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Characterization of perineural immune cell infiltrates in pancreatic neuritis in chronic pancreatitis and pancreatic adenocarcinoma
Abstracts / Pancreatology 13 (2013) S2–S98
PI-10 Abstract id: 103. Autophagic cell death by SPINK insufficiency induces chronic inflammation in the pancreas
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Conclusion: CSE inhibits stimulated pancreatic ductal fluid secretion suggesting a deleterious effect of smoking on fluid secretion. This inhibitory effect may contribute to the pathogenesis of CP, however, further experiments to confirm this needed. Supported by TAMOP and OTKA.
Masaki Ohmuraya, Zhenghua Li, Ken-ichi Yamamura. Institute of Resource Development and Analysis, Kumamoto University, Japan Introduction: The loss-of-function mutations of SPINK1 gene are associated with various forms of human chronic pancreatitis. We previously showed that deletion of Spink3, the mouse homologue of human SPINK1, causes pancreatic acinar cell death by excessive autophagy. Although autophagic cell death is known by morphological study, the role of it in vivo is not clear. Aims: The aim of this study was to rescue the Spink3-/- phenotype by generating Spink3-/- mice with SPINK1 expression, and to analyze the role of autophagic cell death in vivo. Patients & methods: We generated the new mice model which CAG promoter-SPINK1 minigene poly-A (SP1) transgene is inserted into X chromosome. X-inactivation is a process whereby one of the two copies of the X chromosome present in female mammals is inactivated. By utilizing X-inactivation, we were able to create unique mice in which SPINK1 expresses partially. These mice were crossed to Spink3þ/- mice to generate Spink3-/-;SP1 transgenic mice. Results: Female Spink3-/-;SP1 transgenic mice (Spink3-/-;XSP1/þ) in which SP1 transgene is present on only one of the two X chromosomes, contained both normal and degenerated acinar cells with accumulation of autophagic vacuoles at birth. These mice developed pathologic features of chronic pancreatitis, including loss of acinar cells, interstitial fibrosis with activated stellate cells, and inflammatory cell infiltration, and older mice displayed prominent expression of proto-oncogenes Egfr, Her2, and Ras. Conclusion: The results re-inforce the role of SPINK1/Spink3 gene deficiency in the development of chronic pancreatitis and indicate that chronic pancreatitis trigger by autophagic cell death.
PI-11 Abstract id: 283. Cigarette smoke extract inhibits stimulated fluid secretion of isolated guinea pig pancreatic ducts l, Zolta n Rakonczay, P ra Ga Andrea Schnur, Viktoria Venglovecz, Eleono eter Hegyi. First Department of Medicine, University of Szeged, Szeged, Hungary Introduction: Smoking represents an independent risk factor for the development of chronic pancreatitis (CP), however, the pathomechanism remains unclear. Pancreatic ductal epithelial cells (PDEC) secrete an alkaline fluid mediated by anion transport, which is responsible for maintaining the integrity of the gland. Cigarette smoke extract (CSE) was found to modulate anion transport mechanism in human airway epithelial cells, however, no information is available whether smoking has such effects on PDEC. Aims: Our aim was to investigate the effects of CSE on pancreatic ductal fluid secretion. Patients & methods: Intra/interlobular pancreatic ducts were isolated from guinea pig pancreas with enzymatic digestion and microdissection. Basal and forskolin-stimulated fluid secretion into the closed luminal space of the ducts was followed with videomicroscopy. Low magnification (4x objective) bright-field images were acquired at 1-min-intervals using a CCD camera. Relative volume was calculated with Scion Image software. Results: Isolated guinea pig pancreatic ducts were capable of secreting fluid in the presence of bicarbonate, the fluid secretion increased up to 1.550.12 (n¼9). The administration of 5 mM forskolin induced a sustained increase in the relative luminal volume which reached an average value of 1.840.12 (n¼7). Parallel administration of 40mg/ml CSE blocked the stimulatory effect of forskolin (the relative luminal volume decreased to 1.570.11, n¼6).
