Says (R D Systems, USA), according to the manufacturer’s instructions. A total of 100 mL of cell supernatants from the direct and indirect culture groups, respectively, were treated with 20 mL of 1 M Hcl for 10 min, followed by neutralization with 20 mL of 1.2 M NaOH. The samples were then pipetted into microplate wells precoated with a monoclonal antibody specific for TGF-b1, and incubated for 2 h at room temperature. An enzyme-linked polyclonal antibody specific for TGF-b1 was then added to the wells and incubated for a further 2 h to sandwich the TGF-b1 ligand. A substrate solution consisting of hydrogen peroxide and tetramethyl benzidine was added and the intensity of the color was determined using an automicroplate reader (Flexstation 3, Molecular Devices). Each experiment was Title Loaded From File conducted twice and each sample point was assessed in triplicate, and data were averaged.TGF-b Serum LevelsTo further explore the occurrence of TGF-b in the general environment, we compared serum concentrations of TGF-b in patients with EGC or AGC to those in controls. Serum concentrations of TGF-b1 in controls and in patients with EGC and AGC were 27.7866.11, 50.0864.38, and 45.7665.00 ng/ mL, respectively, while the Title Loaded From File corresponding values for TGF-b2 were 59.41615.42, 133.61621.90, and 111.34615.76 ng/mL, respectively. Levels of both TGF-b1 and TGF-b2 were significantly higher in patients with EGC or AGC compared to those in controls (F = 4.745 and P = 0.018; F = 4.939 and P = 0.015). However, there were no significant differences between patients with early and late stage of GC (Figure 2G). These results suggest that the abnormal status of TGF-b in gastric carcinogenesis may be a systemic response involving not only the tumor microenvironment, but also the general circulatory system.Statistical AnalysisStatistical analysis was conducted using SPSS 16.0 for Windows (SPSS, Chicago, USA). Data were presented as means 6 SD. ChiSquare tests were used to analyze the correlation between TGFb staining and clinical pathologic features. Kruskall-Wallis tests were used to compare values among different groups, and MannWhitney tests were used to 1655472 identify specific differences between two groups using a corrected a value. Paired Wilcoxon signed rank tests were performed to compare mRNA levels in tumoral and peritumoral tissues. Concentrations of TGF-b in serum and cell supernatant were analyzed by ANOVA. Bivariate correlation analysis was conducted to examine the associations between mRNA statuses of TGF-b1, TGF-b2, and Smads molecules.Coculture In VitroA coculture model was established to determine if direct cell-tocell contact or indirect cytokine-dependent contact is the main mechanism in a mimicking tumor microenvironment. Firstly,TGF-b Roles in Tumor-Cell Interaction with PBMCsFigure 1. TGF-b1 protein expression in gastric precancer and cancer. (A) Positive staining for TGF-b1 in a case of gastric precancer. (B) Strong cytoplasmic staining in tumor cells limited to the mucosa in a case of early gastric cancer and weak staining in some stromal cells. (C) TGF-b1 expression in a case of intestinal-type advanced gastric cancer, according to Lauren’s classification. (D) Cytoplasmic staining for TGF-b2 in a case of advanced gastric cancer. (All photos are shown at 6200 magnification). doi:10.1371/journal.pone.0054249.gTable 1. Immunoreactivity of TGF-b1 in gastric tissues of precancer and cancer.Parameter Different stage of disease Control Precancer Early GC Advanced GC Hp.Says (R D Systems, USA), according to the manufacturer’s instructions. A total of 100 mL of cell supernatants from the direct and indirect culture groups, respectively, were treated with 20 mL of 1 M Hcl for 10 min, followed by neutralization with 20 mL of 1.2 M NaOH. The samples were then pipetted into microplate wells precoated with a monoclonal antibody specific for TGF-b1, and incubated for 2 h at room temperature. An enzyme-linked polyclonal antibody specific for TGF-b1 was then added to the wells and incubated for a further 2 h to sandwich the TGF-b1 ligand. A substrate solution consisting of hydrogen peroxide and tetramethyl benzidine was added and the intensity of the color was determined using an automicroplate reader (Flexstation 3, Molecular Devices). Each experiment was conducted twice and each sample point was assessed in triplicate, and data were averaged.TGF-b Serum LevelsTo further explore the occurrence of TGF-b in the general environment, we compared serum concentrations of TGF-b in patients with EGC or AGC to those in controls. Serum concentrations of TGF-b1 in controls and in patients with EGC and AGC were 27.7866.11, 50.0864.38, and 45.7665.00 ng/ mL, respectively, while the corresponding values for TGF-b2 were 59.41615.42, 133.61621.90, and 111.34615.76 ng/mL, respectively. Levels of both TGF-b1 and TGF-b2 were significantly higher in patients with EGC or AGC compared to those in controls (F = 4.745 and P = 0.018; F = 4.939 and P = 0.015). However, there were no significant differences between patients with early and late stage of GC (Figure 2G). These results suggest that the abnormal status of TGF-b in gastric carcinogenesis may be a systemic response involving not only the tumor microenvironment, but also the general circulatory system.Statistical AnalysisStatistical analysis was conducted using SPSS 16.0 for Windows (SPSS, Chicago, USA). Data were presented as means 6 SD. ChiSquare tests were used to analyze the correlation between TGFb staining and clinical pathologic features. Kruskall-Wallis tests were used to compare values among different groups, and MannWhitney tests were used to 1655472 identify specific differences between two groups using a corrected a value. Paired Wilcoxon signed rank tests were performed to compare mRNA levels in tumoral and peritumoral tissues. Concentrations of TGF-b in serum and cell supernatant were analyzed by ANOVA. Bivariate correlation analysis was conducted to examine the associations between mRNA statuses of TGF-b1, TGF-b2, and Smads molecules.Coculture In VitroA coculture model was established to determine if direct cell-tocell contact or indirect cytokine-dependent contact is the main mechanism in a mimicking tumor microenvironment. Firstly,TGF-b Roles in Tumor-Cell Interaction with PBMCsFigure 1. TGF-b1 protein expression in gastric precancer and cancer. (A) Positive staining for TGF-b1 in a case of gastric precancer. (B) Strong cytoplasmic staining in tumor cells limited to the mucosa in a case of early gastric cancer and weak staining in some stromal cells. (C) TGF-b1 expression in a case of intestinal-type advanced gastric cancer, according to Lauren’s classification. (D) Cytoplasmic staining for TGF-b2 in a case of advanced gastric cancer. (All photos are shown at 6200 magnification). doi:10.1371/journal.pone.0054249.gTable 1. Immunoreactivity of TGF-b1 in gastric tissues of precancer and cancer.Parameter Different stage of disease Control Precancer Early GC Advanced GC Hp.
