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Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12=side population (SP) cells and OCUM-2MD3=SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12=SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12=SP and OCUM-2MD3=SP cells, but not that of MKN-45=SP and MKN-74=SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12=SP and OCUM-2MD3=SP cells. These stimulatingactivities by CM were significantly decreased by TGFb inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12=SP cells with CAFs was significantly high in comparison with that by OCUM-12=SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFb signaling.

Background:Myofibroblasts in the cancer microenvironment have recently been implicated in tumour growth and metastasis of gastric cancer. However, the mechanisms responsible for the regulation of myofibroblasts in cancer-associated fibroblasts (CAFs) remain unclear. This study was performed to clarify the mechanisms for regulation of myofibroblasts in gastric cancer microenvironment.Methods:Two CAFs (CaF-29 and CaF-33) from the tumoural gastric wall and a normal fibroblast (NF-29) from the nontumoural gastric wall, 4 human gastric cancer cell lines from scirrhous gastric cancer (OCUM-2MD3 and OCUM-12), and non-scirrhous gastric cancer (MKN-45 and MKN-74) were used. Immunofluorescence microscopy by triple-immunofluorescence labelling (α-SMA, vimentin, and DAPI) was performed to determine the presence of α-SMA-positive myofibroblasts. Real-time RT–PCR was performed to examine α-SMA mRNA expression.Results:Immunofluorescence microscopy showed that the frequency of myofibroblasts in CaF-29 was greater than that in NF-29. The number of myofibroblasts in gastric fibroblasts gradually decreased with serial passages. Transforming growth factor-β (TGF-β) significantly increased the α-SMA expression level of CAFs. Conditioned medium from OCUM-2MD3 or OCUM-12 cells upregulated the α-SMA expression level of CAFs, but that from MKN-45 or MKN-74 cells did not. The α-SMA upregulation effect of conditioned medium from OCUM-2MD3 or OCUM-12 cells was significantly decreased by an anti-TGF-β antibody or Smad2 siRNA.Conclusion:Transforming growth factor-β from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs.

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

BACKGROUND: Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear. METHODS: We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen. RESULTS: Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O 2 , whereas that of OCUM-12/ Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells. CONCLUSION: OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.

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 a-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-b 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-b produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells. (Cancer Sci 2012; 103: 797-805)

Fibroblasts, particularly myofibroblasts, affect the malignant progression of cancer cells in vitro. However, to date few reports have addressed the clinical significance of myofibroblasts in the gastric cancer microenvironment. This study examined the correlation between myofibroblast expression and clinicopathological features in gastric carcinoma. A total of 265 primary gastric tumors resected by gastrectomy were stained with antibodies against α-smooth muscle actin and vimentin. Stromal cells positive for vimentin were considered to be fibroblasts. Myofibroblasts were defined as fibroblasts positive for α-smooth muscle staining. Myofibroblast-positive gastric carcinoma was established when myofibroblasts accounted for more than 25% of fibroblasts in the cancer stroma. Myofibroblast expression was positive in 92 (35%) of the 265 gastric carcinomas. Myofibroblast expression showed a significantly (p<0.001) high frequency in advanced gastric cancers (76 of 146), in comparison to the early stage cancers (16 of 119). Taken together, there was a statistically significant correlation between myofibroblast expression and scirrhous type gastric cancer (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001) and peritoneal dissemination (p=0.005). The prognosis of patients with tumors positive for myofibroblast expression was significantly (p<0.001) worse, while a multivariate analysis revealed that myofibroblast expression was not an independent prognostic factor. These findings suggest that myofibroblasts are associated with scirrhous gastric cancer. Overexpression of myofibroblasts may therefore be a useful prognostic indicator of gastric carcinoma.

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