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Heavy alcohol intake is one of the most common causes of acute pancreatitis (AP). We have previously shown that ethanol (EtOH) decreases the expression and activity of the cystic fibrosis transmembrane conductance regulator (CFTR), which plays a key role in alcohol-induced AP development. The prescription drug, Orkambi (a combination of ivacaftor and lumacaftor) can correct impaired CFTR function and expression in cystic fibrosis (CF) patients. Thus, the present study aimed to investigate whether Orkambi can mitigate alcohol-induced AP. Intact guinea-pig pancreatic ducts were pre-treated with different concentrations of ethanol (EtOH; 30, 50 and 100 mm) for 12 h alone or in combination with ivacaftor (VX770) and/or lumacaftor (VX-809), and CFTR expression and activity were evaluated by immunostaining and by the patch clamp technique, respectively. Alcoholic AP was induced in Orkambi-treated guinea-pigs, and standard laboratory and histological parameters were measured. Ivacaftor and lumacaftor alone or in combination dose-dependently restored the apical expression and activity of CFTR after EtOH treatment in vitro. Oral administration of Orkambi reduced the severity of alcohol-induced AP and restored impaired CFTR activity and expression. Orkambi is able to restore the CFTR defect caused by EtOH and decreases the severity of alcohol-induced pancreatitis. This is the first in vivo pre-clinical evidence of Orkambi efficacy in the treatment of alcohol-induced AP. KEY POINTS: Acute pancreatitis is one of the leading causes of hospital admission among gastrointestinal diseases in which the lack of a specific drug therapy plays a crucial role. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role in pancreatic ductal HCO 3 - secretion; inappropriate CFTR function, as seen in heavy alcohol consumption, increases the risk of pancreatitis development. CFTR modulators are able to prevent the inhibitory effect of ethanol and reduce pancreatic ductal injury and the severity of alcohol-induced pancreatitis. CFTR modulators present a novel option in the pharmacotherapy of alcohol-induced pancreatitis by enhancing pancreatic functions or preventing recurrence., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The primary role of pancreatic ductal HCO 3 - secretion is to prevent premature activation of digestive enzymes and to provide a vehicle for the delivery of enzymes to the duodenum. In addition, HCO 3 - is responsible for the neutralization of gastric juice and protect against the formation of protein plugs and viscous mucus. Due to this multifaceted role of HCO 3 - in the pancreas, its altered functioning can greatly contribute to the development of various exocrine diseases. It is well known that the exocrine and endocrine pancreas interact lively with each other, but not all details of this relationship are known. An interesting finding of a recent study by Jo-Watanabe et al. is that the G protein-coupled oestrogen receptor, GPR30, which is expressed in the endocrine pancreas, can be also activated by HCO 3 - . This raises the possibility that ductal cells play a key role not only in the exocrine pancreas, but presumably also in endocrine function through HCO 3 - secretion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter Hegyi reports financial support was provided by CF-Trust. Viktoria Venglovecz reports financial support was provided by National Research Development and Innovation Office. Peter Hegyi reports a relationship with National Research Development and Innovation Office that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background & Aims: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network., Methods: We used single cell RNA sequencing to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models, and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis., Results: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including a Wnt-responsive population, a ciliated population, and Flrt3 + cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples. The expression of Wnt-responsive, interferon-responsive, and epithelial-to-mesenchymal transition population markers increases in chronic pancreatitis and tumor samples., Conclusions: In light of our discovery of previously unidentified ductal populations, we unmask potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis. Thus, novel lineage-tracing models are needed to investigate ductal-specific populations in vivo., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment is distinguished by a high degree of fibrosis and inflammation, known as desmoplasia. Desmoplasia increases the stromal deposition and extracellular matrix (ECM) stiffness observed in the tumor microenvironment, contributing to the dampened penetration of pharmacological agents. The molecular and biophysical composition of the ECM during the earliest cellular changes in the development of PDAC, i.e. acinar ductal metaplasia (ADM), has not been extensively explored. We report that the mRNA expression of key protein components of the ECM increases during ADM in p48 Cre/+ ;LSL-Kras G12D (KC) mouse acinar organoids cultured in Matrigel. Treatment of the organoids with small molecular weight epigenetic modulating compounds that inhibit or reverse ADM (largazole, FK228 and chaetocin) dramatically reduced the tissue mRNA expression of collagens, hyaluronan synthase, laminin and fibronectin. The storage moduli, determined by video tracking of fluorescent nanoparticles embedded into the Matrigel, increased during ADM and was reduced following treatment with the epigenetic modulating compounds. We report that the ECM of mouse organoids stiffens during ADM and is further enhanced by the presence of mutant Kras. Moreover, select HDAC and HMT inhibitors reduced the mRNA expression of ECM components and ECM stiffness during inhibition and reversal of ADM, suggesting that these compounds may be useful as adjuvants to enhance the tumor penetration of agents used to treat PDAC., Competing Interests: Declaration of competing interest HL is co-founder of Oceanyx Pharmaceuticals, Inc., which has optioned and is negotiating licenses for patent applications related to largazole., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The basement membrane (BM) is among the predominant microenvironmental factors of normal epithelia and of precancerous epithelial lesions. Evidence suggests that the BM functions not only as a barrier to tumour invasion but also as an active tumour-suppressing signalling substrate during premalignancy. However, the molecular foundations of such mechanisms have not been elucidated. Here we explore potential tumour-suppressing functions of the BM during precancer evolution, focusing on the expression and function of the extracellular matrix receptor dystroglycan in the pancreas and pancreatic disease. We show that the dystroglycan protein is highly expressed in the acinar compartment of the normal pancreas but lower in the ductal compartment. Moreover, there is a strong suppression of dystroglycan protein expression with acinar-to-ductal metaplasia in chronic pancreatitis and in all stages of pancreatic precancer and cancer evolution, from acinar-to-ductal metaplasia to dysplasia to adenocarcinoma. The conditional knockout of dystroglycan in the murine pancreas produced little evidence of developmental or functional deficiency. However, conditional deletion of dystroglycan expression in the context of oncogenic Kras expression led to a clear acceleration of pancreatic disease evolution, including accelerated dysplasia development and decreased survival. These data establish dystroglycan as a suppressor of pancreatic dysplasia development and one that is muted in chronic pancreatitis and at the earliest stages of oncogene-induced transformation. We conclude that dystroglycan is an important mediator of the tumour-suppressing functions of the BM during precancer evolution and that reduced dystroglycan function increases cancer risk, highlighting the dynamics of cell-BM interactions as important determinants of early cancer progression. © 2024 The Pathological Society of Great Britain and Ireland., (© 2024 The Pathological Society of Great Britain and Ireland.)

