Daiki Eguchi

Pirfenidone Inhibits Pancreatic Cancer Desmoplasia by Regulating Stellate Cells

Pancreatic stellate cells (PSC), which are implicated in desmoplasia in pancreatic cancer, enhance the malignancy of cancer cells and confer resistance to established treatments. We investigated whether the antifibrotic agent pirfenidone can suppress desmoplasia and exert antitumor effects against pancreatic cancer. Primary PSCs were established from pancreatic cancer tissue obtained during surgery. In vitro, pirfenidone inhibited the proliferation, invasiveness, and migration of PSCs in a dose-dependent manner. Although supernatants of untreated PSCs increased the proliferation, invasiveness, and migration of pancreatic cancer cells (PCC), supernatants of pirfenidone-treated PSCs decreased these effects. Exposure to PCC supernatant increased the production of platelet-derived growth factor-A, hepatic growth factor, collagen type I, fibronectin, and periostin in PSCs, which was significantly reduced by pirfenidone. Mice were subcutaneously implanted with PCCs (SUIT-2 cells) and PSCs into the right flank and PCCs alone into the left flank. Oral administration of pirfenidone to these mice significantly reduced tumor growth of co-implanted PCCs and PSCs, but not of PCCs alone. Pirfenidone also decreased the proliferation of PSCs and the deposition of collagen type I and periostin in tumors. In mice with orthotopic tumors consisting of PCCs co-implanted with PSCs, pirfenidone suppressed tumor growth, reduced the number of peritoneal disseminated nodules, and reduced the incidence of liver metastasis. Pirfenidone in combination with gemcitabine more effectively suppressed orthotopic tumor growth compared with pirfenidone or gemcitabine alone. In conclusion, our findings indicate that pirfenidone is a promising antitumor agent for pancreatic cancer, owing to its suppression of desmoplasia through regulating PSCs.

Pancreatic Cancer Cells Enhance the Ability of Collagen Internalization during Epithelial–Mesenchymal Transition

Background Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT). Methodology/Principal Findings We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells. Conclusions/Significance Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer.

MAL2 expression predicts distant metastasis and short survival in pancreatic cancer

BACKGROUND Pancreatic cancer is associated with a devastating prognosis, partially because of its aggressive metastatic ability. Identification of prognostic markers of metastasis would be useful in the clinical management of postoperative patients with pancreatic cancer. Mal, T-cell differentiation protein 2 (MAL2) has been identified as a molecule predictive of metastases; the clinical relevance of MAL2 in pancreatic cancer is unknown. METHODS Orthotopic human pancreatic cancer xenografts from the pancreatic cancer cell line SUIT-2 were established in nude mice. Only liver metastasis was harvested and cultured. These metastatic cycles were repeated 5 times to establish a highly metastatic cell line, termed metastatic SUIT-2 (MS). We investigated proliferation and motility of MS cells compared with those of the parent SUIT-2. Microarray analysis was performed to investigate differences in gene expression. We also performed immunohistochemical analysis of 89 formalin-fixed, paraffin-embedded human pancreatic cancer tissue samples to investigate the clinical significance of MAL2 expression. RESULTS MS cells showed a greater metastatic rate after orthotopic implantation than parental SUIT-2. MS cells had increased motility but decreased proliferation compared with parental SUIT-2. Microarray analyses showed that 26 genes were significantly upregulated (>10-fold) in MS cells compared with parental SUIT-2, particularly MAL2 expression. Immunohistochemical analysis showed that high expression of MAL2 was associated with a lesser survival of postoperative patients (P = .03) and a high rate of distant metastasis (P = .008). CONCLUSION We characterized a newly established pancreatic cancer cell line with highly metastatic potential. MAL2 is a promising predictive marker for distant metastasis and short survival in patients with resected pancreatic cancer.

CD271⁺ subpopulation of pancreatic stellate cells correlates with prognosis of pancreatic cancer and is regulated by interaction with cancer cells.

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271+ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271+ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271+ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271+ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.

