Model Of Chronic Pancreatitis Research Articles

Inhibiting autophagy promotes collagen degradation by regulating matrix metalloproteinases in pancreatic stellate cells

AimsAutophagy is an intracellular metabolic process that degrades and recycles own constituents to maintain homeostasis and supply substrates. Disruption of collagen degradation is one of the pathogenesis of pancreatic fibrosis. In this study, we investigated the effects of inhibiting autophagy on the collagen degradation of PSCs. Main methodsRats were injected dibutyltin dichloride (DBTC) to induce chronic pancreatitis (CP) model. The expression of LC3B was measured by western blotting. Rat PSCs were isolated from pancreas tissues, and the experiments used the primary PSCs. Autophagosome was confirmed by transmission electron microscope. Immunofluorescence for LC3B and α-SMA were applied to assess autophagy and activated PSCs. The effects of autophagy inhibition of 3-MA on the expressions of LC3B, Atg5, and Beclin-1 were investigated by real-time PCR and Western blotting, as well as the α-SMA, TGF-β1, ColI, Col III, FN, MMP-2, MMP-13, TIMP-1 and TIMP-2. Meanwhile, the secretion of ColI, Col III and FN were investigated by ELISA. Key findingsThe LC3-II/I ratio was increased in rat CP model. Autophagosomes and an increased autophagic level were observed during PSCs activation. Inhibiting autophagy could down-regulate the expressions of α-SMA, TGF-β1, FN, ColI, Col III, TIMP-1 and TIMP-2, while the expressions of MMP-2 and MMP-13 were increased. SignificanceThis study confirmed that autophagic level is increased during PSCs activation in vivo and in vitro. Inhibiting autophagy prevents the activation of PSCs, and suppresses fibrosis through promoting extracellular matrix (ECM) degradation by decreasing the expression of TGF-β1 and increasing MMPs/TIMPs ratio.

Early changes in the urine proteome in a diethyldithiocarbamate-induced chronic pancreatitis rat model

Without homeostasis mechanisms, urine accumulates early changes in biomarkers and can be a better and earlier biomarker source than blood, especially for chronic diseases. This study tests whether early changes can be detected in a rat model of chronic pancreatitis induced by intraperitoneal injection of diethyldithiocarbamate. Urinary proteins from three rats were profiled by liquid chromatography coupled with tandem mass spectrometry. Compared with before injection, fifty differential proteins that had human orthologs were significantly changed in the chronic pancreatitis rats. At week 2, fifteen differential proteins were identified when no obvious pathological changes had yet appeared. Among them, twelve proteins were altered at the other two time points, five had previously been associated with chronic pancreatitis. Inflammatory cytokines infiltration, acinar disruption and fibrosis were detected at week 3 and week 4, at which points fourteen identified differential proteins had been reported to be differentially expressed in the serum or pancreatic tissue of chronic pancreatitis patients and other animal model studies. These include proteins that are significant in acute-phase response (FETUA, F2, FINC and REG3G), extracellular matrix organization (COMP, FINC) and tissue remodeling (CSPG4, GAS6). REG3G has been described abnormally expressed in several human digestive system diseases such as chronic pancreatitis. SignificanceThis study demonstrate that changes caused by chronic pancreatitis can be reflected early in urinary proteins. New clues for the early diagnosis of chronic pancreatitis can be found even with only the small number of model animals used.

Genetically induced vs. classical animal models of chronic pancreatitis: a critical comparison.