Acute pancreatitis (AP) is a sudden-onset disease of the digestive system caused by abnormal activation of pancreatic enzymes. Dual oxidase 2 (DUOX2) has been found to be elevated in the progression of a variety of inflammatory diseases. Therefore, we analyzed the specific roles of DUOX2 in AP development. Blood samples were collected from of AP patients and healthy people, and the caerulein- stimulated human pancreatic duct cells (H6C7) were utilized to establish an AP cell model. Cell growth and apoptosis were measured using an MTT assay and TUNEL staining. Additionally, RT-qPCR and western blot assays were conducted to assess the RNA and protein expressions of the cells. ELISA kits were used to determine TNF-α, IL-6, IL-8, and IL-1β levels. The interaction between DUOX2 and miR-605-3p was predicted using the Targetscan database and confirmed by dual-luciferase report assay. We found that DUOX2 increased while miR-605-3p decreased in the blood of AP patients and caerulein-stimulated H6C7 cells. DUOX2 was targeted by miR-605-3p. Furthermore, DUOX2 knockdown or miR-605-3p overexpression promoted cell viability, decreased the TNF-α, IL-6, IL-8, and IL-1β levels, and inhibited apoptosis rate in caerulein-stimulated H6C7 cells. DUOX2 knockdown or miR-605-3p overexpression also increased the Bcl-2 protein levels and down-regulated Bax, cleaved-caspase-1, NLRP3 and p-p65. Interestingly, DUOX2 overexpression reversed the miR-605-3p mimic function in the caerulein-treated H6C7 cells. In conclusion, our research demonstrated that DUOX2 knockdown relieved the injury and inflammation in caerulein-stimulated H6C7 cells., Competing Interests: The authors declare there are no competing interests., (©2024 Zhang et al.)