PI-12 Abstract id: 222. Inflammation contributes to the regression of acinar-to-ductal metaplasia during pancreatitis Kamile Grabliauskaite, Sabrina Sonda, Enrica Saponara, Theresia Reding, Rolf Graf. Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: Acinar-to-ductal metaplasia (ADM) is the de-differentiation of pancreatic acinar cells into progenitor-like cells, a process phenotypically characterised by the formation of tubular complexes expressing ductal cell markers. ADM is transient the regenerative phase after pancreatic injury. However, in the presence of prolonged damage, ADM may progress towards the formation of malignant lesions. The regulation of ADM regression is critical for pancreas homeostasis but still remains unclear. Aims: In this project we aim to investigate the contribution of inflammation to ADM regression. Materials & methods: Pancreatitis was induced by multiple injections of cerulein. The expression of inflammatory markers and immune cells was quantified by immunohistochemistry and qRT-PCR. 60% pancreatectomy was achieved by resecting the pancreatic tail. Recovery from pancreatitis was analysed in mice one week after the termination of cerulein injections in the presence or absence of anti-inflammatory treatment. Results: A higher number of infiltrating leucocytes was observed in ADM areas compared to intact acinar tissue during pancreatitis. A similar level of inflammation was observed in ADM areas after 60% pancreatectomy, while the pancreatic parenchyma remained devoid of inflammatory cells. Further immunohistochemical analyses revealed that the inflammatory response mostly consisted of infiltrating macrophages with a minor proportion of T and B cells. Interestingly, anti-inflammatory treatment administered after ADM establishment prevented ADM dissolution. Conclusion: Our data revealed that ADM is associated with a strong inflammatory response, which is independent from the total level of inflammation in the pancreatic tissue. Notably, the anti-inflammatory treatment suggested that the immunity plays an active role in the regression of ADM.
PI-13 Abstract id: 213. Characterization of perineural immune cell infiltrates in pancreatic neuritis in chronic pancreatitis and pancreatic adenocarcinoma € ralp O. Stephan Schorn, Ihsan Ekin Demir, Kun Wang, Helmut Friess, Gu Ceyhan. Department of Surgery, Klinikum rechts der Isar, Technische € nchen, Munich, Germany Universit€ at Mu Introduction: Pancreatic neuritis is one of the hallmarks of pancreatic neuropathy in chronic pancreatits (CP) and pancreatic adenocarcinoma (PCa) and correlates to pain sensation of CP and PCa patients. However, the exact characteristics of this perineural inflammation in CP and PCa are yet unknown. Aims: To investigate the immune cell subtypes that compose pancreatic neuritis in CP and PCa. Materials & methods: Pancreatic tissues from patients with CP (n¼20) and PCa (n¼20) were immunostained against markers for leukocytes,
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Abstracts / Pancreatology 13 (2013) S2–S98
macrophages, cytotoxic T-lymphocytes, T-helper cells, B-lymphocytes, plasma cells, neutrophils, eosinophils and mast-cells (MC). The inflammatory cells of pancreatic neuritis were colorimetrically quantified and correlated to pain sensation. Expression of the protease activated receptor type 1 (Par-1) and 2 (Par-2) was measured in CP and PCa tissues via QRTPCR and correlated to pain. Results: Cytotoxic T-lymphocytes (PCa: 35%, CP: 33%), macrophages (PCa: 39%, CP: 33%) and MC (PCa: 21%, CP: 27%) were the predominant immune cell subtypes in pancreatic neuritis. However, only the amount of activated mast cells (PCa without pain: 14% vs. PCa with pain: 31%; CP without pain: 19% vs. CP with pain: 34%), not other of cell subtypes, was significantly elevated in patients with neuropathic pain. Par-1 and Par-2 expression did not correlate with pain in CP and PCa. Conclusion: The majority of immune cells in pancreatic neuritis are cytotoxic T-cells, macrophages and mast cells. The particular elevation of MCTC-type mast cells around intrapancreatic nerves suggests that these cells play an important role in pancreatic neuropathic pain.