Chronic pancreatitis (CP) is an utmost complex disease that is pathogenetically linked to pancreas-intrinsic ( e.g., duct obstruction), environmental-toxic ( e.g., alcohol, smoking), and genetic factors. Studying such a complex disease naturally requires validated experimental models. In the past 2 decades, the various animal models of CP usually addressed either the pancreas-intrinsic ( e.g., the caerulein model), the environmental-toxic ( e.g., diet-induced models), or the genetic component of CP. As such, these models were far from mirroring CP in its full spectrum, and the correct choice of models was vital for valid scientific conclusions on CP. The quest for mechanistic, genetic models gave rise to models based on gene modification and transgene insertion, such as the PRSS1 and the IL-1β/IL-1β models. Recently, we witnessed the development of highly exciting models that rely on the importance of autophagy in CP, that is, the murine pancreas-specific Atg5 and LAMP2 knockout models. Today, critical comparison of these several models is more important than ever for guiding research on CP in an efficient direction. The present review outlines the characteristics of the new genetic models in comparison with the well-known classic models for CP, notes the caveats in the choice of models, and also indicates novel directions for model development.-Klauss, S., Schorn, S., Teller, S., Steenfadt, H., Friess, H., Ceyhan, G. O., Demir, I. K. Genetically induced vs. classical animal models of chronic pancreatitis: a critical comparison.

Ductal Mucus Obstruction and Reduced Fluid Secretion Are Early Defects in Chronic Pancreatitis.

Objective: Defective mucus production in the pancreas may be an important factor in the initiation and progression of chronic pancreatitis (CP), therefore we aimed to (i) investigate the qualitative and quantitative changes of mucus both in human CP and in an experimental pancreatitis model and (ii) to correlate the mucus phenotype with epithelial ion transport function.Design: Utilizing human tissue samples and a murine model of cerulein induced CP we measured pancreatic ductal mucus content by morphometric analysis and the relative expression of different mucins in health and disease. Pancreatic fluid secretion in CP model was measured in vivo by magnetic resonance cholangiopancreatography (MRCP) and in vitro on cultured pancreatic ducts. Time-changes of ductal secretory function were correlated to those of the mucin production.Results: We demonstrate increased mucus content in the small pancreatic ducts in CP. Secretory mucins MUC6 and MUC5B were upregulated in human, Muc6 in mouse CP. In vivo and in vitro fluid secretion was decreased in cerulein-induced CP. Analysis of time-course changes showed that impaired ductal ion transport is paralleled by increased Muc6 expression.Conclusion: Mucus accumulation in the small ducts is a combined effect of mucus hypersecretion and epithelial fluid secretion defect, which may lead to ductal obstruction. These results suggest that imbalance of mucus homeostasis may have an important role in the early-phase development of CP, which may have novel diagnostic and therapeutic implications.

Luteolin Ameliorates Experimental Chronic Pancreatitis Induced by Trinitrobenzenesulfonic Acid in Rats.

The purpose of this study is to assess the effect and possible mechanism of luteolin on chronic pancreatitis (CP). Trinitrobenzenesulfonic acid-induced CP was used as CP models in vivo. After the intervention of luteolin for 28 days, chronic pancreatic injury was assessed by serum hydroxyproline and pancreatic histology. α-Smooth muscle actin (α-SMA) expression was performed to detect the activation of pancreatic stellate cells (PSCs). Pancreatic stellate cells were also isolated and cultured in vitro, and the effect of luteolin on PSCs was evaluated. Transforming growth factor β (TGF-β1) signaling and its regulated mRNA expression was tested by Western blot and quantitative real-time polymerase chain reaction, respectively. The protective role of luteolin on CP was confirmed by increased pancreas/body weight ratio, decreased pancreas hydroxyproline level, and reduced fibrosis. α-SMA expressions in PSCs were significantly decreased both in vitro and in vivo after the management of luteolin. Pancreas TGF-β1 expression was significantly decreased by luteolin. Luteolin inhibited the proliferation and activation of PSCs in a dose-dependent manner. Luteolin played a protective role in CP in many aspects, partly by regulating release of inflammatory cytokines through TGF-β1 signaling pathway.

