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Do microvesicles represent potential novel diagnostic markers in pancreatic cancer?
Abstracts / Pancreatology 12 (2012) 502–597
Conclusion: Nilotinib inhibits proliferation in both gemcitabine sensitive and resistant cells and inhibits ABC-transporter expression and function. This new mechanism could be useful in combination therapies against ABC-transporter expressing tumors.
P165. Secretin-stimulated duodenal markers associated with increased risk for pancreatic ductal adenocarcinoma H. Raeder 1, 2, F.E. McAllister 4, E. Tjora 3, S. Bhatt 1, I. Haldorsen 5,6, J. Hu 1, S.M. Willems 4, M. Vesterhus 7, A. ElOuamaari 1, M. Liu 8, M.T.B. Raeder 3, H. Immervoll 9,10, D. Hoem 11, G. Dimsevski 7, P.R. Njolstad 2, 3, A. Molven 9,10, S.P. Gygi 4, R.N. Kulkarni 1. 1 Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA 2 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway 3 Department of Clinical Medicine, University of Bergen, Bergen, Norway 4 Department of Cell Biology, Harvard Medical School, Boston, MA, USA 5 Department of Radiology, Haukeland University Hospital, Bergen, Norway 6 Section for Radiology, Department of Surgical Sciences, University of Bergen 7 Department of Medicine, Haukeland University Hospital, Bergen, Norway 8 Department of Biomedical Engineering, Boston University, Boston, MA, USA 9 Section for Pathology, The Gade Institute, University of Bergen, Norway 10 Department of Pathology, Haukeland University Hospital, Bergen, Norway 11 Department of Surgery, Haukeland University Hospital, Bergen, Norway
Only a small minority of patients manifests surgically treatable disease at the diagnosis of pancreatic cancer. The deficiency of clinically useful biomarkers in pancreatic cancer led us to study CEL-MODY, a monogenic form of diabetes including clinical features associated with increased risk of pancreatic ductal adenocarcinoma. We applied an “-omics” approach to study secretin-stimulated duodenal juice from several family members with a common germline cause of pancreatic disease in an attempt to identify biomarkers along the disease course leading to increased risk for ductal adenocarcinoma. We combined these studies with “-omics” studies of pancreatic tissue from both CEL mutation carrying and non-carrying subjects with pancreatic ductal adenocarcinoma, and we propose some candidate markers potentially involved in the early stages of pancreatic cancer.
P166. Do microvesicles represent potential novel diagnostic markers in pancreatic cancer?
551
Materials and methods: All experiments were performed using monolayer cell cultures of pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1 and MiaPaCa-2). Cell lines were grown in serum-free medium for the period of time of MV production. MVs were isolated from the cell culture supernatants using a new isolation procedure. Later, MVs were subjected to analyses carried out by fluorescence-activated cell sorting (FACS), electron microscopy (EM) and dynamic light scattering (DLS). Results: Viability and apoptosis/necrosis of the cells were analyzed by FACS. The membraneous character of MVs was checked by staining with the lipophilic membrane marker (PKH) and annexin V binding. After the isolation procedure, visual confirmation of the presence of MVs was provided by EM. Size distribution and heterogenity of MVs was examined by DLS, while intravesicular protein content was analyzed by immunofluorescence staining. Conclusion: Our method seems suitable to isolate and detect MVs in cell culture supernatants of PDAC cells. In further experiments we plan to use xenograft settings for in vivo results. Our hope is that this approach may lead to important findings related to tumor behavior, and MVs could be used in routine clinical work as potential diagnostic markers.
