S). HE staining and immunohistochemistry for ARG2, CAIX, and SLC2A

S). HE staining and immunohistochemistry for ARG2, CAIX, and SLC2A1 in serial tissue sections. 23388095 Necrotic areas are surrounded by star marks in the upper HE photo and the rectangle (light blue) corresponds to the area of the lower column. Double immunostaining (the right-most columns) reveals that most of the granular ARG2 staining (brown) is present in spindle-shaped cells stained for CAIX (purple). Inset is a very highpower view. doi:10.1371/journal.pone.0055146.gArginase II in Pancreatic CancerFigure 5. Hypoxia induces expression of ARG2 in CAFs extracted from PDC tissue. (A) Relative gene expression is measured by real-time RT-PCR in fibroblasts extracted from PDC tissue after 48 hrs of exposure to hypoxia (Hypoxia) or culture under normoxic control conditions (Normoxia). Expression of the genes in the fibroblasts after 48 hrs of culture under hypoxic conditions followed by 48 hrs of culture under normoxic conditions (Re-oxygenation) was also analyzed. Each data column represents the mean relative expression standardized with 18SrRNA 6 SE for triplicate determinations. The SLC2A1 (alternatively known as glucose transporter type 1 or GLUT1) gene is hypoxia-inducible. Significance value (MedChemExpress GLPG0187 Student’s t test) of P,0.01 (*). Some genes are expressed in CAFs at an MedChemExpress GS-9973 extremely lower level than in a normal tissue that is the major tissue of expressing the gene. In such cases, comparison of gene expression is shown in the insets with 1/50 to 1/10000 scales. (B) Western blot analysis (upper column) reveals that ARG2 protein expression is induced in CAFs used in (A) upon exposure to hypoxia. N and H indicate the cells cultured under normoxic and hypoxic conditions, respectively. ARG2 protein induced in CAFs has arginase activity (lower column). One unit of arginase converts 1 mmol of L-arginine to ornithine and urea per minute at pH 9.5 and 37uC. Each data column represents the mean activity 6 SE for triplicate determinations. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). (C) Expression of genes in CAFs from PDC tissue during cultivation under hypoxic conditions detected by real-time RT-PCR. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.01 (*) and P,0.001 (**). (D) Western blot analysis of HIF-1a protein. Accumulation of HIF-1a protein is observed in CAFs after 6 hrs of exposure to hypoxic conditions. N and H indicate cells cultured under normoxic and hypoxic conditions, respectively. doi:10.1371/journal.pone.0055146.gPDCs showed that the presence of ARG2-expressing CAFs was significantly correlated with higher infiltration of CD68+ macrophages (P = 0.05) and CD66b+ neutrophils (P,0.0001), and lower infiltration of CD4+ T cells (P = 0.003) and CD8+ T cells (P = 0.036). In addition, tumor-infiltrating CD3+ T cells aroundARG2-expressing CAFs were few and showed less proliferation (Figure 6). These findings suggest that the presence of ARG2expressing CAFs is closely related to the immunosuppressive microenvironment. This situation can be explained in terms of tissue hypoxia, [24] although the extent to which local expressionArginase II in Pancreatic CancerFigure 6. ARG2-expressing CAFs potentially affect the immune reaction. (A) T cell proliferation assay. CFSE-labeled CD3+ T cells were stimulated with anti-CD3/CD28 antibody-conjugated beads in fresh medium supplemented with the indicating L-arginine (control) or in conditioned medium after culture of CAFs under normoxic conditi.S). HE staining and immunohistochemistry for ARG2, CAIX, and SLC2A1 in serial tissue sections. 23388095 Necrotic areas are surrounded by star marks in the upper HE photo and the rectangle (light blue) corresponds to the area of the lower column. Double immunostaining (the right-most columns) reveals that most of the granular ARG2 staining (brown) is present in spindle-shaped cells stained for CAIX (purple). Inset is a very highpower view. doi:10.1371/journal.pone.0055146.gArginase II in Pancreatic CancerFigure 5. Hypoxia induces expression of ARG2 in CAFs extracted from PDC tissue. (A) Relative gene expression is measured by real-time RT-PCR in fibroblasts extracted from PDC tissue after 48 hrs of exposure to hypoxia (Hypoxia) or culture under normoxic control conditions (Normoxia). Expression of the genes in the fibroblasts after 48 hrs of culture under hypoxic conditions followed by 48 hrs of culture under normoxic conditions (Re-oxygenation) was also analyzed. Each data column represents the mean relative expression standardized with 18SrRNA 6 SE for triplicate determinations. The SLC2A1 (alternatively known as glucose transporter type 1 or GLUT1) gene is hypoxia-inducible. Significance value (Student’s t test) of P,0.01 (*). Some genes are expressed in CAFs at an extremely lower level than in a normal tissue that is the major tissue of expressing the gene. In such cases, comparison of gene expression is shown in the insets with 1/50 to 1/10000 scales. (B) Western blot analysis (upper column) reveals that ARG2 protein expression is induced in CAFs used in (A) upon exposure to hypoxia. N and H indicate the cells cultured under normoxic and hypoxic conditions, respectively. ARG2 protein induced in CAFs has arginase activity (lower column). One unit of arginase converts 1 mmol of L-arginine to ornithine and urea per minute at pH 9.5 and 37uC. Each data column represents the mean activity 6 SE for triplicate determinations. Significance value (Student’s t test) of P,0.05 (*) and P,0.01 (**). (C) Expression of genes in CAFs from PDC tissue during cultivation under hypoxic conditions detected by real-time RT-PCR. Data represent one of three independent experiments. Significance value (Student’s t test) of P,0.01 (*) and P,0.001 (**). (D) Western blot analysis of HIF-1a protein. Accumulation of HIF-1a protein is observed in CAFs after 6 hrs of exposure to hypoxic conditions. N and H indicate cells cultured under normoxic and hypoxic conditions, respectively. doi:10.1371/journal.pone.0055146.gPDCs showed that the presence of ARG2-expressing CAFs was significantly correlated with higher infiltration of CD68+ macrophages (P = 0.05) and CD66b+ neutrophils (P,0.0001), and lower infiltration of CD4+ T cells (P = 0.003) and CD8+ T cells (P = 0.036). In addition, tumor-infiltrating CD3+ T cells aroundARG2-expressing CAFs were few and showed less proliferation (Figure 6). These findings suggest that the presence of ARG2expressing CAFs is closely related to the immunosuppressive microenvironment. This situation can be explained in terms of tissue hypoxia, [24] although the extent to which local expressionArginase II in Pancreatic CancerFigure 6. ARG2-expressing CAFs potentially affect the immune reaction. (A) T cell proliferation assay. CFSE-labeled CD3+ T cells were stimulated with anti-CD3/CD28 antibody-conjugated beads in fresh medium supplemented with the indicating L-arginine (control) or in conditioned medium after culture of CAFs under normoxic conditi.

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