E0.423 0.836 0.0008 0.115 0.001 ,0.0001 0.026 0.161 0.012 0.0005 ,0.0001 0.003 0.221 0.HR (95 CI)P value2.446 (1.511?.959)0.1.459 (1.030?.065)0.1.991 (1.408?.815),0.1.715 (1.244?.364)0.W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated. tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma. *Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society. doi:10.1371/journal.pone.0055146.tmolecules, it is unlikely that such small amounts of induced transcripts would be biologically important. Rather, it is speculated that small amounts of contaminating cells such as endothelial cells might have responded to the hypoxic conditions. Peroxynitrite is a reactive nitrogen intermediate produced when both NOS and ARG are present. Since these cells did not express nitrotyrosine examined by immunohistochemistry (data not shown) that is generated by the nitration of tyrosine residues by peroxynitrite, NOS2 expression was not induced in CAFs within and around necrotic areas in PDC tissue. These results suggest that NOS2 is not induced in ARG2-expressing CAFs.ARG2-expressing CAFs Potentially Affect the Immune ReactionThe normal physiological concentration of L-arginine in serum is around 100 mM (50?50 mM). We determined that 12?5 mM L-arginine would be required for T cell proliferation induced by T cell receptor stimulation under the experimental Pentagastrin conditions we employed (Figures 6A and 6B). Next, we tried to examine if ARG2 induced by exposure to hypoxia affects the proliferation of T cells in vitro. In contrary to the expectation, the conditioned medium that had been used for culturing CAFs under hypoxic conditions did not suppress T cell proliferation significantly in comparison to medium that had been used for culturing CAFs under normoxic conditions (Figure 6A and B). The discrepancy of the findings to our results of the induction of ARG2 protein with a certain enzymatic activity in CAFs by hypoxic exposure might be caused by the secretion of undetermined molecules that can support T cell proliferation from the CAFs. In order to determine whether CD3+ T cells are proliferating around ARG2-expressing CAFs in PDC tissue, we performed double immunohistochemistry for CD3 and Ki-67 and compared tumor-infiltrating CD3+ T cells with their proliferating index in the area around ARG2-expressing CAFs to the area within the tumor except for necrotic tissue. It was surprised that there werefew CD3+ T cells around ARG2-expressing CAFs. Both the absolute number of tumor-infiltrating CD3+ T cells (Figure 6C) and the proportion of Ki-67-positive proliferating cells among the CD3+ T cells (Figure 6D) observed in the area around ARG2expressing CAFs were significantly lower than those observed in the other area. These findings suggest that the adaptive immune response is suppressed in areas around ARG2-expressing CAFs. The direct effect of ARG2 induced by exposure to hypoxia in CAFs against cancer cells was investigated using an in vitro system in which CAFs MedChemExpress PD1-PDL1 inhibitor 1 extracted from PDC tissues were co-cultured with pancreatic cancer MiaPaCa-2 cells. The proliferation of cancer cells was not significantly affected by ARG2 induced by hypoxia (Figure 7A), and oxidative stress-induced apoptosis of cancer cells but CAFs themselves was not prevented by polyamine produced by ARG2 in response to hypoxia (Figure 7C).DiscussionARG plays key roles in regulating most aspects of arginine meta.E0.423 0.836 0.0008 0.115 0.001 ,0.0001 0.026 0.161 0.012 0.0005 ,0.0001 0.003 0.221 0.HR (95 CI)P value2.446 (1.511?.959)0.1.459 (1.030?.065)0.1.991 (1.408?.815),0.1.715 (1.244?.364)0.W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated. tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma. *Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society. doi:10.1371/journal.pone.0055146.tmolecules, it is unlikely that such small amounts of induced transcripts would be biologically important. Rather, it is speculated that small amounts of contaminating cells such as endothelial cells might have responded to the hypoxic conditions. Peroxynitrite is a reactive nitrogen intermediate produced when both NOS and ARG are present. Since these cells did not express nitrotyrosine examined by immunohistochemistry (data not shown) that is generated by the nitration of tyrosine residues by peroxynitrite, NOS2 expression was not induced in CAFs within and around necrotic areas in PDC tissue. These results suggest that NOS2 is not induced in ARG2-expressing CAFs.ARG2-expressing CAFs Potentially Affect the Immune ReactionThe normal physiological concentration of L-arginine in serum is around 100 mM (50?50 mM). We determined that 12?5 mM L-arginine would be required for T cell proliferation induced by T cell receptor stimulation under the experimental conditions we employed (Figures 6A and 6B). Next, we tried to examine if ARG2 induced by exposure to hypoxia affects the proliferation of T cells in vitro. In contrary to the expectation, the conditioned medium that had been used for culturing CAFs under hypoxic conditions did not suppress T cell proliferation significantly in comparison to medium that had been used for culturing CAFs under normoxic conditions (Figure 6A and B). The discrepancy of the findings to our results of the induction of ARG2 protein with a certain enzymatic activity in CAFs by hypoxic exposure might be caused by the secretion of undetermined molecules that can support T cell proliferation from the CAFs. In order to determine whether CD3+ T cells are proliferating around ARG2-expressing CAFs in PDC tissue, we performed double immunohistochemistry for CD3 and Ki-67 and compared tumor-infiltrating CD3+ T cells with their proliferating index in the area around ARG2-expressing CAFs to the area within the tumor except for necrotic tissue. It was surprised that there werefew CD3+ T cells around ARG2-expressing CAFs. Both the absolute number of tumor-infiltrating CD3+ T cells (Figure 6C) and the proportion of Ki-67-positive proliferating cells among the CD3+ T cells (Figure 6D) observed in the area around ARG2expressing CAFs were significantly lower than those observed in the other area. These findings suggest that the adaptive immune response is suppressed in areas around ARG2-expressing CAFs. The direct effect of ARG2 induced by exposure to hypoxia in CAFs against cancer cells was investigated using an in vitro system in which CAFs extracted from PDC tissues were co-cultured with pancreatic cancer MiaPaCa-2 cells. The proliferation of cancer cells was not significantly affected by ARG2 induced by hypoxia (Figure 7A), and oxidative stress-induced apoptosis of cancer cells but CAFs themselves was not prevented by polyamine produced by ARG2 in response to hypoxia (Figure 7C).DiscussionARG plays key roles in regulating most aspects of arginine meta.