320 - A Novel Genetic Model of Progressive Chronic Pancreatitis Caused by Impairment of the Endoplasmic Reticulum Acetyl-COA Transporter AT-1

320 - A Novel Genetic Model of Progressive Chronic Pancreatitis Caused by Impairment of the Endoplasmic Reticulum Acetyl-COA Transporter AT-1

Changes of urinary proteins in DDC‐induced chronic pancreatitis rat model

Biomarkers are changes associated with the disease. Without homeostasis mechanisms, theoretically urine can be better and earlier biomarker source than blood especially for chronic diseases. Chronic pancreatitis is a chronic inflammatory disease characterized by pancreatic fibrosis as a major pathological change. If diagnosed earlier, effective preventive measures may be used. In this study, we test if early changes can be detected in rat model of chronic pancreatitis induced by intraperitoneal injection of sodium diethyldithiocarbamate. Urine samples before injection and 2 weeks, 3 weeks and 4 weeks after injection were collected. Urinary proteins from three rats were profiled by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). Compared with before injection, the urinary patterns have changed significantly in all three rats. At week 2, fifteen differential proteins were identified when there was no obvious pathological changes yet. Twelve of these differential proteins were altered at other two time points and five proteins had previously been associated with chronic pancreatitis. At week 3, twenty‐nine differential proteins were identified, when inflammatory cytokines infiltration, acinar disruption, and slight fibrosis occurred. Significant fibrosis was detected at week 4 and thirty‐eight differential proteins were identified. At the stages of week 3 and week 4, fourteen urinary proteins had been reported to deferentially expressed in the serum or pancreatic tissue of chronic pancreatitis patients and other animal model studies. These findings demonstrate that changes caused by chronic pancreatitis can be reflected early in urinary proteins. Interesting clues can be found with only small number of modeled animals for further validation.Support or Funding InformationThis work was supported by the National Key Research and Development Program of China (2016YFC1306300), Beijing Natural Science Foundation (7173264, 7172076), and funds from Beijing Normal University (11100704, 10300‐310421102).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuronal Transforming Growth Factor beta Signaling via SMAD3 Contributes to Pain in Animal Models of Chronic Pancreatitis

Chronic pancreatitis (CP) is characterized by pancreatic inflammation and fibrosis, associated with increased pancreatic expression of transforming growth factor beta (TGFB). It is not clear how these might contribute to pain. We investigated whether TGFB signaling via SMAD induces sensitization of pancreatic sensory neurons to increase nociception. CP was induced in Sprague-Dawley rats by infusion of trinitrobenzene sulfonic acid; some rats were given intrathecal infusions of TGFB1. CP was induced in control mice by administration of cerulein; we also studied β1glo/Ptf1acre-ER mice, which on induction overexpress TGFB1 in pancreatic acinar cells, and TGFBr1f/f-CGRPcreER mice, which have inducible disruption of TGFBr1 in calcitonin gene-related peptide-positive neurons. Dominant negative forms of human TGFBR2 and SMAD3 were overexpressed from viral vectors in rat pancreas. Some rats were given the SMAD3 inhibitors SIS3 or halofuginone. After induction of CP, mice were analyzed for pain in behavior tests or electrophysiologic studies of sensory neurons. Pancreatic nociceptor excitability was examined by patch-clamp techniques and nociception was measured by Von Frey Filament tests for referred somatic hyperalgesia and behavioral responses to pancreatic electrical stimulation. Pancreata were collected from mice and rats and analyzed histologically and by enzyme-linked immunosorbent assay and immunohistochemistry. Overexpression of TGFB in pancreatic acinar cells of mice and infusion of TGFB1 into rats resulted in sensory neuron hyperexcitability, SMAD3 activation, and increased nociception. This was accompanied by a reduction in the transient A-type current in pancreas-specific sensory neurons in rats, a characteristic of nociceptive sensitization in animal models of CP. Conversely, pancreata from TGFBr1f/f-CGRPcreER mice, rats with pancreatic expression of dominant negative forms of human TGFBR2 or SMAD3, and rats given small molecule inhibitors of SMAD3 had attenuated neuronal sensitization and pain behavior following induction of CP. In contrast to findings from peripheral administration of TGFB1, intrathecal infusion of TGFB1 reduced hyperalgesia in rats with CP. In pancreata of mice and rats, TGFB promotes peripheral nociceptive sensitization via a direct effect on primary sensory neurons mediated by intra-neuronal SMAD3. This is distinct from the central nervous system, where TGFB reduces nociception. These results provide an explanation for the link between fibrosis and pain in patients with CP. This signaling pathway might be targeted therapeutically to reduce pain in patients with CP.