PI-14 Abstract id: 23. Endogenous nitrogen losses (praecaecal and total) in case of exocrine pancreatic insufficiency (induced by pancreatic duct ligation) in pigs – Used as a model for humans €sseler 1, Harry Loock 1, Jessamyn Classen 1, Peter Gregory 2, Josef Anne Mo Kamphues 1. 1 University of Veterinary Medicine Hannover, Institute for Animal Nutrition, Germany 2 Abbott Laboratories GmbH (Germany), Germany
Introduction: Exocrine pancreatic insufficiency (EPI) causes a distinct reduction in digestibility and absorption of nutrients but information regarding impact on endogenous losses of protein in those patients is scarce. Aims: This study aimed to quantify endogenous N losses in pancreaticduct-ligated (PL) pigs - used as a model for human EPI - and healthy control (C) pigs. € ttinger minipigs (4 PL, 4 controls) were Materials & methods: 8 Go fitted with an ileo-caecal re-entrant fistula. The diet fed was almost N-free [crude protein content (cp): 0.3 %]. 222 g dry matter (DM) of the diet were given twice a day. Ileal chyme was collected over 12 hours on 7 consecutive days. Feces were collected in a separate study for 10 days. Results: In PL the amount of chyme was higher (factor 5.5) and DMcontent of chyme was markedly higher, while cp-content of chyme was higher in C. Mass of feces did not differ but cp-concentration was higher in PL. Basal ileocaecal cp-flux (g/kg DM intake) was higher in PL (34.6 7.15) compared to control (12.5 3.68). Endogenous N losses (g/kg DM intake) via feces were also higher in PL (18.3 6.66) than in controls (7.99 4.36). Conclusion: In controls endogenous N losses were comparable with those of other studies, while in PL-pigs endogenous N-losses were 2 to 3 times higher. Therefore higher cp-recommendations for maintenance in case of EPI might be reasonable to compensate higher endogenous losses.
PI-15 Abstract id: 191. Lymphotoxin-associated inflammation as an etiological factor of pancreatic carcinogenesis Gitta Seleznik 1, Theresia Reding 1, Sabrina Sonda 1, Mathias €lder 2, Rolf Graf 1. Heikenwa 1 Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland 2 ~ 1/4 nchen, Germany Institute for Virology, Helmholtz-Zentrum MA
Introduction: Pancreatic inflammation is a well-known risk factor for pancreatic ductal adenocarcinoma (PDAC) development in humans, and its
initiation is linked to activating mutations in KRAS oncogene. Recent studies propose a stepwise process starting from acinar cells undergoing dsuctal reprogramming (acinar-to-ductal metaplasia, ADM) throughout premalignant PanIN (pancreatic intraepithelial neoplasia) lesions leading to tumor formation. Aims: To investigate the mechanisms how inflammatory damage promotes ADM and PanIN progression. Patients & methods: Here we establish a new genetic model by intercrossing the commonly used p48þ/Cre;Krasþ/G12D (KP) model for pancreatic tumorigenesis, to a novel transgenic mouse developing spontaneous pancreatic inflammation, due to pancreas specific overexpression of lymphotoxin (LT). Immunohistochemistry along with RT-PCR were used to obtain an inflammatory signature. Results: Overexpression of lymphotoxin in mice harbouring a constitutively active form of Kras mutation in the pancreas (LTKP) dramatically accelerates the development of premalignant PanIN lesions compared to KP animals. Already at 6 weeks of age we observed highly proliferating cells, development of ADM and PanIN in LTKP mice. This coincided with a significant upregulation of pro-inflammatory cytokines and cell-cycle inhibitors. This type of molecular and phenotypic change was only observed around 16 weeks of age in Kras animals. Similarly, earlier activation of downstream targets of Kras was observed in LTKP mice. Furthermore, in the KP model we detected a significant elevation of ligands and receptors of the LTbR-signaling pathway during PanIN progression. Conclusion: Our data point towards the involvement of the LTbRsignaling pathway in the initiation of pancreatic cancer, revealing lymphotoxin as a critical component of spontaneous and pancreatitis-accelerated PDAC precursor formation.
