomal membrane markers CD63 and TSG101. (d) Schematic illustration of the permeability analysis of human umbilical vein endothelial cells (HUVECs). Upper panel: Permeability assessment was conducted using rhodamine-labeled dextrose molecular probes capable of passing through a developing HUVEC monolayer membrane on a filter. Lower panel: The number of tumor cells that infiltrated the HUVEC monolayer on the filter was calculated. (e) HUVEC monolayer membranes were pretreated with Nor-HCT116/EMT-HCT116-derived exosomes (50 µg; 10 µg/mL per 1 × 105 cultured cells) for 24 h. Then, rhodamine–dextran was added to the upper chamber and incubated for 60 min. Subsequently, dextran content was detected in the lower chamber at an optical density (OD) of 590 nm. (f, g) HUVEC monolayer membranes were pretreated with Nor-HCT116/EMT-HCT116-derived exosomes (50 µg; 10 µg/mL per 1 × 105 cultured cells) for 24 h; then, the TEER and CI values were calculated. (h) HUVECs were incubated with Nor-HCT116/EMT-HCT116-derived exosomes (50 µg; 10 µg/mL per 1 × 105 cultured cells) for 24 h. The number of tumor cells that invaded through the HUVEC monolayer was determined. (i, j, k) HUVECs were incubated with Nor-HCT116/EMT-HCT116-derived exosomes (50 µg; 10 µg/mL per 1 × 105 cultured cells) for 4 h. The cell membrane VE-cadherin, ZO-1, and occludin levels were measured using western blotting, flow cytometry, and immunofluorescence (scale bar = 25 μm). Data are expressed as mean ± standard deviation (n = 5). **P < 0.01, compared with the control group; ##P < 0.01, compared with the Nor-HCT116 group. (l, m) HUVECs were incubated with exosomes derived from EMT HCT116 or EMT HCT116 + Annexin V cells. Dextran content was detected (OD 590 nm) in the lower chamber, and the number of tumor cells invading the HUVEC monolayer was determined. (n, o) HUVECs incubated with exosomes derived from EMT HCT116 or EMT HCT116 + Annexin V cells. TEER and CI values were calculated. (p–r) HUVECs were incubated with exosomes derived from EMT HCT116 or EMT HCT116 + Annexin V cells. Cell membrane VE-cadherin levels were measured using western blotting, flow cytometry, and immunofluorescence (scale bar = 25 μm). Data are expressed as mean ± standard deviation (n = 5). **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the EMT-HCT116 group