Upregulation of cancer-associated myofibroblasts by TGF-β from scirrhous gastric carcinoma cells

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 (alpha-SMA, vimentin, and DAPI) was performed to determine the presence of alpha-SMA-positive myofibroblasts. Real-time RT-PCR was performed to examine alpha-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-beta (TGF-beta) significantly increased the alpha-SMA expression level of CAFs. Conditioned medium from OCUM-2MD3 or OCUM-12 cells upregulated the alpha-SMA expression level of CAFs, but that from MKN-45 or MKN-74 cells did not. The alpha-SMA upregulation effect of conditioned medium from OCUM-2MD3 or OCUM-12 cells was significantly decreased by an anti-TGF-beta antibody or Smad2 siRNA. CONCLUSION: Transforming growth factor-beta from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs. British Journal of Cancer (2011) 105, 996-1001. doi:10.1038/bjc.2011.330 www.bjcancer.com Published online 23 August 2011 (C) 2011 Cancer Research UK