PI-16 Abstract id: 159. K-Ras-dependent signalling modulates the activation of infiltrating macrophages associated with a distinct miRNA profile in pancreatic cancer Leonie Muehlberg, Benjamin Kuehnemuth, Heidi Griesmann, Malte Buchholz, Thomas Gress, Patrick Michl. University of Marburg, Germany Introduction: Tumour-associated macrophages (TAM) play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages have been identified in invasive tumours, but also in early preinvasive pancreatic intraepithelial neoplasias (PanIN lesions) harbouring activating K-Ras mutations. Aims: We hypothesized that, by modulating secreted effectors, K-Ras modulates polarisation and transcriptional program of infiltrating macrophages, thereby facilitating tumorigenesis and tumour progression. Materials & methods: To study the interaction between preinvasive pancreatic cells and macrophages in vitro, we used human macrophages differentiated from buffy coats which were cultured with conditioned media from human pancreatic ductal epithelial (HPDE) cell lines stably expressing mutant or wild-type K-Ras. Markers for classically (M1) or alternatively (M2) activated macrophages were investigated by FACS and qRT-PCR. Furthermore, a comprehensive analysis of changes in miRNA expression levels was performed by using miRNA PCR profilers comprising 754 miRNA’s. Results: Conditioned media of HPDE cells with constitutively active KRas led to a marked upregulation of M2-markers in macrophages such as CD206. In contrast, M1-markers such as CD197 or MHCII were not significantly altered. miRNA profiling revealed a distinct set of miRNA’s up- or downregulated upon incubation with conditioned media of mutant or wild-type K-Ras-expressing HPDE cells. Several of the miRNA’s upregulated by K-Ras have been implicated in tumour promotion and angiogenesis, whereas several miRNA’s downregulated by K-Ras have been described as tumour suppressive. Conclusion: The data suggest that activating K-Ras mutations in preinvasive pancreatic precursor lesions are able to trigger an alternatively
PI-10 Abstract id: 103. Autophagic cell death by SPINK insufficiency induces chronic inflammation in the pancreas
S23
Conclusion: CSE inhibits stimulated pancreatic ductal fluid secretion suggesting a deleterious effect of smoking on fluid secretion. This inhibitory effect may contribute to the pathogenesis of CP, however, further experiments to confirm this needed. Supported by TAMOP and OTKA.
Masaki Ohmuraya, Zhenghua Li, Ken-ichi Yamamura. Institute of Resource Development and Analysis, Kumamoto University, Japan Introduction: The loss-of-function mutations of SPINK1 gene are associated with various forms of human chronic pancreatitis. We previously showed that deletion of Spink3, the mouse homologue of human SPINK1, causes pancreatic acinar cell death by excessive autophagy. Although autophagic cell death is known by morphological study, the role of it in vivo is not clear. Aims: The aim of this study was to rescue the Spink3-/- phenotype by generating Spink3-/- mice with SPINK1 expression, and to analyze the role of autophagic cell death in vivo. Patients & methods: We generated the new mice model which CAG promoter-SPINK1 minigene poly-A (SP1) transgene is inserted into X chromosome. X-inactivation is a process whereby one of the two copies of the X chromosome present in female mammals is inactivated. By utilizing X-inactivation, we were able to create unique mice in which SPINK1 expresses partially. These mice were crossed to Spink3þ/- mice to generate Spink3-/-;SP1 transgenic mice. Results: Female Spink3-/-;SP1 transgenic mice (Spink3-/-;XSP1/þ) in which SP1 transgene is present on only one of the two X chromosomes, contained both normal and degenerated acinar cells with accumulation of autophagic vacuoles at birth. These mice developed pathologic features of chronic pancreatitis, including loss of acinar cells, interstitial fibrosis with activated stellate cells, and inflammatory cell infiltration, and older mice displayed prominent expression of proto-oncogenes Egfr, Her2, and Ras. Conclusion: The results re-inforce the role of SPINK1/Spink3 gene deficiency in the development of chronic pancreatitis and indicate that chronic pancreatitis trigger by autophagic cell death.