Objective: Acute pancreatitis (AP) stands as a frequent cause for clinical emergency hospital admissions. The X-box binding protein 1 (XBP1) was found to be implicated in pancreatic acinar cell apoptosis. The objective is to unveil the potential mechanisms governed by XBP1 and SIRT6 in the context of AP., Methods: Caerulein-treated human pancreatic duct epithelial (HPDE) cells to establish an in vitro research model. The levels and regulatory role of SIRT6 in the treated cells were evaluated, including its effects on inflammatory responses, oxidative stress, apoptosis, and endoplasmic reticulum stress. The relationship between XBP1 and SIRT6 was explored by luciferase and ChIP experiments. Furthermore, the effect of XBP1 overexpression on the regulatory function of SIRT6 on cells was evaluated., Results: Caerulein promoted the decrease of SIRT6 and the increase of XBP1 in HPDE cells. Overexpression of SIRT6 slowed down the secretion of inflammatory factors, oxidative stress, apoptosis level, and endoplasmic reticulum stress in HPDE cells. However, XBP1 negatively regulated SIRT6, and XBP1 overexpression partially reversed the regulation of SIRT6 on the above aspects., Conclusion: Our study illuminates the role of XBP1 in downregulating SIRT6 in HPDE cells, thereby promoting cellular injury. Inhibiting XBP1 or augmenting SIRT6 levels holds promise in preserving cell function and represents a potential therapeutic avenue in the management of AP., (© 2024 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Background and Aims: Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP., Methods: We measured sweat chloride (Cl - ) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO 3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis., Results: Sweat samples from smokers, both with and without CP, exhibited elevated Cl - concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO 3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca 2+ ] i ), depletion of intracellular ATP (ATP i ) and mitochondrial membrane depolarisation., Conclusion: Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking., Key Points: Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO 3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Inversin-deficient (inv) mice have anomalies in liver and pancreatic development in addition to an inverted left-right axis of the body. The present study was undertaken to unveil mechanisms of bile and pancreatic duct development from immunohistochemical analyses of anomalies in inv mice. Intrahepatic bile ducts having proximodistal polarity in size and the height of their epithelia, and ductules were formed in livers of wild-type neonates. By contrast, in inv mice, ductal plates, precursor structures of intrahepatic bile ducts and ductules, persisted without the proximodistal polarity. Their epithelial cells did not acquire planar cell polarity (PCP) in terms of expression of tight junction proteins although they expressed bile duct markers, HNF1β and SOX9. They had an apicobasal polarity from expression of basal laminar components. Enlargement of the hepatic artery and poor connective tissue development, including the abnormal deposition of the extracellular matrices, were also noted in inv mice, suggesting that bile duct development was coupled to that of the hepatic artery and portal vein. In pancreata of inv neonates, neither the main pancreatic duct was formed, nor dilated duct-like structures had the morphological polarity from the connecting point with the common bile duct. Lumina of acini was dilated, and centroacinar cells changed their position in the acini to their neck region. Immunohistochemical analyses of tight junction proteins suggested that epithelial cells of the duct-like structures did not have a PCP. Thus, Invs may be required for the establishment of the PCP of the whole duct system in the liver and pancreas., (© 2023 American Association for Anatomy.)

Implications: The study identifies an opportunity to discover a PKA-independent pathway downstream of oncogene GNAS for managing IPMN lesions and their progression to PDAC., (©2024 American Association for Cancer Research.)

Introduction: Inflammation of the pancreas contributes to the development of diabetes mellitus. Although it is well-accepted that local inflammation leads to a progressive loss of functional beta cell mass that eventually causes the onset of the disease, the development of islet inflammation remains unclear., Methods: Here, we used single-cell RNA sequencing to explore the cell type-specific molecular response of primary human pancreatic cells exposed to an inflammatory environment., Results: We identified a duct subpopulation presenting a unique proinflammatory signature among all pancreatic cell types., Discussion: Overall, the findings of this study point towards a role for duct cells in the propagation of islet inflammation, and in immune cell recruitment and activation, which are key steps in the pathophysiology of diabetes mellitus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Muñoz García, Juksar, Groen, Zaldumbide, de Koning and Carlotti.)

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)-cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)-cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets., (© 2024. The Author(s).)

