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

DNA Methylation Profiles at Precancerous Stages Associated with Recurrence of Lung Adenocarcinoma

<div><p>The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average β-values based on Infinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (Δβ_T–N>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the <i>ADCY5, EVX1, GFRA1, PDE9A,</i> and <i>TBX20</i> genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2′-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.</p> </div

The 28 probes for which the average β-value in N samples (β_N) was higher in recurrence-positive patients than in recurrence-negative patients, for which the average β-value in C samples (β_C) was less than 0.2, and for which the average β-value in T samples minus that in corresponding N samples (Δβ_T–N) was more than 0.1.

a<p>Probe ID for the Infinium HumanMethylation27 Bead Array (Illumina).</p>b<p>National Center for Biotechnology Information database (Genome Build 37).</p>c<p>Non-adjusted <i>P</i>-values and.</p>d<p>Benjamini-Hochberg-adjusted <i>P</i>-values for the Cox regression model used for evaluation of correlation with recurrence.</p

Correlation between DNA methylation levels and mRNA expression levels.

<p>DNA methylation levels (average β-values) (<b>A</b>) and mRNA expression levels (<b>B</b>) for the <i>ADCY5, CNTNAP, EVX1, GFRA1, PDE9A</i> and <i>TBX20</i> genes in samples of non-cancerous lung tissue (N) from patients with lung adenocarcinomas and samples of the corresponding tumorous tissue (T) were determined by Infinium assay and quantitative real-time reverse transcription-PCR analysis, respectively. DNA methylation levels for all six genes were significantly higher in T samples than in N samples, and levels of expression of mRNAs for the <i>ADCY5, EVX1, GFRA1, PDE9A</i> and <i>TBX20</i> genes were significantly lower in T samples than in N samples, although the reduction in the expression of the <i>CNTNAP2</i> gene did not reach statistical significance. These results suggested that DNA hypermethylation of the <i>ADCY5, EVX1, GFRA1, PDE9A</i> and <i>TBX20</i> genes may result in reduced mRNA expression in tissue samples from the same cohort.</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

Hazard ratio (HR) obtained from the Cox regression model.

<p>Correlation between DNA methylation status (average β-values) and recurrence was examined in 145 samples of tumorous tissue (<b>A</b>) and 145 samples of the corresponding non-cancerous lung tissue (<b>B</b>) obtained from patients with lung adenocarcinomas who had undergone complete resection and had not received any adjuvant therapy after surgery. All of the examined 26,455 probes of the Infinium array are shown along the chromosomes. Red color means that higher β-values of the probes were observed in recurrence-positive patients than in recurrence-negative patients (<i>P</i><0.001). Blue color means that lower β-values of the probes were observed in recurrence-positive patients than in recurrence-negative patients (<i>P</i><0.001).</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

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