PI-11 Abstract id: 283. Cigarette smoke extract inhibits stimulated fluid secretion of isolated guinea pig pancreatic ducts l, Zolta n Rakonczay, P ra Ga Andrea Schnur, Viktoria Venglovecz, Eleono eter Hegyi. First Department of Medicine, University of Szeged, Szeged, Hungary Introduction: Smoking represents an independent risk factor for the development of chronic pancreatitis (CP), however, the pathomechanism remains unclear. Pancreatic ductal epithelial cells (PDEC) secrete an alkaline fluid mediated by anion transport, which is responsible for maintaining the integrity of the gland. Cigarette smoke extract (CSE) was found to modulate anion transport mechanism in human airway epithelial cells, however, no information is available whether smoking has such effects on PDEC. Aims: Our aim was to investigate the effects of CSE on pancreatic ductal fluid secretion. Patients & methods: Intra/interlobular pancreatic ducts were isolated from guinea pig pancreas with enzymatic digestion and microdissection. Basal and forskolin-stimulated fluid secretion into the closed luminal space of the ducts was followed with videomicroscopy. Low magnification (4x objective) bright-field images were acquired at 1-min-intervals using a CCD camera. Relative volume was calculated with Scion Image software. Results: Isolated guinea pig pancreatic ducts were capable of secreting fluid in the presence of bicarbonate, the fluid secretion increased up to 1.550.12 (n¼9). The administration of 5 mM forskolin induced a sustained increase in the relative luminal volume which reached an average value of 1.840.12 (n¼7). Parallel administration of 40mg/ml CSE blocked the stimulatory effect of forskolin (the relative luminal volume decreased to 1.570.11, n¼6).
PI-12 Abstract id: 222. Inflammation contributes to the regression of acinar-to-ductal metaplasia during pancreatitis Kamile Grabliauskaite, Sabrina Sonda, Enrica Saponara, Theresia Reding, Rolf Graf. Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: Acinar-to-ductal metaplasia (ADM) is the de-differentiation of pancreatic acinar cells into progenitor-like cells, a process phenotypically characterised by the formation of tubular complexes expressing ductal cell markers. ADM is transient the regenerative phase after pancreatic injury. However, in the presence of prolonged damage, ADM may progress towards the formation of malignant lesions. The regulation of ADM regression is critical for pancreas homeostasis but still remains unclear. Aims: In this project we aim to investigate the contribution of inflammation to ADM regression. Materials & methods: Pancreatitis was induced by multiple injections of cerulein. The expression of inflammatory markers and immune cells was quantified by immunohistochemistry and qRT-PCR. 60% pancreatectomy was achieved by resecting the pancreatic tail. Recovery from pancreatitis was analysed in mice one week after the termination of cerulein injections in the presence or absence of anti-inflammatory treatment. Results: A higher number of infiltrating leucocytes was observed in ADM areas compared to intact acinar tissue during pancreatitis. A similar level of inflammation was observed in ADM areas after 60% pancreatectomy, while the pancreatic parenchyma remained devoid of inflammatory cells. Further immunohistochemical analyses revealed that the inflammatory response mostly consisted of infiltrating macrophages with a minor proportion of T and B cells. Interestingly, anti-inflammatory treatment administered after ADM establishment prevented ADM dissolution. Conclusion: Our data revealed that ADM is associated with a strong inflammatory response, which is independent from the total level of inflammation in the pancreatic tissue. Notably, the anti-inflammatory treatment suggested that the immunity plays an active role in the regression of ADM.