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO 3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO 3 - secretion, CFTR activity, and the expression of CFTR, Na + /H + exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO 3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO 3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO 3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO 3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO 3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) are grossly visible (typically > 5 mm) intraductal epithelial neoplasms of mucin-producing cells, arising in the main pancreatic duct or its branches. According to the current 2-tiered grading scheme, these lesions are categorized as having either low-grade (LG) dysplasia, which has a benign prognosis, or high-grade (HG) dysplasia, which formally represents a carcinoma in situ and thus can transform to pancreatic ductal adenocarcinoma (PDAC). Because both entities require different treatments according to their risk of becoming malignant, a precise pretherapeutic diagnostic differentiation is inevitable for adequate patient management. Recently, our group has demonstrated that 68 Ga-fibroblast activation protein (FAP) inhibitor (FAPI) PET/CT shows great potential for the differentiation of LG IPMNs, HG IPMNs, and PDAC according to marked differences in signal intensity and tracer dynamics. The purpose of this study was to biologically validate FAP as a target for PET imaging by analyzing immunohistochemical FAP expression in LG IPMNs, HG IPMNs, and PDAC and comparing with SUV and time to peak (TTP) measured in our prior study. Methods: To evaluate the correlation of the expression level of FAP and α-smooth muscle actin (αSMA) in neoplasm-associated stroma depending on the degree of dysplasia in IPMNs, 98 patients with a diagnosis of LG IPMN, HG IPMN, PDAC with associated HG IPMN, or PDAC who underwent pancreatic surgery at the University Hospital Heidelberg between 2017 and 2023 were identified using the database of the Institute of Pathology, University Hospital Heidelberg. In a reevaluation of hematoxylin- and eosin-stained tissue sections of formalin-fixed and paraffin-embedded resection material from the archive, which was originally generated for histopathologic routine diagnostics, a regrading of IPMNs was performed by a pathologist according to the current 2-tiered grading scheme, consequently eliminating the former diagnosis of "IPMN with intermediate-grade dysplasia." For each case, semithin tissue sections of 3 paraffin blocks containing neoplasm were immunohistologically stained with antibodies directed against FAP and αSMA. In a masked approach, a semiquantitative analysis of the immunohistochemically stained slides was finally performed by a pathologist by adapting the immunoreactive score (IRS) and human epidermal growth factor receptor 2 (Her2)/neu score to determine the intensity and percentage of FAP- and αSMA-positive cells. Afterward, the IRS of 14 patients who underwent 68 Ga-FAPI-74 PET/CT in our previous study was compared with their SUV max , SUV mean , and TTP for result validation. Results: From 98 patients, 294 specimens (3 replicates per patient) were immunohistochemically stained for FAP and αSMA. Twenty-three patients had LG IPMNs, 11 had HG IPMNs, 10 had HG IPMNs plus PDAC, and 54 had PDAC. The tumor stroma was in all cases variably positive for FAP. The staining intensity, percentage of FAP-positive stroma, IRS, and Her2/neu score increased with higher malignancy. αSMA expression could be shown in normal pancreatic stroma as well as within peri- and intraneoplastic desmoplastic reaction. No homogeneous increase in intensity, percentage, IRS, and Her2/neu score with higher malignancy was observed for αSMA. The comparison of the mean IRS of FAP with the mean SUV max , SUV mean , and TTP of 68 Ga-GAPI-74 PET/CT showed a matching value increasing with higher malignancy in 68 Ga-FAPI-74 PET imaging and immunohistochemical FAP expression. Conclusion: The immunohistochemical staining of IPMNs and PDAC validates FAP as a biology-based stromal target for in vivo imaging. Increasing expression of FAP in lesions with a higher degree of malignancy matches the expectation of a stronger FAP expression in PDAC and HG IPMNs than in LG IPMNs and corroborates our previous findings of higher SUVs and a longer TTP in PDAC and HG IPMNs than in LG IPMNs., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO 3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease., Competing Interests: Declaration of Competing Interest Dr. Uc receives grant support from NIH R01 DK118752 and U01 DK108334. She is a consultant for Cystic Fibrosis Foundation and Abbvie Pharmaceuticals, Inc. Other authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background & Aims: Pancreatic ductal adenocarcinoma can develop from precursor lesions, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN). Previous studies indicated that loss of Acvr1b accelerates the Kras-mediated development of papillary IPMN in the mouse pancreas; however, the cell type predominantly affected by these genetic changes remains unclear., Methods: We investigated the contribution of cellular origin by inducing IPMN associated mutations (KRAS G12D expression and Acvr1b loss) specifically in acinar (Ptf1a CreER ;Kras LSL-G12D ;Acvr1b fl/fl mice) or ductal (Sox9CreER;Kras LSL-G12D ;Acvr1b fl/fl mice) cells in mice. We then performed magnetic resonance imaging and a thorough histopathologic analysis of their pancreatic tissues., Results: The loss of Acvr1b increased the development of pancreatic intraepithelial neoplasia and IPMN-like lesions when either acinar or ductal cells expressed a Kras mutation. Magnetic resonance imaging, immunohistochemistry, and histology revealed large IPMN-like lesions in these mice that exhibited features of flat, gastric epithelium. In addition, cyst formation in both mouse models was accompanied by chronic pancreatitis. Experimental acute pancreatitis accelerated the development of large mucinous cysts and pancreatic intraepithelial neoplasia when acinar, but not ductal, cells expressed mutant Kras and lost Acvr1b., Conclusions: These findings indicate that loss of Acvr1b in the presence of the Kras oncogene promotes the development of large and small precancerous lesions from both ductal and acinar cells. However, the IPMN-like phenotype was not equivalent to that observed when these mutations were made in all pancreatic cells during development. Our study underscores the significance of the cellular context in the initiation and progression of precursor lesions from exocrine cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1 K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1 K700E alone is insufficient to induce malignant transformation of the murine pancreas, but that it increases aggressiveness of PDAC if it co-occurs with mutated KRAS and p53. We further show that Sf3b1 K700E already plays a role during early stages of pancreatic tumor progression and reduces the expression of TGF-β1-responsive epithelial-mesenchymal transition (EMT) genes. Moreover, we found that SF3B1 K700E confers resistance to TGF-β1-induced cell death in pancreatic organoids and cell lines, partly mediated through aberrant splicing of Map3k7 . Overall, our findings demonstrate that SF3B1 K700E acts as an oncogenic driver in PDAC, and suggest that it promotes the progression of early stage tumors by impeding the cellular response to tumor suppressive effects of TGF-β., Competing Interests: PS, EI, TM, KM, SA, KV, AK, TT, CS, NA, GR, MS, GS No competing interests declared, (© 2023, Simmler et al.)

