Figure 1.
Exosomes characterization and MSC internalization.
A. The morphology of exosomes and exosomal marker expression. (a) Morphologic analysis of gastric cancer derived exosomes. Scale bar = 100 nm. (b) CD9 and CD81 expression in exosomes. B. Exosomes were uptaken by hucMSCs. Exosomes labeled with CM-Dil (red) at 37°C for 1 h were added into hucMSCs and incubated for 4 h. Effluent from a filtered suspension of CM-Dil-labeled exosomes (control) and the CM-Dil-labeled withdrawn exosomes fraction (e-exos) were used as controls. Cells were fixed and stained for cytoplasm (cy2-actin, green) and nuclei (DAPI, blue). Scale bar = 20 µm.
Figure 2.
Gastric cancer cell derived exosomes induces the expression of CAF markers in hucMSCs.
A.Quantitative analyses of the FAP, IL-6 and α-SMA mRNA expression in hucMSCs. HucMSCs were treated with various concentrations of SGC7901 exosomes or GES-1 exosomes for 36 hours. B. Quantitative analyses of the FAP, IL-6 and N-Cadherin expression in hucMSCs. HucMSCs were treated with various concentrations of SGC7901 exosomes or GES-1 exosomes for 14 days. C. Western blotting analyses of the FAP, α-SMA, N-Cadherin and Vimentin protein expression in hucMSCs treated with SGC7901 exosomes (800 µg/ml) for different times. (a–d) Density analysis of Western blotting bands. *P<0.05 and # P<0.01, compared to the relative control group (n = 3).
Figure 3.
Gastric cancer cell derived exosomes promote hucMSCs migration.
A. HucMSCs were treated with gastric cancer cell or normal gastric epithelial cell derived exosomes (800 µg/mL) in the presence or absence of SD208 (2 µM) for 6 h. Transwell migration assay was performed to analyze the migratory ability of the cells. B. The number of migrated cells above was evaluated. These experiments were repeated for three times. *P<0.05 and # P<0.01, n = 3. Scale bar = 50 µm.
Figure 4.
Gastric cancer cell derived exosomes induce Smad2/3 and p38 phosphorylation in hucMSCs.
A. Western blotting analyses of TGF-β expression in gastric cancer cell (SGC7901) and normal gastric epithelial cell (GES-1) derived exosomes. B. HucMSCs were treated with SGC7901 derived exosomes (800 µg/mL) for different times as indicated. The levels of p-Smad2/3, t-Smad2/3, p-p38and t-p38 were analyzed by Western blotting. TGF-β served as a positive control. C. HucMSCs were treated with GES-1 derived exosomes (800 µg/mL) for different times as indicated. The levels of p-Smad2/3, t-Smad2/3, p-p38 and t-p38 were analyzed by Western blotting.
Figure 5.
TGF-β inhibition reverses gastric cancer cell- exosomes induced Smad2 and p38 activation in hucMSCs.
A. HucMSCs were treated with SGC7901 derived exosomes (800 µg/mL) in the presence or absence of SD208 (2 µM) for 1 hour. The levels of phosphorylated Smad2 and p38 were examined by Western blotting. B. Gastric cancer cell (SGC7901) derived exosomes were pre-incubated with anti-TGF-β antibody (20 µg/mL) for 2 h, and then added to hucMSCs. Six hours later, the cells were collected and the levels of phosphorylated Smad2 and p38 proteins were examined by Western blotting.
Figure 6.
TGF-β inhibition attenuates gastric cancer cell derived exosomes-induced differentiation of hucMSCs to CAFs.
A. HucMSCs were treated with gastric cancer cell derived exosomes in the presence or absence of SD208 (2 µM) for 2 weeks. The expression of FAP, α-SMA, Vimentin and N-cadherin proteins was determined by Western blotting. (a) SGC7901; (b) HGC27. B. HucMSCs were treated with gastric cancer cell (SGC7901) derived exosomes in the presence or absence of TGF-β antibody (20 µg/mL) for 2 weeks. Rabbit IgG was used as the control. The expression of FAP and α-SMA proteins was determined by Western blotting.