PI-13 Abstract id: 213. Characterization of perineural immune cell infiltrates in pancreatic neuritis in chronic pancreatitis and pancreatic adenocarcinoma € ralp O. Stephan Schorn, Ihsan Ekin Demir, Kun Wang, Helmut Friess, Gu Ceyhan. Department of Surgery, Klinikum rechts der Isar, Technische € nchen, Munich, Germany Universit€ at Mu Introduction: Pancreatic neuritis is one of the hallmarks of pancreatic neuropathy in chronic pancreatits (CP) and pancreatic adenocarcinoma (PCa) and correlates to pain sensation of CP and PCa patients. However, the exact characteristics of this perineural inflammation in CP and PCa are yet unknown. Aims: To investigate the immune cell subtypes that compose pancreatic neuritis in CP and PCa. Materials & methods: Pancreatic tissues from patients with CP (n¼20) and PCa (n¼20) were immunostained against markers for leukocytes,
S24
Abstracts / Pancreatology 13 (2013) S2–S98
macrophages, cytotoxic T-lymphocytes, T-helper cells, B-lymphocytes, plasma cells, neutrophils, eosinophils and mast-cells (MC). The inflammatory cells of pancreatic neuritis were colorimetrically quantified and correlated to pain sensation. Expression of the protease activated receptor type 1 (Par-1) and 2 (Par-2) was measured in CP and PCa tissues via QRTPCR and correlated to pain. Results: Cytotoxic T-lymphocytes (PCa: 35%, CP: 33%), macrophages (PCa: 39%, CP: 33%) and MC (PCa: 21%, CP: 27%) were the predominant immune cell subtypes in pancreatic neuritis. However, only the amount of activated mast cells (PCa without pain: 14% vs. PCa with pain: 31%; CP without pain: 19% vs. CP with pain: 34%), not other of cell subtypes, was significantly elevated in patients with neuropathic pain. Par-1 and Par-2 expression did not correlate with pain in CP and PCa. Conclusion: The majority of immune cells in pancreatic neuritis are cytotoxic T-cells, macrophages and mast cells. The particular elevation of MCTC-type mast cells around intrapancreatic nerves suggests that these cells play an important role in pancreatic neuropathic pain.
PI-14 Abstract id: 23. Endogenous nitrogen losses (praecaecal and total) in case of exocrine pancreatic insufficiency (induced by pancreatic duct ligation) in pigs – Used as a model for humans €sseler 1, Harry Loock 1, Jessamyn Classen 1, Peter Gregory 2, Josef Anne Mo Kamphues 1. 1 University of Veterinary Medicine Hannover, Institute for Animal Nutrition, Germany 2 Abbott Laboratories GmbH (Germany), Germany
Introduction: Exocrine pancreatic insufficiency (EPI) causes a distinct reduction in digestibility and absorption of nutrients but information regarding impact on endogenous losses of protein in those patients is scarce. Aims: This study aimed to quantify endogenous N losses in pancreaticduct-ligated (PL) pigs - used as a model for human EPI - and healthy control (C) pigs. € ttinger minipigs (4 PL, 4 controls) were Materials & methods: 8 Go fitted with an ileo-caecal re-entrant fistula. The diet fed was almost N-free [crude protein content (cp): 0.3 %]. 222 g dry matter (DM) of the diet were given twice a day. Ileal chyme was collected over 12 hours on 7 consecutive days. Feces were collected in a separate study for 10 days. Results: In PL the amount of chyme was higher (factor 5.5) and DMcontent of chyme was markedly higher, while cp-content of chyme was higher in C. Mass of feces did not differ but cp-concentration was higher in PL. Basal ileocaecal cp-flux (g/kg DM intake) was higher in PL (34.6 7.15) compared to control (12.5 3.68). Endogenous N losses (g/kg DM intake) via feces were also higher in PL (18.3 6.66) than in controls (7.99 4.36). Conclusion: In controls endogenous N losses were comparable with those of other studies, while in PL-pigs endogenous N-losses were 2 to 3 times higher. Therefore higher cp-recommendations for maintenance in case of EPI might be reasonable to compensate higher endogenous losses.
