Gastric cancer exosomes trigger differentiation of umbilical cord derived mesenchymal stem cells to carcinoma-associated fibroblasts through TGF-β/Smad pathway

Abstract

Background: Mesenchymal stem cells (MSCs) promote tumor growth by differentiating into carcinoma-associated fibroblasts (CAFs) and composing the tumor microenvironment. However, the mechanisms responsible for the transition of MSCs to CAFs are not well understood. Exosomes regulate cellular activities by mediating cell-cell communication. In this study, we aimed to investigate whether cancer cell-derived exosomes were involved in regulating the differentiation of human umbilical cord-derived MSCs (hucMSCs) to CAFs.

Methodology/principal findings: We first showed that gastric cancer cell-derived exosomes induced the expression of CAF markers in hucMSCs. We then demonstrated that gastric cancer cell-derived exosomes stimulated the phosphorylation of Smad-2 in hucMSCs. We further confirmed that TGF-β receptor 1 kinase inhibitor attenuated Smad-2 phosphorylation and CAF marker expression in hucMSCs after exposure to gastric cancer cell-derived exosomes.

Conclusion/significance: Our results suggest that gastric cancer cells triggered the differentiation of hucMSCs to CAFs by exosomes-mediated TGF-β transfer and TGF-β/Smad pathway activation, which may represent a novel mechanism for MSCs to CAFs transition in cancer.

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and incubated for 4 h. Effluent from a filtered suspension of CM-Dil-labeled exosomes (control) and the CM-Dil-labeled withdrawn exosomes fraction (e-exos) were used as controls.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.