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid-base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na + /H + exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO 3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid-base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO 3 , especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO 3 . We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO 3 gradients similar to that expected in the tumor.

The ubiquitin-proteasome system (UPS) is a major pathway for cellular protein degradation. The molecular function of the UPS is the removal of damaged proteins, and this function is applied in many biological processes, including inflammation, proliferation, and apoptosis. Accumulating evidence also suggests that the UPS also has a key role in pancreatic β-cell transdifferentiation in diabetes and can be targeted for treatment of diabetic diseases. In this review, we summarized the mechanistic roles of the UPS in the biochemical activities of pancreatic β-cells, including the role of the UPS in insulin synthesis and secretion, as well as β-cell degradation. Also, we discuss how the UPS mediates the transdifferentiation of pancreatic duct epithelial cells into β-cells as the experimental basis for the development of new strategies for the treatment of diabetes in regenerative medicine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pancreas ductal adenocarcinoma (PDAC) remains a malignant tumor with very poor prognosis and low 5-year overall survival. Here, we aimed to simultaneously target mitochondria and lysosomes as a new treatment paradigm of malignant pancreas cancer in vitro and in vivo. We demonstrate that the clinically used sphingosine analog FTY-720 together with PAPTP, an inhibitor of mitochondrial Kv1.3, induce death of pancreas cancer cells in vitro and in vivo. The combination of both drugs results in a marked inhibition of the acid sphingomyelinase and accumulation of cellular sphingomyelin in vitro and in vivo in orthotopic and flank pancreas cancers. Mechanistically, PAPTP and FTY-720 cause a disruption of both mitochondria and lysosomes, an alteration of mitochondrial bioenergetics and accumulation of cytoplasmic Ca 2+ , events that collectively mediate cell death. Our findings point to an unexpected cross-talk between lysosomes and mitochondria mediated by sphingolipid metabolism. We show that the combination of PAPTP and FTY-720 induces massive death of pancreas cancer cells, thereby leading to a substantially delayed and reduced PDAC growth in vivo. KEY MESSAGES: FTY-720 inhibits acid sphingomyelinase in pancreas cancer cells (PDAC). FTY-720 induces sphingomyelin accumulation and lysosomal dysfunction. The mitochondrial Kv1.3 inhibitor PAPTP disrupts mitochondrial functions. PAPTP and FTY-720 synergistically kill PDAC in vitro. The combination of FTY-720 and PAPTP greatly delays PDAC growth in vivo., (© 2023. The Author(s).)

Pancreatic cancer (PC) is a devastating malignant tumor of gastrointestinal (GI) tumors characterized by late diagnosis, low treatment success and poor prognosis. The most common pathological type of PC is pancreatic ductal adenocarcinoma (PDAC), which accounts for approximately 95% of PC. PDAC is primarily driven by the Kirsten rat sarcoma virus (KRAS) oncogene. Ferroptosis was originally described as ras-dependent cell death but is now defined as a regulated cell death caused by iron accumulation and lipid peroxidation. Recent studies have revealed that ferroptosis plays an important role in the development and therapeutic response of tumors, especially PDAC. As the non-apoptotic cell death, ferroptosis may minimize the emergence of drug resistance for clinical trials of PDAC. This article reviews what has been learned in recent years about the mechanisms of ferroptosis in PDAC, introduces the association between ferroptosis and the KRAS target, and summarizes several potential strategies that are capable of triggering ferroptosis to suppress PDAC progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by intra-tumoral heterogeneity, and patients are always diagnosed after metastasis. Thus, finding out how to effectively estimate metastatic risk underlying PDAC is necessary. In this study, we proposed scMetR to evaluate the metastatic risk of tumor cells based on single-cell RNA sequencing (scRNA-seq) data. First, we identified diverse cell types, including tumor cells and other cell types. Next, we grouped tumor cells into three sub-populations according to scMetR score, including metastasis-featuring tumor cells (MFTC), transitional metastatic tumor cells (TransMTC), and conventional tumor cells (ConvTC). We identified metastatic signature genes (MSGs) through comparing MFTC and ConvTC. Functional enrichment analysis showed that up-regulated MSGs were enriched in multiple metastasis-associated pathways. We also found that patients with high expression of up-regulated MSGs had worse prognosis. Spatial mapping of MFTC showed that they are preferentially located in the cancer and duct epithelium region, which was enriched with the ductal cells' associated inflammation. Further, we inferred cell-cell interactions, and observed that interactions of the ADGRE5 signaling pathway, which is associated with metastasis, were increased in MFTC compared to other tumor sub-populations. Finally, we predicted 12 candidate drugs that had the potential to reverse expression of MSGs. Taken together, we have proposed scMetR to estimate metastatic risk in PDAC patients at single-cell resolution which might facilitate the dissection of tumor heterogeneity.