Histone deacetylase 2 is involved in µ‑opioid receptor suppression in the spinal dorsal horn in a rat model of chronic pancreatitis pain.

Chronic pain occurs in ~85–90% of chronic pancreatitis (CP) patients. However, as the pathogenesis of CP pain remains to be fully understood, the current therapies for CP pain remain inadequate. Emerging evidence has suggested that the epigenetic modulations of genes are involved in chronic pain. In the present study, intrapancreatic trinitrobenzene sulfonic acid infusions were used to establish a CP model in rats. Mechanical allodynia was measured with von Frey filaments. Immunofluorescent staining analysis was used to observe the expression changes of histone deacetylase 2 (HDAC2) and µ-opioid receptor (MOR), and intrathecal administration of the selective HDAC2 inhibitor AR-42 was used to assess the underlying mechanisms. The expression levels of c-Jun N-terminal kinase (JNK) in the thoracic spinal cord were detected by western blotting, and the mRNA expression levels of interleukin (IL)1-β, IL-6 and tumor necrosis factor (TNF)-α were detected by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that HDAC2 expression was upregulated during the course of CP induction, while MOR activity in the thoracic spinal dorsal horn was significantly suppressed. Intrathecal infusion of AR-42 significantly attenuated CP-induced mechanical allodynia, with rescued MOR activity. Additionally, HDAC2 facilitated the release of inflammatory cytokines, including IL-1β, IL-6 and TNF-α. These results suggested that the underlying mechanisms of HDAC2 regulating MOR activity under CP induction may occur via promoting the release of inflammatory cytokines, thus activating the JNK signaling pathway. The present study suggested that the epigenetic-regulated disturbance of MOR is dependent on the endogenous analgesia system in CP, which may a provide novel therapeutic strategy for treating pain in CP.

Modified Xiaochaihu Decoction () Promotes Collagen Degradation and Inhibits Pancreatic Fibrosis in Chronic Pancreatitis Rats.

To investigate the effect of Modified Xiaochaihu Decoction (MXD, ) on collagen degradation in rats with chronic pancreatitis (CP). Rats were injected dibutyltin dichloride (DBTC, 7 mg/kg of body weight) into the right caudal vein to induce CP model. Thirty heallhy male Wistar rats were randomly divided into three groups by a random number table: the control, the model and the treatment groups. Rats of treatment group were administered MXD (10 g/kg of body weight) orally once daily starting from the day post-model establishment. Pancreatic tissues were harvested after 28-day feeding and fibrosis was evaluated by picro-sirius red staining. The contents of collagen type I and III were detected using enzymelinked immunosorbent assay (ELISA), the expression of matrix metalloproteinase 13 (MMP13) and tissue inhibitor of metalloproteinase 1 (TIMP1) was analyzed by Western blot and real-time polymerase chain reaction (PCR). The fibrosis scoring of pancreatic tissues, the concentrations of collagen type I and III, the expression levels of MMP13 and TIMP1 proteins and mRNA in the model group were all increased compared with the control group (P<0.05). After treatment with MXD, the fibrosis scoring of pancreatic tissues, the concentrations of collagen type I and III, the expression levels of MMP13 proteins and mRNA in the teatment group were all decreased compared with the model group (P<0.05), but there were no significant differences in the expression levels of TIMP1 proteins and mRNA (P>0.05). MXD could promote collagen degradation and reverse pancreatic fibrosis in CP rats via a mechanism involve up-regulation of MMP13 expression.

Pancreatic HIF2α Stabilization Leads to Chronic Pancreatitis and Predisposes to Mucinous Cystic Neoplasm.