P167. Role of S100A8/A9 in the cross-talk between cancer cells and stromaassociated cells Taoufik Nedjadi, John Neoptolemos, Eithne Costello. Department of Molecular and Clinical Cancer Medicine, School of Cancer Studies, University of Liverpool, United Kingdom Introduction: Pancreatic cancer is characterized by the presence of dense desmoplastic stroma harbouring a variety of cells including S100A8 and S100A9-secreting monocytes. We previously reported that exogenous S100A8/A9 proteins increase cancer cell proliferation and migration. In other cancers, these proteins are known to contribute to the formation of a pre-metastatic niche at distant sites. The aim of the current stuhellody was to further characterize the influence of S100A8 and S100A9 on pancreatic cancer cells and tumour-derived stellate cells. Methods: Profiling of 27 cytokines secreted into cell-culture medium by pancreatic cancer cell lines and tumour-derived primary stellate cells was performed using Luminex-based Multiplex assays. Reporter assays and Western blotting were undertaken to unravel signaling mechanisms involved in S100A8/S100A9-mediated effects on pancreatic cancer cells. Results: Recombinant S100A8 and S100A9 proteins stimulated secretion of specific cytokines (e.g. IL-8, FGF and TNF-alpha), whereas, PDGF secretion was stimulated by S100A8 only. S100A8/A9 activated phosphop38 and phospho-p44/42 MAPK and enhanced NF-kB activity through RAGE. S100A8 and S100A9 also induced Smad4 signaling as evidenced by phosphorylation of Smad2/3 and activation of the Smad4 luciferase. Baseline cytokine profiles for pancreatic stellate cells have been obtained, and the effects of S100A8 and S100A9 are currently under assessment. Conclusion: S100A8 and S100A9 promote specific cytokine secretion from pancreatic cancer cells. Interestingly, a number of these cytokines, in turn, induce the secretion of S100A8 and S100A9 from monocytic cells, creating a paracrine loop. These events may create a favorable environment for tumour development and metastases.
S. Szanyi, S. Békási, Z. Szabó, K. Pálóczi, É. Pállinger, S. Tóth, E. Buzás, A. Falus, Z. Darvas. Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary Introduction: Microvesicles (MVs), one of the major types of extracellular vesicles, represent a novel form of cell-to-cell communication involved in several physiological and pathological functions, including tumorigenesis and metastatic activity. Objectives: Our main goal was to set up a method for isolation and detection of MVs, and to establish a protocol for examination of the intravesicular protein content.
P168. Acute pancreatitis as the first manifestation of duodenal MALT lymphoma I. Simková 1, *, K. Urbánek 2, V. Procházka 1, M. Konecný 1, J. Gregar 1, M. Geierová 3.
* Corresponding author. II. interní klinika LF UP a FN Olomouc, I.P, Pavlova 6, Olomouc 775 20, Czech Republic.
Conclusion: Nilotinib inhibits proliferation in both gemcitabine sensitive and resistant cells and inhibits ABC-transporter expression and function. This new mechanism could be useful in combination therapies against ABC-transporter expressing tumors.
P165. Secretin-stimulated duodenal markers associated with increased risk for pancreatic ductal adenocarcinoma H. Raeder 1, 2, F.E. McAllister 4, E. Tjora 3, S. Bhatt 1, I. Haldorsen 5,6, J. Hu 1, S.M. Willems 4, M. Vesterhus 7, A. ElOuamaari 1, M. Liu 8, M.T.B. Raeder 3, H. Immervoll 9,10, D. Hoem 11, G. Dimsevski 7, P.R. Njolstad 2, 3, A. Molven 9,10, S.P. Gygi 4, R.N. Kulkarni 1. 1 Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA 2 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway 3 Department of Clinical Medicine, University of Bergen, Bergen, Norway 4 Department of Cell Biology, Harvard Medical School, Boston, MA, USA 5 Department of Radiology, Haukeland University Hospital, Bergen, Norway 6 Section for Radiology, Department of Surgical Sciences, University of Bergen 7 Department of Medicine, Haukeland University Hospital, Bergen, Norway 8 Department of Biomedical Engineering, Boston University, Boston, MA, USA 9 Section for Pathology, The Gade Institute, University of Bergen, Norway 10 Department of Pathology, Haukeland University Hospital, Bergen, Norway 11 Department of Surgery, Haukeland University Hospital, Bergen, Norway
Only a small minority of patients manifests surgically treatable disease at the diagnosis of pancreatic cancer. The deficiency of clinically useful biomarkers in pancreatic cancer led us to study CEL-MODY, a monogenic form of diabetes including clinical features associated with increased risk of pancreatic ductal adenocarcinoma. We applied an “-omics” approach to study secretin-stimulated duodenal juice from several family members with a common germline cause of pancreatic disease in an attempt to identify biomarkers along the disease course leading to increased risk for ductal adenocarcinoma. We combined these studies with “-omics” studies of pancreatic tissue from both CEL mutation carrying and non-carrying subjects with pancreatic ductal adenocarcinoma, and we propose some candidate markers potentially involved in the early stages of pancreatic cancer.
