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. doi:10.1371/journal.pone.0055146.gtissue and co-expressed CAIX, SLC2A1, and HIF-1a. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 after exposure to hypoxia. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not 1531364 a general event and that the presence of ARG2-expressing CAFs is an independent prognostic factor for PDC patients, and also Memory Th1 repertoire.Persisting bim2/2 SMARTA “Memory” Cells are Functionally DefectiveThe reflects tissue hypoxia. Reduced oxygen tension has been reported to affect ARG activity in several tissues and cell types including lung, brain, endothelial cells, and macrophages, [14,15,16] although the effects on ARGs differ among tissues, cells, and species. In humans, it has been shown that hypoxia induces the expression of ARG2 in pulmonary artery smooth muscle cells (PASMC) [17] and lung microvascular endothelial cells. [18,19] To our knowledge, the effects of hypoxia on ARG expression in cancer tissues or fibroblasts have not been demonstrated previously. Our study together with the study that hypoxia induced expression of ARG2 but not ARG1 in PASMCs suggests that ARG2 is the hypoxiainducible isoform. [17] It is consistent with our findings that accumulation of HIF-1a was found in CAFs under hypoxic conditions and also that the 5′ flanking region of the first exon of the ARG2 gene has potential binding sites for HIF-1. Ornithine generated by ARG can be further metabolized to polyamines and proline, which are critical for cell proliferation,differentiation and tissue repair, thus potentially preventing cell death. [20,21,22] Therefore, induction of ARG2 might be a defensive response of CAFs in PDC tissue under severe hypoxic stress potentially leading to cell death. Indeed, our results showed that ARG2-expressing CAFs ameliorated oxidative Title Loaded From File stress-induced apoptosis of CAFs themselves (Figure 7C). This is consistent with that fact that these CAFs showed a high level of ARG and a low level of NOS, indicating extensive generation of polyamines and proline in them. These CAFs can also induce fibrosis, since proline is an essential component of collagen. As it has been observed that necrotic tissue is often remodeled to fibrous tissue 1662274 as a form of healing response, [23] it is suggested that ARG2 induction is involved in fibrosis in hypoxic areas to facilitate post-necrotic fibrosis. Although it is still unclear if ARG2-expressing CAFs promote tumor progression, no significant effects on the proliferation of cancer cells and the protection of oxidative stress-induced apoptosis of cancer cells were found in our in vitro experiments (Figures 7A and 7C). Most of the PDC cells did not express ARG2. Lung cancer expresses ARG2, although immune suppression mediated by ARG2-expressing cancer cells has not been observed. [6] Thus, it is suggested that cancer cell-mediated immune suppression by ARG2 (and NOS2) in prostate cancer [5] is probably tissuedependent, and not a general event in the cancer microenvironment. Our investigation of tumor-infiltrating immune cells inFigure 4. ARG2 was expressed mostly in CAFs under hypoxic conditions. Histology of PDC tissue in low- (upper columns) and high-power view (lower column.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. doi:10.1371/journal.pone.0055146.gtissue and co-expressed CAIX, SLC2A1, and HIF-1a. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 after exposure to hypoxia. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not 1531364 a general event and that the presence of ARG2-expressing CAFs is an independent prognostic factor for PDC patients, and also reflects tissue hypoxia. Reduced oxygen tension has been reported to affect ARG activity in several tissues and cell types including lung, brain, endothelial cells, and macrophages, [14,15,16] although the effects on ARGs differ among tissues, cells, and species. In humans, it has been shown that hypoxia induces the expression of ARG2 in pulmonary artery smooth muscle cells (PASMC) [17] and lung microvascular endothelial cells. [18,19] To our knowledge, the effects of hypoxia on ARG expression in cancer tissues or fibroblasts have not been demonstrated previously. Our study together with the study that hypoxia induced expression of ARG2 but not ARG1 in PASMCs suggests that ARG2 is the hypoxiainducible isoform. [17] It is consistent with our findings that accumulation of HIF-1a was found in CAFs under hypoxic conditions and also that the 5′ flanking region of the first exon of the ARG2 gene has potential binding sites for HIF-1. Ornithine generated by ARG can be further metabolized to polyamines and proline, which are critical for cell proliferation,differentiation and tissue repair, thus potentially preventing cell death. [20,21,22] Therefore, induction of ARG2 might be a defensive response of CAFs in PDC tissue under severe hypoxic stress potentially leading to cell death. Indeed, our results showed that ARG2-expressing CAFs ameliorated oxidative stress-induced apoptosis of CAFs themselves (Figure 7C). This is consistent with that fact that these CAFs showed a high level of ARG and a low level of NOS, indicating extensive generation of polyamines and proline in them. These CAFs can also induce fibrosis, since proline is an essential component of collagen. As it has been observed that necrotic tissue is often remodeled to fibrous tissue 1662274 as a form of healing response, [23] it is suggested that ARG2 induction is involved in fibrosis in hypoxic areas to facilitate post-necrotic fibrosis. Although it is still unclear if ARG2-expressing CAFs promote tumor progression, no significant effects on the proliferation of cancer cells and the protection of oxidative stress-induced apoptosis of cancer cells were found in our in vitro experiments (Figures 7A and 7C). Most of the PDC cells did not express ARG2. Lung cancer expresses ARG2, although immune suppression mediated by ARG2-expressing cancer cells has not been observed. [6] Thus, it is suggested that cancer cell-mediated immune suppression by ARG2 (and NOS2) in prostate cancer [5] is probably tissuedependent, and not a general event in the cancer microenvironment. Our investigation of tumor-infiltrating immune cells inFigure 4. ARG2 was expressed mostly in CAFs under hypoxic conditions. Histology of PDC tissue in low- (upper columns) and high-power view (lower column.