Background & AimsTissue hypoxia controls cell differentiation in the embryonic pancreas, and promotes tumor growth in pancreatic cancer. The cellular response to hypoxia is controlled by the hypoxia-inducible factor (HIF) proteins, including HIF2α. Previous studies of HIF action in the pancreas have relied on loss-of-function mouse models, and the effects of HIF2α expression in the pancreas have remained undefined.MethodsWe developed several transgenic mouse models based on the expression of an oxygen-stable form of HIF2α, or indirect stabilization of HIF proteins though deletion of von Hippel-Lindau, thus preventing HIF degradation. Furthermore, we crossed both sets of animals into mice expressing oncogenic KrasG12D in the pancreas.ResultsWe show that HIF2α is not expressed in the normal human pancreas, however, it is up-regulated in human chronic pancreatitis. Deletion of von Hippel-Lindau or stabilization of HIF2α in mouse pancreata led to the development of chronic pancreatitis. Importantly, pancreatic HIF1α stabilization did not disrupt the pancreatic parenchyma, indicating that the chronic pancreatitis phenotype is specific to HIF2α. In the presence of oncogenic Kras, HIF2α stabilization drove the formation of cysts resembling mucinous cystic neoplasm (MCN) in humans. Mechanistically, we show that the pancreatitis phenotype is linked to expression of multiple inflammatory cytokines and activation of the unfolded protein response. Conversely, MCN formation is linked to activation of Wnt signaling, a feature of human MCN.ConclusionsWe show that pancreatic HIF2α stabilization disrupts pancreatic homeostasis, leading to chronic pancreatitis, and, in the context of oncogenic Kras, MCN formation. These findings provide new mouse models of both chronic pancreatitis and MCN, as well as illustrate the importance of hypoxia signaling in the pancreas.

Amygdalin improves microcirculatory disturbance and attenuates pancreatic fibrosis by regulating the expression of endothelin-1 and calcitonin gene-related peptide in rats

BackgroundThe pathogenesis of chronic pancreatitis (CP) is a complex process of interaction between tissue injury and repair, which involves microcirculatory disturbance. Amygdalin, an effective component extracted from Semen Persicae (a kind of Chinese herbal medicine), can decrease blood viscosity and improve microcirculation. In this study, we investigated the therapeutic effects of amygdalin on pancreatic fibrosis in rats with CP. MethodsThe rat CP model was induced by injecting dibutyltin dichloride (DBTC) into the right caudal vein. Amygdalin was administrated via the penile vein at a dose of 10 mg/(kg d) from the next day, after the induction of CP, once a day for the previous 3 days, and then once every 2 days, until the end of the experiment. Body weight was observed every 7 days. Pancreatic blood flow and histopathological changes were assessed at 28 days. The activation of pancreatic stellate cells (PSCs) was estimated by the expression of α-smooth muscle actin (α-SMA). At the same time, the expression of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β-1 (TGFβ-1), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP) of pancreatic tissues were detected. ResultsTreatment of CP rats with amygdalin improved body weight and pancreatic blood flow, as well as alleviated pancreatic fibrosis and acinar destruction, accompanied by the down-regulation of the expressions of α-SMA, PDGF-BB, TGFβ-1, and ET-1, and the up-regulation of the CGRP's expression. ConclusionAmygdalin could reduce the production of pro-fibrotic cytokines, inhibit the activation of PSCs, and attenuate pancreatic fibrosis in a rat with CP. The mechanism probably includes improving microcirculatory disturbance by regulating the production of ET-1 and CGRP.

P2X7R Blockade Prevents NLRP3 Inflammasome Activation and Pancreatic Fibrosis in a Mouse Model of Chronic Pancreatitis.