Peritoneal myofibroblasts at metastatic foci promote dissemination of pancreatic cancer

Myofibroblasts in the stroma of pancreatic cancers promote tumor proliferation, invasion and metastasis by increasing extracellular matrix and secretion of several growth factors. In contrast, the role of myofibroblasts at peritoneally disseminated sites of pancreatic cancer has not yet been determined. This study was designed to assess the role of myofibroblasts at peritoneally disseminated sites of pancreatic cancer. Three primary cultures of human peritoneal myofibroblasts (hPMFs) were established from disseminated sites of pancreatic cancer and their interactions with the SUIT-2 and CAPAN-1 human pancreatic cancer cell lines were analyzed in vitro. Using a model in BALB/c nu/nu mice, we compared the dissemination ability of intraperitoneally implanted pancreatic cancer cells, with and without hPMFs, and examined the presence of green fluorescent protein (GFP)-labeled hPMFs at peritoneally disseminated sites in mice. hPMFs significantly promoted the migration and invasion of pancreatic cancer cells (P<0.05), while the cancer cells significantly promoted the migration and invasion of hPMFs (P<0.05). In vivo, the number of peritoneally disseminated nodules, more than 3 mm in size, was significantly greater in mice implanted with cancer cells plus hPMFs compared to mice implanted with cancer cells alone, with GFP-labeled hPMFs surviving in the peritoneal cavity of the former. hPMFs promote the peritoneal dissemination of pancreatic cancer. The cancer-stromal cell interaction in the peritoneal cavity may be a new therapeutic target to prevent the dissemination of pancreatic cancer.

Single-incision laparoscopy-assisted surgery for bowel obstruction: Report of three cases

We applied single-incision laparoscopy-assisted surgery for several different types of bowel obstruction in selected patients. Before the operation, a long nasal tube was inserted for intestinal decompression and assessment of a stenotic lesion. A specially-designed instrument for single-incision laparoscopic surgery, the SILS Port, was introduced at the umbilicus or proposed ileostomy site. After intracorporeal procedures, extracorporeal resection and reconstruction of the intestine was performed as needed. Three patients with bowel obstruction due to jejunal carcinoma, colonic stenosis, and adhesion underwent single-incision laparoscopy-assisted surgery. The port site was used for subsequent extracorporeal resection and anastomosis of the jejunum in two patients, and for ileostomy in the remaining patient. All of the procedures were completed safely, and there were no postoperative complications. Single-incision laparoscopy can therefore be applied for selected patients with bowel obstruction. In such cases, the preoperative insertion of a long nasal tube for decompression of intestinal contents and assessment of the stenotic lesion is necessary.

Tu1759 A CD166 Negative Subpopulation of Pancreatic Cancer Cells Has Strong Invasive and Migratory Activity

BACKGROUND: CD166 expression is correlated with prognosis in several cancers. However, its significance in pancreatic cancer is not clear. The aim of this study is to clarify the significance of CD166 expression in pancreatic cancer. METHODS: We performed flow cytometry to analyze expression of CD166 in pancreatic cancer cell lines. We also analyzed the functional differences between CD166+ and CD166cells using invasion, migration and proliferation assays. We performed immunohistochemistry to investigate CD166 expression in surgically resected pancreatic cancer tissues. RESULTS: In flow cytometry, CD166 was expressed in pancreatic cancer cells in wide range (0-99.5%). In invasion assay, the invasiveness of CD166cells was greater than that of CD166+ cancer cells (p ,0.05). .In migration assay, the migratory activity of CD166cells was greater than that of CD166+ cancer cells (p,0.05). In proliferation assay, there was no significant difference between CD166+ pancreatic cancer cells and CD166cells. The analysis of real-time quantitative RT-PCR revealed that epithelial-mesenchymal transition activator Zeb1 mRNA was over-expressed in CD166cells (p,0.001 compared with that in CD166 + cells). In immunohistochemistry, there was no significant difference in prognosis between CD166 high staining group (15 patients; 48.4%) and CD166 low staining group (16 patients; 51.6%). CONCLUSION: Our findings suggest that CD166cells exhibit more aggressive behavior and activation of Zeb1 may play a role in this behavior, although further investigation is needed.