PI-15 Abstract id: 191. Lymphotoxin-associated inflammation as an etiological factor of pancreatic carcinogenesis Gitta Seleznik 1, Theresia Reding 1, Sabrina Sonda 1, Mathias €lder 2, Rolf Graf 1. Heikenwa 1 Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland 2 ~ 1/4 nchen, Germany Institute for Virology, Helmholtz-Zentrum MA
Introduction: Pancreatic inflammation is a well-known risk factor for pancreatic ductal adenocarcinoma (PDAC) development in humans, and its
initiation is linked to activating mutations in KRAS oncogene. Recent studies propose a stepwise process starting from acinar cells undergoing dsuctal reprogramming (acinar-to-ductal metaplasia, ADM) throughout premalignant PanIN (pancreatic intraepithelial neoplasia) lesions leading to tumor formation. Aims: To investigate the mechanisms how inflammatory damage promotes ADM and PanIN progression. Patients & methods: Here we establish a new genetic model by intercrossing the commonly used p48þ/Cre;Krasþ/G12D (KP) model for pancreatic tumorigenesis, to a novel transgenic mouse developing spontaneous pancreatic inflammation, due to pancreas specific overexpression of lymphotoxin (LT). Immunohistochemistry along with RT-PCR were used to obtain an inflammatory signature. Results: Overexpression of lymphotoxin in mice harbouring a constitutively active form of Kras mutation in the pancreas (LTKP) dramatically accelerates the development of premalignant PanIN lesions compared to KP animals. Already at 6 weeks of age we observed highly proliferating cells, development of ADM and PanIN in LTKP mice. This coincided with a significant upregulation of pro-inflammatory cytokines and cell-cycle inhibitors. This type of molecular and phenotypic change was only observed around 16 weeks of age in Kras animals. Similarly, earlier activation of downstream targets of Kras was observed in LTKP mice. Furthermore, in the KP model we detected a significant elevation of ligands and receptors of the LTbR-signaling pathway during PanIN progression. Conclusion: Our data point towards the involvement of the LTbRsignaling pathway in the initiation of pancreatic cancer, revealing lymphotoxin as a critical component of spontaneous and pancreatitis-accelerated PDAC precursor formation.
PI-16 Abstract id: 159. K-Ras-dependent signalling modulates the activation of infiltrating macrophages associated with a distinct miRNA profile in pancreatic cancer Leonie Muehlberg, Benjamin Kuehnemuth, Heidi Griesmann, Malte Buchholz, Thomas Gress, Patrick Michl. University of Marburg, Germany Introduction: Tumour-associated macrophages (TAM) play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages have been identified in invasive tumours, but also in early preinvasive pancreatic intraepithelial neoplasias (PanIN lesions) harbouring activating K-Ras mutations. Aims: We hypothesized that, by modulating secreted effectors, K-Ras modulates polarisation and transcriptional program of infiltrating macrophages, thereby facilitating tumorigenesis and tumour progression. Materials & methods: To study the interaction between preinvasive pancreatic cells and macrophages in vitro, we used human macrophages differentiated from buffy coats which were cultured with conditioned media from human pancreatic ductal epithelial (HPDE) cell lines stably expressing mutant or wild-type K-Ras. Markers for classically (M1) or alternatively (M2) activated macrophages were investigated by FACS and qRT-PCR. Furthermore, a comprehensive analysis of changes in miRNA expression levels was performed by using miRNA PCR profilers comprising 754 miRNA’s. Results: Conditioned media of HPDE cells with constitutively active KRas led to a marked upregulation of M2-markers in macrophages such as CD206. In contrast, M1-markers such as CD197 or MHCII were not significantly altered. miRNA profiling revealed a distinct set of miRNA’s up- or downregulated upon incubation with conditioned media of mutant or wild-type K-Ras-expressing HPDE cells. Several of the miRNA’s upregulated by K-Ras have been implicated in tumour promotion and angiogenesis, whereas several miRNA’s downregulated by K-Ras have been described as tumour suppressive. Conclusion: The data suggest that activating K-Ras mutations in preinvasive pancreatic precursor lesions are able to trigger an alternatively