Cancer-associated orthotopic myofibroblasts stimulates the motility of gastric carcinoma cells

Abstract

Tumor progression has been recognized as the product of evolving crosstalk between cancer cells and the surrounding stromal cells. Cancer-associated orthotopic myofibroblasts may be linked to the progression of gastric carcinomas. To understand the significance of orthotopic myofibroblasts, we examined the effects of cancer-associated orthotopic myofibroblasts on the malignant phenotype of gastric cancer cells. Three human gastric cancer cell lines (OCUM-2MD3, OCUM-12, MKN-45) and four human gastric fibroblast cell lines (cancer-associated orthotopic fibroblast [CaF]-29, CaF-33, normal orthotopic fibroblast [NF]-29, NF-33) were used. The cancer-associated orthotopic fibroblast cell lines CaF-29 and CaF-33 were established from a tumoral gastric wall, and normal orthotopic fibroblast NF-29 and NF-33 were established from a non-tumoral gastric wall. Fibroblasts that were α-smooth muscle actin-positive were defined as myofibroblasts. We examined the effects of cancer-associated orthotopic myofibroblasts on the aggressiveness of gastric cancer cells by wound-healing assay, invasion assay, and RT-PCR. The ratios of myofibroblasts in CaF-29 (33%) and CaF-33 (46%) were significantly (P < 0.001) greater than those in NF-29 (11%) or NF-33 (13%). Although all four orthotopic fibroblast lines increased the motility of gastric cancer cells, including migration and invasion ability, the motility-stimulating activity of cancer-associated fibroblasts (CaF-29 and CaF-33) was significantly higher than that of normal fibroblasts (NF-29 and NF-33). These motility-stimulating activities of cancer-associated orthotopic fibroblasts were downregulated by Smad2 siRNA treatment and anti-transforming growth factor-β neutralizing antibody. These findings suggest that cancer-associated orthotopic myofibroblasts may play an important role in the progression of gastric cancers and that transforming growth factor-β produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells.

Figure 1

α‐Smooth muscle actin (αSMA) expression in fibroblasts. (a) αSMA immunostaining in CaF‐29 and NF‐29 fibroblasts at the fifth passage. αSMA‐positive myofibroblasts (arrows) were frequently found in CaF‐29, relative to NF‐29. (b) Immunofluorescence of NF‐29 fibroblasts and CaF‐29 fibroblasts. Fibroblasts were stained with αSMA (red) and vimentin (green), and cell nuclei were stained with DAPI (blue). The percentage of αSMA‐positive myofibroblasts (arrows) in CaF‐29 was higher than in NF‐29. (c) The myofibroblast content of CaF‐29 was significantly higher than that of NF‐29. The percentages of αSMA‐positive myofibroblast cells among the total fibroblasts were significantly higher (P < 0.001) in CaF‐29 (33%) and CaF‐33 (46%) than in NF‐29 (11%) and NF‐33(13%). Data are presented as mean ± SD (four samples per group). **P < 0.01. (d) A significant correlation (r = 0.965, P < 0.001) was found between the αSMA mRNA level and myofibroblast frequency among fibroblasts by Pearson's correlation coefficient test. CaF, cancer‐associated orthotopic fibroblast NF, normal orthotopic fibroblast; N.S., not significant.

Figure 2

Effect of gastric fibroblasts on the wound‐healing ability of cancer cells. (a) Representative pictures of wound‐healing assay. The number of OCUM‐2MD3 cells able to migrate over the wound line (dotted line) increased in the presence of conditioned medium from fibroblasts but did not do so in the presence of the control. (b) Conditioned medium from fibroblasts significantly stimulated the migration by gastric cancer cells. The migration‐stimulating abilities of the cancer‐associated fibroblasts, CaF‐29 and CaF‐33, were higher than that of the normal fibroblasts, NF‐29 and NF‐33. Data are presented as mean ± SD (four samples per group). *P < 0.05; **P < 0.01. (c) Smad2 siRNA significantly inhibited the migration of OCUM‐2MD3 cells stimulated by conditioned medium from CaF‐33. CaF, cancer‐associated orthotopic fibroblast; CM, conditioned medium; NF, normal orthotopic fibroblast; N.S., not significant.

