Ons (normoxia) or hypoxic conditions (hypoxia) in the medium with the indicating L-arginine (including 2.0 mM arginine of serum contents) for four days, and their proliferation profiles were analyzed by flow cytometry. Non-proliferated cells showed the highest fluorescence intensity and the intensity of the labeled T cells was halved with every cell division. The numbers Bromopyruvic acid represent the ratio of numbers of cells having undergone multiple divisions relative to the original number of cells. Data represent one of six independent experiments. (B) Percentages of cells having undergone cell divisions. Data represent 1531364 one of six independent experiments. (C, D) Comparison of absolute number of tumor-infiltrating CD3+ T cells (C) with their proliferating index (the proportion of Ki-67-positive proliferating cells among the CD3+ T cells) (D) between the area around ARG2-expressing CAFs (white) and the area within the tumor except for necrotic tissue (speckled). We counted tumorinfiltrating CD3+ T cells and their Ki-67 positivity in each twenty different high-power fields per area categories using two PDC cases. Each data column represents the mean6 SE for triplicate determinations. Significance value (Student’s t test) of P,0.05 (*) and P,0.001 (**). doi:10.1371/journal.pone.0055146.gArginase II in Pancreatic CancerFigure 7. Pancreatic cancer cells and ARG2-expressing CAFs. (A) ARG2-expressing CAFs do not support proliferation of pancreatic cancer cells. CAFs extracted from PDC tissues and MiaPaCa-2 cells were co-cultured in medium with or without 2 mM DFMO under normoxic or hypoxic conditions for 48 hrs and the numbers of living cells were calculated the basis of data obtained by flow cytometry. The absolute number of MiaPaCa2 cells cultured under hypoxic conditions decreased significantly in comparison with normoxic conditions, although this effect was not significantly affected by the presence of DFMO in the culture medium. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). (B) Oxidative stress-induced apoptosis was induced in MiaPaCa-2 cells by exposure to various concentrations (0?500 mM) of H2O2 for 7 hrs. The dead cells and living cells were detected by flow cytometry after staining with Annexin V and PI. (C) ARG2-expressing CAFs did not protect pancreatic cancer cells from oxidative-induced apoptosis. After 48 hrs of co-culture of CAFs extracted from PDC tissues and MiaPaCa-2 cells in medium with 1662274 or without 2 mM DFMO under normoxic or hypoxic conditions, all the cells were cultured for another 4 hrs under oxidative stress (50 mM H2O2) using the same conditions as before. The percentages of living cells were measured by flow cytometry (left column). In order to evaluate the effect of oxidative stress, the percentages of living cells after exposure to oxidative stress were divided by the percentages of living cells cultured under the same conditions Sermorelin web before oxidative stress (right column). The ratio of living cells before and after oxidative stress decreased significantly in both MiaPaCa-2 cells and CAFs cultured under hypoxic conditions. Blocking the synthesis of polyamines with DFMO increased significantly the degree of oxidative stress-induced apoptosis in the CAFs. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). doi:10.1371/journal.pone.0055146.gof ARG2 in CAFs contributes to this immunosuppression.Ons (normoxia) or hypoxic conditions (hypoxia) in the medium with the indicating L-arginine (including 2.0 mM arginine of serum contents) for four days, and their proliferation profiles were analyzed by flow cytometry. Non-proliferated cells showed the highest fluorescence intensity and the intensity of the labeled T cells was halved with every cell division. The numbers represent the ratio of numbers of cells having undergone multiple divisions relative to the original number of cells. Data represent one of six independent experiments. (B) Percentages of cells having undergone cell divisions. Data represent 1531364 one of six independent experiments. (C, D) Comparison of absolute number of tumor-infiltrating CD3+ T cells (C) with their proliferating index (the proportion of Ki-67-positive proliferating cells among the CD3+ T cells) (D) between the area around ARG2-expressing CAFs (white) and the area within the tumor except for necrotic tissue (speckled). We counted tumorinfiltrating CD3+ T cells and their Ki-67 positivity in each twenty different high-power fields per area categories using two PDC cases. Each data column represents the mean6 SE for triplicate determinations. Significance value (Student’s t test) of P,0.05 (*) and P,0.001 (**). doi:10.1371/journal.pone.0055146.gArginase II in Pancreatic CancerFigure 7. Pancreatic cancer cells and ARG2-expressing CAFs. (A) ARG2-expressing CAFs do not support proliferation of pancreatic cancer cells. CAFs extracted from PDC tissues and MiaPaCa-2 cells were co-cultured in medium with or without 2 mM DFMO under normoxic or hypoxic conditions for 48 hrs and the numbers of living cells were calculated the basis of data obtained by flow cytometry. The absolute number of MiaPaCa2 cells cultured under hypoxic conditions decreased significantly in comparison with normoxic conditions, although this effect was not significantly affected by the presence of DFMO in the culture medium. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). (B) Oxidative stress-induced apoptosis was induced in MiaPaCa-2 cells by exposure to various concentrations (0?500 mM) of H2O2 for 7 hrs. The dead cells and living cells were detected by flow cytometry after staining with Annexin V and PI. (C) ARG2-expressing CAFs did not protect pancreatic cancer cells from oxidative-induced apoptosis. After 48 hrs of co-culture of CAFs extracted from PDC tissues and MiaPaCa-2 cells in medium with 1662274 or without 2 mM DFMO under normoxic or hypoxic conditions, all the cells were cultured for another 4 hrs under oxidative stress (50 mM H2O2) using the same conditions as before. The percentages of living cells were measured by flow cytometry (left column). In order to evaluate the effect of oxidative stress, the percentages of living cells after exposure to oxidative stress were divided by the percentages of living cells cultured under the same conditions before oxidative stress (right column). The ratio of living cells before and after oxidative stress decreased significantly in both MiaPaCa-2 cells and CAFs cultured under hypoxic conditions. Blocking the synthesis of polyamines with DFMO increased significantly the degree of oxidative stress-induced apoptosis in the CAFs. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). doi:10.1371/journal.pone.0055146.gof ARG2 in CAFs contributes to this immunosuppression.