Background and Purpose: The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma., Experimental Approach: We used patch-clamp electrophysiology, supported by Ca 2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc-1 and MiaPaCa-2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line., Key Results: DCPIB markedly increased whole-cell currents in Panc-1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by K Ca 1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induced a rapid Ca 2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca 2+ -dependent channels. However, Ca 2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding., Conclusion and Implications: DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca 2+ . Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB-based modulators of ion channels and other membrane proteins., (© 2022 The British Pharmacological Society.)

Chronic pancreatitis (CP) is a fibroinflammatory disorder of the pancreas. Our understanding of CP pathogenesis is partly limited by the incomplete characterization of pancreatic cell types. Here, we performed single-cell RNA sequencing on 3825 cells from the pancreas of one control mouse and mice with caerulein-induced CP. An analysis of the single-cell transcriptomes revealed 16 unique clusters and cell type-specific gene expression patterns in the mouse pancreas. Sub-clustering of the pancreatic mesenchymal cells from the control mouse revealed four clusters of cells with specific gene expression profiles (combinatorial expressions of Smoc2 , Cxcl14 , Tnfaip6 , and Fn1 ). We observed that immune cells in the pancreas of the CP mice were abundant and diverse in cellular type. Compared to the control, 547 upregulated genes (including Mmp7 , Ttr , Rgs5 , Adh1 , and Cldn2 ) and 257 downregulated genes were identified in ductal cells from the CP group. The elevated expression levels of MMP7 and TTR were further verified in the pancreatic ducts of CP patients. This study provides a preliminary description of the single-cell transcriptome profiles of mouse pancreata and accurately demonstrates the characteristics of pancreatic ductal cells in CP. The findings provide insight into novel disease-specific biomarkers and potential therapeutic targets of CP.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplasm with high metastatic potential and poor clinical outcome. Like other solid tumors, PDAC in the early stages is often asymptomatic, and grows very slowly under a distinct acidic pHe (extracellular pH) microenvironment. However, most previous studies have only reported the fate of cancerous cells upon cursory exposure to acidic pHe conditions. Little is known about how solid tumors-such as the lethal PDAC originating within the pancreatic duct-acinar system that secretes alkaline fluids-evolve to withstand and adapt to the prolonged acidotic microenvironmental stress., Methods: Representative PDAC cells were exposed to various biologically relevant periods of extracellular acidity. The time effects of acidic pHe stress were determined with respect to tumor cell proliferation, phenotypic regulation, autophagic control, metabolic plasticity, mitochondrial network dynamics, and metastatic potentials., Results: Unlike previous short-term analyses, we found that the acidosis-mediated autophagy occurred mainly as an early stress response but not for later adaptation to microenvironmental acidification. Rather, PDAC cells use a distinct and lengthy process of reversible adaptive plasticity centered on the early fast and later slow mitochondrial network dynamics and metabolic adjustment. This regulates their acute responses and chronic adaptations to the acidic pHe microenvironment. A more malignant state with increased migratory and invasive potentials in long-term acidosis-adapted PDAC cells was obtained with key regulatory molecules being closely related to overall patient survival. Finally, the identification of 34 acidic pHe-related genes could be potential targets for the development of diagnosis and treatment against PDAC., Conclusions: Our study offers a novel mechanism of early rapid response and late reversible adaptation of PDAC cells to the stress of extracellular acidosis. The presence of this distinctive yet slow mode of machinery fills an important knowledge gap in how solid tumor cells sense, respond, reprogram, and ultimately adapt to the persistent microenvironmental acidification., (© 2022. The Author(s).)

Immune responses are an integral part of the pathogenesis of pancreatitis. Studies applying the mouse model of pancreatitis induced by partial ligation of the pancreatic duct to explore the pancreatic immune microenvironment are still lacking. The aim of the present study is to explore the macrophage profile and associated regulatory mechanisms in mouse pancreatitis, as well as the correlation with human chronic pancreatitis (CP). In the present study, the mouse model of pancreatitis was induced by partial ligation of the pancreatic duct. Mice in the acute phase were sacrificed at 0, 4, 8, 16, 32, 72 h after ligation, while mice in the chronic phase were sacrificed at 7, 14, 21, 28 days after ligation. We found that the pancreatic pathological score, expression of TNF-α and IL-6 were elevated over time and peaked at 72h in the acute phase, while in the chronic phase, the degree of pancreatic fibrosis peaked at day 21 after ligation. Pancreatic M1 macrophages and pyroptotic macrophages showed a decreasing trend over time, whereas M2 macrophages gradually rose and peaked at day 21. IL-4 is involved in the development of CP and is mainly derived from pancreatic stellate cells (PSCs). The murine pancreatitis model constructed by partial ligation of the pancreatic duct, especially the CP model, can ideally simulate human CP caused by obstructive etiologies in terms of morphological alterations and immune microenvironment characteristics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peng, Tu, Yu, Wu, Zhang, Li, Li and Yu.)