The aim of this study was to investigate the role of P2X7R (purinergic 2X7 receptor) and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). Chronic pancreatitis was induced by repeated intraperitoneal injections of 50 μg/kg cerulein for 6 weeks in mice. P2X7R antagonist oxidized ATP (OxATP) or brilliant blue G (BBG) was administered after the last cerulein injection for 2 weeks. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining. We further determined pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, interleukin 1β (IL-1β), and IL-18. The pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, IL-1β, and IL-18 were all reduced significantly in both the OxATP and BBG groups (P < 0.05). The pancreatic chronic inflammation and the fibrosis indices were all remarkably attenuated (P < 0.05). P2X7R antagonist OxATP and BBG significantly decreased pancreatic chronic inflammation and fibrosis in a mouse CP model and suggested that blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.

Adipose Stem Cell Therapy for Chronic Pancreatitis

Chronic pancreatitis (CP) is a persistent inflammation of the pancreas that disrupts normal structure and functions. The absence of well-defined criteria for early diagnosis of CP and the multi-factorial features of the disease render it difficult to develop an effective treatment. Stem cell therapy has emerged as a promising therapy for autoimmune diseases and may represent a therapeutic option for CP treatment. In this issue of Molecular Therapy, Sun et al.1 report that the infusion of adipose-derived mesenchymal stem cells (ASCs) into an experimental murine model of CP inhibited the progression of CP, greatly attenuated pancreatic damage, and reduced fibrosis and cell death.

Role of eosinophils in the initiation and progression of pancreatitis pathogenesis.

Eosinophilic pancreatitis (EP) is reported in humans; however, the etiology and role of eosinophils in EP pathogenesis are poorly understood and not well explored. Therefore, it is interesting to examine the role of eosinophils in the initiation and progression of pancreatitis pathogenesis. Accordingly, we performed anti-major basic protein immunostaining, chloroacetate esterase, and Masson's trichrome analyses to detect eosinophils, mast cells, and collagen in the tissue sections of mouse and human pancreas. Induced eosinophils accumulation and degranulation were observed in the tissue sections of human pancreatitis, compared with no eosinophils in the normal pancreatic tissue sections. Similarly, we observed induced tissue eosinophilia along with mast cells and acinar cells atrophy in cerulein-induced mouse model of chronic pancreatitis. Additionally, qPCR and ELISA analyses detected induced transcript and protein levels of proinflammatory and profibrotic cytokines, chemokines like IL 5, IL-18, eotaxin-1, eotaxin-2, TGF-β1, collagen-1, collagen-3, fibronectin, and α-SMA in experimental pancreatitis. Mechanistically, we show that eosinophil-deficient GATA1 and endogenous IL-5-deficient mice were protected from the induction of proinflammatory and profibrotic cytokines, chemokines, tissue eosinophilia, and mast cells in a cerulein-induced murine model of pancreatitis. These human and experimental data indicate that eosinophil accumulation and degranulation may have a critical role in promoting pancreatitis pathogenesis including fibrosis. Taken together, eosinophil tissue accumulation needs appropriate attention to understand and restrict the progression of pancreatitis pathogenesis in humans. NEW & NOTEWORTHY The present study for the first time shows that eosinophils accumulate in the pancreas and promote disease pathogenesis, including fibrosis in earlier reported cerulein-induced experimental models of pancreatitis. Importantly, we show that GATA-1 and IL-5 deficiency protects mice form the induction of eosinophil active chemokines, and profibrotic cytokines, including accumulation of tissue collagen in an experimental model of pancreatitis. Additionally, we state that cerulein-induced chronic pancreatitis is independent of blood eosinophilia.

A Mouse Model of Chronic Pancreatitis Induced by an Alcohol and High Fat Diet.

