Correlation of expression of ARG2 in stromal cells with clinicopathological characteristics.

<p>W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated.</p><p>tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma.</p>*<p>Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society.</p>**<p>Comparisons of qualitative variables are performed using the χ² test and otherwise Fisher’s exact test.</p>***<p>Total number of patients are 211 and otherwise 214.</p

ARG2 was expressed mostly in CAFs under hypoxic conditions.

<p>Histology of PDC tissue in low- (upper columns) and high-power view (lower columns). HE staining and immunohistochemistry for ARG2, CAIX, and SLC2A1 in serial tissue sections. 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 high-power view.</p

Univariate and multivariate analyses of prognostic factors associated with disease-free survival in patients with ductal carcinoma of the pancreas.

<p>W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated.</p><p>tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma.</p>*<p>Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society.</p

Kaplan-Meier survival curves.

<p>(A, C) Kaplan-Meier survival curve showing a comparison of overall survival between the presence (grades 1 and 2) and absence (grade 0) of ARG2 expression in stromal cells (<i>P</i>  = 0.003) in A and that among expression grades (grades 0 to 2) of ARG2 in stromal cells (grade 0 vs. grade 1, log-rank test, <i>P</i>  = 2.08; grade 0 vs. grade 2, <i>P</i><0.0001; grade 1 vs. grade 2, <i>P</i>  = 0.0009) in C. (B, D) Kaplan-Meier survival curve showing a comparison of disease-free survival between the presence (grades 1 and 2) and absence (grade 0) of ARG2 expression in stromal cells (<i>P</i>  = 0.0006) in B and that among expression grades (grades 0 to 2) of ARG2 in stromal cells (grade 0 vs. grade 1, <i>P</i>  = 0.069; grade 0 vs. grade 2, <i>P</i><0.0001; grade 1 vs. grade 2, <i>P</i>  = 0.013) in D. Black circle and white circle represent censoring and failure, respectively.</p

ARG2-expressing CAFs potentially affect the immune reaction.

<p>(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 conditions (normoxia) or hypoxic conditions (hypoxia) in the medium with the indicating L-arginine (including 2.0 µM 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 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 tumor-infiltrating 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 mean± SE for triplicate determinations. Significance value (Student’s <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.001 (**).</p

Immunohistochemical expression of ARG2 in stromal cells within and around necrotic areas in PDC tissue.

<p>(A) Histology of PDC tissue in low- (upper columns) and high-power view (lower columns). HE staining (left columns) and immunohistochemistry for ARG2 (center columns) and cytokeratins (right columns) in serial tissue sections. Necrotic areas are surrounded by star marks (large area of necrosis is located at bottom right) in the upper HE photo and the rectangle (light blue) corresponds to the area of the lower column. (B) Immunohistochemistry for ARG2 (left upper column) and for mitochondria (left lower column) in high-power views. Positive staining of ARG2 is visualized as a dot-like or coarse granular pattern in the cytoplasm of spindle cells. This positive staining pattern was compatible with mitochondrial antigen. Double immunofluorescence (right column) shows ARG2 (green), mitochondria (red), and nuclei (white). Almost all ARG2-positive staining is co-localized with mitochondria (yellow). (C) Immunohistochemical expression of ARG1 was observed only in neutrophils. Upper and lower columns are middle- and high-power views, respectively.</p

Pancreatic cancer cells and ARG2-expressing CAFs.

<p>(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 MiaPaCa-2 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 <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.01 (**). (B) Oxidative stress-induced apoptosis was induced in MiaPaCa-2 cells by exposure to various concentrations (0–500 µM) 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 or without 2 mM DFMO under normoxic or hypoxic conditions, all the cells were cultured for another 4 hrs under oxidative stress (50 µM 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 <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.01 (**).</p

ARG2 was expressed mostly in CAFs within and around necrotic areas in PDC tissue.

<p>(A) Triple immunofluorescence shows that most of the dot-like staining of ARG2 (red) is present in α-SMA-positive fibroblasts (green). Cancer cells are positive for cytokeratins (blue). Nuclei are stained by DAPI (white). (B) Histology and immunohistochemistry of PDC tissue in low- (upper-photo of each pair of photos) and high-power view (lower-photo of each pair of photos). HE staining and immunohistochemistry for several antigens in serial tissue sections. Necrotic areas are surrounded by star marks in the low-power HE photo and the rectangle (light blue) corresponds to the area of the high-power view.</p

Univariate and multivariate analyses of prognostic factors associated with overall survival in patients with ductal carcinoma of the pancreas.

<p>W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated.</p><p>tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma.</p>*<p>Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society.</p

Hypoxia induces expression of <i>ARG2</i> in CAFs extracted from PDC tissue.

<p>(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 ± SE for triplicate determinations. The <i>SLC2A1</i> (alternatively known as glucose transporter type 1 or GLUT1) gene is hypoxia-inducible. Significance value (Student’s <i>t</i> test) of <i>P</i><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 µmol of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C. Each data column represents the mean activity ± SE for triplicate determinations. Significance value (Student’s <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><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 <i>t</i> test) of <i>P</i><0.01 (*) and <i>P</i><0.001 (**). (D) Western blot analysis of HIF-1α protein. Accumulation of HIF-1α 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.</p