P166. Do microvesicles represent potential novel diagnostic markers in pancreatic cancer?
551
Materials and methods: All experiments were performed using monolayer cell cultures of pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1 and MiaPaCa-2). Cell lines were grown in serum-free medium for the period of time of MV production. MVs were isolated from the cell culture supernatants using a new isolation procedure. Later, MVs were subjected to analyses carried out by fluorescence-activated cell sorting (FACS), electron microscopy (EM) and dynamic light scattering (DLS). Results: Viability and apoptosis/necrosis of the cells were analyzed by FACS. The membraneous character of MVs was checked by staining with the lipophilic membrane marker (PKH) and annexin V binding. After the isolation procedure, visual confirmation of the presence of MVs was provided by EM. Size distribution and heterogenity of MVs was examined by DLS, while intravesicular protein content was analyzed by immunofluorescence staining. Conclusion: Our method seems suitable to isolate and detect MVs in cell culture supernatants of PDAC cells. In further experiments we plan to use xenograft settings for in vivo results. Our hope is that this approach may lead to important findings related to tumor behavior, and MVs could be used in routine clinical work as potential diagnostic markers.
P167. Role of S100A8/A9 in the cross-talk between cancer cells and stromaassociated cells Taoufik Nedjadi, John Neoptolemos, Eithne Costello. Department of Molecular and Clinical Cancer Medicine, School of Cancer Studies, University of Liverpool, United Kingdom Introduction: Pancreatic cancer is characterized by the presence of dense desmoplastic stroma harbouring a variety of cells including S100A8 and S100A9-secreting monocytes. We previously reported that exogenous S100A8/A9 proteins increase cancer cell proliferation and migration. In other cancers, these proteins are known to contribute to the formation of a pre-metastatic niche at distant sites. The aim of the current stuhellody was to further characterize the influence of S100A8 and S100A9 on pancreatic cancer cells and tumour-derived stellate cells. Methods: Profiling of 27 cytokines secreted into cell-culture medium by pancreatic cancer cell lines and tumour-derived primary stellate cells was performed using Luminex-based Multiplex assays. Reporter assays and Western blotting were undertaken to unravel signaling mechanisms involved in S100A8/S100A9-mediated effects on pancreatic cancer cells. Results: Recombinant S100A8 and S100A9 proteins stimulated secretion of specific cytokines (e.g. IL-8, FGF and TNF-alpha), whereas, PDGF secretion was stimulated by S100A8 only. S100A8/A9 activated phosphop38 and phospho-p44/42 MAPK and enhanced NF-kB activity through RAGE. S100A8 and S100A9 also induced Smad4 signaling as evidenced by phosphorylation of Smad2/3 and activation of the Smad4 luciferase. Baseline cytokine profiles for pancreatic stellate cells have been obtained, and the effects of S100A8 and S100A9 are currently under assessment. Conclusion: S100A8 and S100A9 promote specific cytokine secretion from pancreatic cancer cells. Interestingly, a number of these cytokines, in turn, induce the secretion of S100A8 and S100A9 from monocytic cells, creating a paracrine loop. These events may create a favorable environment for tumour development and metastases.
S. Szanyi, S. Békási, Z. Szabó, K. Pálóczi, É. Pállinger, S. Tóth, E. Buzás, A. Falus, Z. Darvas. Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary Introduction: Microvesicles (MVs), one of the major types of extracellular vesicles, represent a novel form of cell-to-cell communication involved in several physiological and pathological functions, including tumorigenesis and metastatic activity. Objectives: Our main goal was to set up a method for isolation and detection of MVs, and to establish a protocol for examination of the intravesicular protein content.
P168. Acute pancreatitis as the first manifestation of duodenal MALT lymphoma I. Simková 1, *, K. Urbánek 2, V. Procházka 1, M. Konecný 1, J. Gregar 1, M. Geierová 3.
* Corresponding author. II. interní klinika LF UP a FN Olomouc, I.P, Pavlova 6, Olomouc 775 20, Czech Republic.