Background/Aims:Study of acute pancreatitis in chemically-induced rodent models has provided useful data; models of alcoholic chronic pancreatitis have not been available in mice. The aim of the present study was to characterize a mouse model of chronic pancreatitis induced solely with an alcohol and high fat (AHF) diet.Methods:Mice were fed a liquid high fat diet containing 6% alcohol as well as a high fat supplement (57% total dietary fat) over a period of five months or as control, normal chow ad libitum. Pain related measures utilized as an index of pain included mechanical sensitivity of the hind paws determined using von Frey filaments and a smooth/rough textured plate. A modified hotplate test contributed information about higher order behavioral responses to visceral hypersensitivity. Mice underwent mechanical and thermal testing both with and without pharmacological treatment with a peripherally restricted μ-opioid receptor agonist, loperamide.Results:Mice on the AHF diet exhibited mechanical and heat hypersensitivity as well as fibrotic histology indicative of chronic pancreatitis. Low dose, peripherally restricted opiate loperamide attenuated both mechanical and heat hypersensitivity.Conclusion:Mice fed an alcohol and high fat diet develop histology consistent with chronic pancreatitis as well as opioid sensitive mechanical and heat hypersensitivity.

Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression

Reg3g is a potential risk for pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that Reg3g promoted pancreatic carcinogenesis via a STAT3 signaling pathway in a murine model of chronic pancreatitis. Whether the immune response is involved in tumorigenesis induced by Reg3g remains unknown. In this study, Reg3g-regulated tumor immunity was evaluated in tumor-implanted murine models, immune cells, and tumor microenvironment. In mice that had been orthotopically or ectopically implanted with Panc02 cells, Reg3g overexpression increased EGFR and Ki67, diminished MHC-I and caspase-3 expression, and accelerated growth of tumors. By interacting with PD-1/PD-L1, Reg3g also promoted differentiation of Tregs and recruitment of MDSC, retarded maturation of DCs and inactivation of CD8+ T cells, and suppressed cross-priming of CD8+ T-cell responses by DCs in tumor-bearing mice. Knockdown of Reg3g delayed tumor development in normal mice, but not in CD8+ T-cell-deficient mice. In vitro, Reg3g upregulated EGFR in DCs, activated heme oxygenase-1 (Hmox1) involved JAK2/STAT3 signaling, raised levels of Th2 cytokines in and suppressed maturation of DCs, and enhanced tumor cell proliferation. These results reveal a novel role of Reg3g as an immunosuppressive promoter that weakens tumor-specific antigenicity and suppresses antitumor effects of CD8+ T cells in a murine model of pancreatic cancer. Reg3g produces these effects by activating the JAK2/STAT3 signaling pathway in DCs, triggering the generation of an immunosuppressive tumor microenvironment.

Abstract 2981: Identification of circulating stroma-related biomarkers for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive epithelial malignancies characterized by a relevant amount of tumor stroma. It is usually diagnosed late and has limited treatment options. Plasmatic markers capable of detecting the disease in its early stages and monitoring treatment effectiveness might change the fate of PDAC patients. This study investigates the potential value of circulating stroma-related molecules as PDAC biomarkers capable to detect the disease and to monitor treatment response. Thirty-eight stroma-related candidate biomarkers were selected from published proteomic studies on PDAC. Levels of these molecules were tested by ELISA and Multiplex Luminex assays in a first cohort of plasma samples from PDAC patients and healthy subjects. Thirteen molecules -extracellular matrix proteins and proteolytic fragments, matrix-degrading enzymes and their inhibitors, growth factors and adhesion molecules-, were found to be differentially expressed in PDAC plasma compared to healthy subjects. A second analysis in an independent cohort of PDAC patients, chronic pancreatitis patients and healthy controls confirmed the previous findings and allowed restricting the number of biomarkers up-regulated in the plasma of PDAC patients. The plasma analysis of two genetically engineered mouse models of pancreatic cancer [Pdx-1-Cre;LSL-KrasG12D and Pdx-1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+] revealed an up-regulation of four markers in association with PanIN development, indicating their potential role in the early diagnosis of pancreatic cancer. These markers were also elevated in the plasma of mice bearing patient derived orthotopic PDAC xenografts (PDAC-PDX), but not in a mouse model of caerulein-induced chronic pancreatitis. Their level was associated with PDAC-PDX tumor growth and response to gemcitabine combined with nab-paclitaxel. In conclusion this analysis has identified a panel of stroma-related potential biomarkers associated with tumor progression and drug response of PDAC. Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC 5 per mille grant n. 12182) and Fondazione “Eugenio Morandi” ONLUS per lo studio e la cura dei tumori del pancreas. Citation Format: Andrea Resovi, Luca Porcu, Alessia Anastasia, Paola Allavena, Paola Cappello, Anna Falanga, Giulia Taraboletti, Mariarosa Bani, Dorina Belotti, Raffaella Giavazzi. Identification of circulating stroma-related biomarkers for pancreatic ductal adenocarcinoma [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 2981. doi:10.1158/1538-7445.AM2017-2981