Background & Aims: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression., Methods: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, Kras G12D -induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. Kras G12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining., Results: scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with Kras G12D -induced ADM identifies populations associated with disease progression. Activation of Kras G12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype., Conclusions: Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. Kras G12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Cystic fibrosis (CF) related diabetes is the most common comorbidity for CF patients and associated with islet dysfunction. Exocrine pancreas remodeling in CF alters the microenvironment in which islets reside. Since CFTR is mainly expressed in pancreatic ductal epithelium, we hypothesized altered CF ductal secretions could impact islet function through paracrine signals., Method: We evaluated the secretome and cellular proteome of polarized WT and CF ferret ductal epithelia using quantitative ratiometric mass spectrometry. Differentially secreted proteins (DSPs) or expressed cellular proteins were used to mine pathways, upstream regulators and the CFTR interactome to map candidate CF-associated alterations in ductal signaling and phenotype. Candidate DSPs were evaluated for their in vivo pancreatic expression patterns and their functional impact on islet hormone secretion., Results: The secretome and cellular proteome of CF ductal epithelia was significantly altered relative to WT and implicated dysregulated TGFβ, WNT, and BMP signaling pathways. Cognate receptors of DSPs from CF epithelia were equally distributed among endocrine, exocrine, and stromal pancreatic cell types. IGFBP7 was a downregulated DSP in CF ductal epithelia in vitro and exhibited reduced CF ductal expression in vivo. IGFBP7 also altered WT islet insulin secretion in response to glucose. Many CFTR-associated proteins, including SLC9A3R1, were differentially expressed in the CF cellular proteome. Upstream regulators of the differential CF ductal proteome included TGFβ, PDX1, AKT/PTEN, and INSR signaling. Data is available via ProteomeXchange with identifier PXD025126., Conclusion: These findings provide a proteomic roadmap for elucidating disturbances in autocrine and paracrine signals from CF pancreatic ducts and how they may alter islet function and maintenance., Competing Interests: Declaration of competing interest The authors declare no existing conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Ferroptosis, a new form of programmed cell death, provides a new option for anti-tumor treatment. However, the roles of ferroptosis-related (FR) genes in pancreatic adenocarcinoma (PAAD) were not fully elaborated. In the present study, 185 TCGA samples and 81 ICGC samples were used as training and validation cohorts, respectively. A novel FR risk signature (ALOX5, ALOX12, PTGS2, SAT1, STEAP3 and SQLE) was constructed via the Lasso regression analysis. In TCGA cohort, the risk signature was identified as an independent prognostic factor. Decision curve analysis (DCA) indicated that FR risk score could increase the net benefit when making clinical-decision. In addition, we constructed a nomogram to predict the overall survival rate (OSR) of individual at 1,2,3 year. Meanwhile, the prognostic value was partly validated in ICGC cohort. Through immune analyses, we found that high FR risk could affect the immune abundances of five lymphocytes but not effectively affect the activities of immune-related pathways. The expressions of most FR risk genes did not correlate with that of PD-L1(CD274) and CTLA4. Further, through RT-qPCR tests, the expressions of PTGS2 and SQLE were proven to be significantly upregulated in normal pancreatic duct epithelia cell (HPDE6-C7) compared to pancreatic cancer cells (SW1990 and BxPC-3). MTT, wound-healing and transwell assays revealed that silencing PTGS2 and SQLE could inhibit the proliferation, migration and invasion of pancreatic cancer cells. Besides, western-blot assays showed that blocking PTGS2 and SQLE expressions could suppress the protein expressions of cyclin D1 and N-cadherin, but facilitate that of E-cadherin, which suggested that they were involved in the epithelial to mesenchymal transition (EMT). Collectively, FR risk signature provides an important complement for PAAD prognostic analysis. High FR risk level can adversely affect anti-tumor immune process, but may not serve as a predictive marker of ICIs efficacy. PTGS2 and SQLE are proven to possess cancer-promoting abilities in PAAD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The production of pancreatic β cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Intraductal papillary mucinous neoplasm (IPMN) constitutes up to 20% of all pancreatic resections, and has been increasing in recent years. Histomorphological findings of IPMN are well established; however, there are not many published papers regarding the cytological findings of IPMN on fine needle aspiration (FNA) specimens. We review the cytomorphological features, molecular profile, imaging findings, and prognosis of IPMN., Methods: The English literature was thoroughly searched with key phrases containing IPMN., Observations: IPMN is a rare entity, affecting men and women equally and is usually diagnosed at the age of 60-70 years. The characteristic imaging features include a cystic lesion with associated dilatation of the main or branch pancreatic duct, and atrophy of surrounding pancreatic parenchyma. Cytomorphological features of IPMN include papillary fragments of mucinous epithelium in a background of abundant thick extracellular mucin, a hallmark feature. IPMNs should be evaluated for high-grade dysplasia, which manifests with nuclear atypia, nuclear moulding, prominent nucleoli, nuclear irregularity, and cellular crowding. Molecular profiling of IPMN along with carcinoembryonic antigen and amylase levels is useful in predicting malignancy or high-grade dysplasia arising in IPMN. Overall, the prognosis of IPMN is excellent except in those cases with high-grade dysplasia and malignant transformation. Postoperative surveillance is required for resected IPMNs., Conclusion: IPMN requires a multidisciplinary approach for management. Cytomorphological findings of IPMN on FNA, in conjunction with tumour markers in pancreatic fluid cytology and imaging findings, are of paramount importance in clinical decision-making for IPMN., (© 2021 John Wiley & Sons Ltd.)

