Dissemination Of Pancreatic Cancer Cells Research Articles

Abstract 3912: Pancreatic cancer cell interaction with platelets impedes cell cycle progression and increases viability

Abstract Background: Metastasizing cancer cells rapidly associate with platelets in circulation, but the nature and functional consequences of this interaction have yet to be fully elucidated. Here we investigated the physical interaction of platelets with pancreatic cancer (PC) cells and the contribution of platelet-derived platelet factor 4 (PF4) mediated signaling in PC cells. Methods: Immunohistochemistry (IHC) and western blot analysis were performed to characterize the expression of PF4 receptor, CXCR3, in 42 Whipple samples and 8 PC cell lines respectively. Physical interaction of human platelets with PC cell lines and its impact on PC cell proliferation in low attachment conditions was analyzed by flow cytometry. Cell viability was assayed by MTT analysis. Immunofluorescence analysis was performed to evaluate PF4 induced autophagy. Results: IHC and western blot analysis in Whipple samples showed CXCR3 expression in 86 percent of the pancreatic tumors and on all the tested PC cell lines respectively. Flow cytometry demonstrated that 24 percent of Capan1 cells interacted with platelets. Interestingly, incubation of platelets with cell-cycle-synchronized Capan1 cells under low attachment conditions inhibited the proliferation of PC cells (~10 percent) at 24 but not 48 hours. This effect was abrogated by pharmacological inhibition of CXCR3 with AMG487. Concurrently, the viability of Capan1 cells grown in low attachment conditions and treated with platelets for 72 hours was greater than that of control and AMG487 with platelets treated groups as assessed by MTT assay. Mechanistic studies suggested that recombinant PF4 augments autophagosome formation as an early event and increases the expression of p21 as a late event. Of note, both LC3 punctation and p21 expression are abrogated by concomitant incubation with PF4 and AMG487. Conclusion: Platelets appear to rapidly interact with cancer cells in suspension. Additionally, in a PF4-CXCR3 signaling dependent manner, platelets inhibit cell cycle progression and increase PC cell viability in low attachment conditions. Cumulatively, these data suggest that platelets aid in the metastatic dissemination of pancreatic cancer cells. Citation Format: Andrew C. Cannon, Sushil Kumar, Bradley Hall, Rakesh Bhatia, Rakesh Singh, Surinder Batra. Pancreatic cancer cell interaction with platelets impedes cell cycle progression and increases viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3912. doi:10.1158/1538-7445.AM2017-3912

S100P is a metastasis-associated gene that facilitates transendothelial migration of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is the 5th most common cause of cancer death in the UK and the 4th in the US. The vast majority of deaths following pancreatic cancer are due to metastatic spread, hence understanding the metastatic process is vital for identification of critically needed novel therapeutic targets. An enriched set of 33 genes differentially expressed in common between primary PDAC and liver metastases, when compared to normal tissues, was obtained through global gene expression profiling. This metastasis-associated gene set comprises transcripts from both cancer (S100P, S100A6, AGR2, etc.) and adjacent stroma (collagens type I, III, and V, etc.), thus reinforcing the concept of a continuous crosstalk between the two compartments in both primary tumours and their metastases. The expression of S100P, SFN, VCAN and collagens was further validated in additional primary PDACs and matched liver metastatic lesions, while the functional significance of one of the most highly expressed genes, S100P, was studied in more detail. We show that this protein increases the transendothelial migration of PDAC cancer cells in vitro, which was also confirmed in vivo experiments using a zebrafish embryo model. Thus S100P facilitates cancer cell intravasation/extravasation, critical steps in the hematogenous dissemination of pancreatic cancer cells.

AGR2 Is a Novel Surface Antigen That Promotes the Dissemination of Pancreatic Cancer Cells through Regulation of Cathepsins B and D

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB) and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management.

Reovirus inhibits the peritoneal dissemination of pancreatic cancer cells in an immunocompetent animal model

The prognosis of pancreatic cancer with peritoneal dissemination has not improved. The aim of this study was to clarify whether oncolytic reovirus is effective against the peritoneal dissemination of pancreatic cancer in an immunocompetent animal model. The hamster pancreatic cancer cells HaP-T1 were inoculated into the peritoneal cavity of the hamster and reovirus (1x10(8) plaque-forming units) was administered into the peritoneal cavity on days 1, 3, 5 and 7 after HaP-T1 inoculations. The number and weight of the disseminated nodules in each group were recorded. Reovirus protein in the disseminated nodules was examined by immunohistochemical staining. The tumor volumes of peritoneal dissemination in the treatment group were significantly less than those in the control group (p<0.05). In addition, the amount of ascites was decreased in the treatment group in comparison to the control group. Immunohistochemical examination revealed that reovirus replication was seen only in the disseminated nodules but not in surrounding normal tissues. There were no serious side effects observed in this study. These data suggested that intraperitoneal administration of reovirus might be an effective form of oncolytic viral therapy for peritoneal dissemination of pancreatic cancer.

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells.

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5′ splice site (5′ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5′ free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5′ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells.British Journal of Cancer (2002) 87, 898–904. doi:10.1038/sj.bjc.6600563 www.bjcancer.com© 2002 Cancer Research UK

Two types of peritoneal dissemination of pancreatic cancer cells in a hamster model.

Peritoneal dissemination has an unfavorable impact on the prognosis of pancreatic cancer, and a peritoneal dissemination model was created in hamsters by using an experimental pancreatic cancer to clarify its pathological characteristics. PGHAM-1, a cancer cell line we established from BOP induced pancreatic cancer in Syrian golden hamsters, was inoculated into the abdominal cavity of Syrian golden hamsters. After inoculation, sequential changes in the diaphragm, omentum, and parietal peritoneum, and the metastatic patterns of the PGHAM-1 cells were morphologically investigated by macroscopical, microscopical, and ultrastructural observation. The cancer cells were easily absorbed at the stomata in the diaphragm and milky spots in the omentum, which were absorptive lymphatic structures, and lymphatic metastasis occurred 4 days after inoculation. In the parietal peritoneum, however, the cancer cells attached to and proliferated on the parietal peritoneum where mesothelial cells had exfoliated and the basement membrane was exposed. This process was comparatively time-consuming, and metastasis occurred in the parietal peritoneum at 7 days after inoculation. This study suggested that there might be two patterns of peritoneal dissemination of hamster pancreatic cancer. One route is lymphatic metastasis via stomata in the diaphragm and milky spots in the omentum, and the other is direct metastasis on the parietal peritoneum; each metastasis forms independently.

Detection of ras gene mutations in perioperative peripheral blood with pancreatic adenocarcinoma.

Surgeons wish to know of any correlation between an operation and the incidence of metastasis. In perioperative periods, pancreatic cancer cells were identified by detecting mutant K‐ras gene by two‐step PCR and RFLP analysis in blood samples taken from peripheral blood. In no case was K‐ras point mutation detected in blood before operation, although the mutant band was observed in all cases at the time the lesion was resected. Surprisingly, in five of ten cases, positive bands were identified just after laparotomy, before we had reached the primary lesion. In almost all cases, mutant K‐ras was detected until the fourteenth postoperative day. These findings suggest that cancer cells exist in the circulation, and have a potential for hematogenous metastasis during the perioperative period. In conclusion, surgical stress causes hematogenous dissemination of pancreatic cancer cells, and surgeons should employ the appropriate anti‐metastasis therapy in the perioperative period.