Figure 3

Invasion‐stimulating effect of cancer‐associated fibroblasts on gastric cancer cells. (a) Conditioned medium from cancer‐associated fibroblasts, CaF‐29 or CaF‐33, significantly inhibited invasion by scirrhous gastric cancer cells, but were not affected by the normal fibroblasts, NF‐29 and NF‐33. Data are presented as mean ± SD (four samples per group). *P < 0.05; **P < 0.01. (b) Compared with the negative siRNA control, Smad2 siRNA significantly reduced the invasion of OCUM‐2MD3 cells stimulated by conditioned medium from CaF‐33. CaF, cancer‐associated orthotopic fibroblast; CM, conditioned medium; NF, normal orthotopic fibroblast; N.S., not significant.

Figure 4

Representative phase‐contrast photographs of gastric cancer cells. The numbers of OCUM‐12, OCUM‐2MD3, and MKN45 cells displaying EMT transition significantly increased in the presence of CM from cancer‐associated fibroblasts, CaF‐29 and CaF‐33, when compared to those in the presence of the control. In contrast, a few gastric cancer cells showed EMT in the presence of CM from NF‐29 and NF‐33. CaF, cancer‐associated orthotopic fibroblast; CM, conditioned medium; EMT, epithelial‐to‐mesenchymal; NF, normal orthotopic fibroblast.

Figure 5

Effect of fibroblasts on the vimentin and E‐cadherin expression of gastric cancer cells. (a) Vimentin expression of all gastric cancer cells was significantly increased by the CM from the cancer‐associated fibroblasts, CaF‐29 and CaF‐33, but not by the CM from NF‐29 and NF‐33. E‐cadherin expression of all gastric cancer cells was significantly decreased by the CM from the cancer‐associated fibroblasts, CaF‐29 or CaF‐33, but not by the CM from NF‐29 and NF‐33. *P < 0.05; **P < 0.01. (b) TGF‐β stimulated the vimentin expression of gastric cancer cells. The neutralizing anti‐TGF‐β antibody downregulated vimentin expression, which was stimulated by TGF‐β. Smad2 siRNA (30 nmol/L) downregulated vimentin expression, which was stimulated by TGF‐β in OCUM‐2MD3 cells. However, the treatment of negative control siRNA (30 nmol/L) had no effect on the expression. The vimentin expression level was relative to the control of MKN45. (c) TGFβ decreased the E‐cadherin expression of gastric cancer cells. The neutralizing anti‐TGFβ antibody up‐regulated E‐cadherin expression decreased by TGFβ. Smad2 siRNA (30 nmol/L) up‐regulated E‐cadherin expression decreased by TGFβ in OCUM‐2MD3 cells, whereas treatment of negative control siRNA (30 nmol/L) had no effect on the expression. The E‐cadherin expression level was relative to the control of MKN45. (d) Smad2 siRNA and anti‐TGF‐β antibody decreased vimentin expression of gastric cancer cells induced by CM from the cancer‐associated fibroblast CaF‐33, but did not affect that of cancer cells in the presence of CM from NF‐29. (e) Smad2 siRNA and anti‐TGFβ antibody increased E‐cadherin expression of gastric cancer cells induced by CM from the cancer‐associated fibroblast CaF‐33, but did not affect that of cancer cells in the presence of CM from NF‐29. CaF, cancer‐associated orthotopic fibroblast; CM, conditioned medium; NF, normal orthotopic fibroblast; TGF‐β, transforming growth factor‐β.

Figure 6

TGF‐β production from αSMA‐positive myofibroblasts. Immunofluorescence microscopic findings showed that TGF‐β expression was found in αSMA‐positive fibroblasts (arrows). αSMA‐positive fibroblasts were frequently found in CaF‐29 relative to in NF‐29. CaF, cancer‐associated orthotopic fibroblast; NF, normal orthotopic fibroblast; SMA, smooth muscle actin; TGF‐β, transforming growth factor‐β.