Imaging Macrophage Accumulation in a Murine Model of Chronic Pancreatitis with 125I-Iodo-DPA-713 SPECT/CT.

Pancreatitis remains a diagnostic challenge in patients with mild to moderate disease, with current imaging modalities being inadequate. Given the prominent macrophage infiltration in chronic pancreatitis, we hypothesized that 125I-iodo-DPA-713, a small-molecule radiotracer that specifically targets macrophages, could be used with SPECT/CT to image pancreatic inflammation in a relevant experimental model. Methods: Chronic pancreatitis was induced with cerulein in C57BL/6 mice, which were contrasted with saline-injected control mice. The animals were imaged at 7 wk after induction using N,N-diethyl-2-(2-(3-125I-iodo-4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (125I-iodo-DPA-713) SPECT/CT or 18F-FDG PET/CT. The biodistribution of 125I-iodo-DPA-713 was determined under the same conditions, and a pair of mice was imaged using a fluorescent analog of 125I-iodo-DPA-713, DPA-713-IRDye800CW, for correlative histology. Results: Pancreatic 125I-iodo-DPA-713 uptake was significantly higher in treated mice than control mice (5.17% ± 1.18% vs. 2.41% ± 0.34% injected dose/g, P = 0.02), as corroborated by imaging. Mice imaged with 18F-FDG PET/CT showed cerulein-enhanced pancreatic uptake in addition to a moderate signal from healthy pancreas. Near-infrared fluorescence imaging with DPA-713-IRDye800CW showed strong pancreatic uptake, focal liver uptake, and gastrointestinal uptake in the treated mice, whereas the control mice showed only urinary excretion. Ex vivo fluorescence microscopy revealed a large influx of macrophages in the pancreas colocalizing with the retained fluorescent probe in the treated but not the control mice. Conclusion: These data support the application of both 125I-iodo-DPA-713 SPECT/CT and DPA-713-IRDye800CW near-infrared fluorescence to delineate pancreatic, liver, or intestinal inflammation in living mice.

Inhibition of Jak/STAT signaling reduces the activation of pancreatic stellate cells in vitro and limits caerulein-induced chronic pancreatitis in vivo

Chronic pancreatitis (CP) is a fibro-inflammatory disease leading to pain, maldigestion, and pancreatic insufficiency. No therapeutic options exist due to a limited understanding of the biology of CP pathology. Recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of inflammatory and fibrotic processes during CP. Here, we utilized primary and immortalized PSC obtained from mice and patients with CP or pancreatic cancer to examine the effect of Jak/STAT and MAPK pathway inhibition in vitro. The well-characterized caerulein model of CP was used to assess the therapeutic efficacy of Jak1/2 inhibition in vivo. Treatment of cultured PSC with the Jak1/2 inhibitor ruxolitinib reduced STAT3 phosphorylation, cell proliferation, and expression of alpha-smooth muscle actin (α-SMA), a marker of PSC activation. Treatment with the MAPK inhibitor, MEK162, had less consistent effects on PSC proliferation and no impact on activation. In the caerulein-induced murine model of CP, administration of ruxolitinib for one week significantly reduced biomarkers of inflammation and fibrosis. These data suggest that the Jak/STAT pathway plays a prominent role in PSC proliferation and activation. In vivo treatment with the Jak1/2 inhibitor ruxolitinib reduced the severity of experimental CP, suggesting that targeting Jak/STAT signaling may represent a promising therapeutic strategy for CP.