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding 1 . Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types 2 . Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis., Competing Interests: AH, AR, LL, SA, JS, SG, XP, HJ, DB, MP, LL, JL, EC, MA, GF, JY, CY, GK, VW, MH No competing interests declared, (© 2021, Hendley et al.)

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR tm1HGU ) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis-mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Recent studies have proven that the overall pathophysiology of pancreatitis involves not only the pancreatic acinar cells but also duct cells, however, pancreatic duct contribution in acinar cells homeostasis is poorly known and the molecular mechanisms leading to acinar insult and acute pancreatitis (AP) are unclear. Our previous work also showed that S100A9 protein level was notably increased in AP rat pancreas through iTRAQ-based quantitative proteomic analysis. Therefore, we investigated the actions of injured duct cells on acinar cells and the S100A9-related effects and mechanisms underlying AP pathology in the present paper. Methods: In this study, we constructed S100A9 knockout (s100a9 -/- ) mice and an in vitro coculture system for pancreatic duct cells and acinar cells. Moreover, a variety of small molecular inhibitors of S100A9 were screened from ChemDiv through molecular docking and virtual screening methods. Results: We found that the upregulation of S100A9 induces cell injury and inflammatory response via NLRP3 activation by targeting VNN1-mediated ROS release; and loss of S100A9 decreases AP injury in vitro and in vivo . Moreover, molecular docking and mutant plasmid experiments proved that S100A9 has a direct interaction with VNN1 through the salt bridges formation of Lys57 and Glu92 residues in S100A9 protein. We further found that compounds C 42 H 60 N 4 O 6 and C 28 H 29 F 3 N 4 O 5 S can significantly improve AP injury in vitro and in vivo through inhibiting S100A9-VNN1 interaction. Conclusions: Our study showed the important regulatory effect of S100A9 on pancreatic duct injury during AP and revealed that inhibition of the S100A9-VNN1 interaction may be a key therapeutic target for this disease., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.

Aims: We investigated the in vitro differentiation of adult rat PDESCs into β-like cells through supplementation of different combinations of GABA, BMP7, and Activin A in basic culture media., Materials and Methods: The PDESCs were cultured using different inducement combinations for 28 days and microscopy, dithizone (DTZ) staining, immunohistochemical staining, real-time PCR, and glucose-stimulated insulin secretion (GSIS) assay were used to delineate the differentiation inducement potential of these combinations., Key Findings: The results show that after 28 days, the PDESCs were differentiated into ICCs containing insulin-secreting β-like cells in different groups treated with A + B, A + G, B + G, and A + B + G but not in the control group. Upon DTZ staining the cells in ICCs were stained crimson red, demonstrating the presence of β-like cells in ICCs and the immunohistochemistry showed the expression of Pdx1 and insulin in ICCs. Further, on 28 d the expression of Pdx1 and insulin mRNA was high in inducement groups as compared to the control group and β-like cells in ICCs also secreted insulin and C-peptide upon glucose stimulation. Thus, the supplementation of GABA, BMP7, and Activin A in different combinations in basic culture media can induce the in vitro differentiation of PDESCs into ICCs containing β-like cells., Significance: The in vitro development of β-like cells is a herald for cell therapy of diabetic patients and our results are a step closer towards finding the cure for diabetes., (Copyright © 2020. Published by Elsevier Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Kras mut ) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-Kras G12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreER T2 mediated recombination. Hnf1b:CreER T2 ;Kras G12V (cKras Hnf1b/+ ) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKras Low ), moderate (cKras Mod ), and high (cKras High ) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKras High mice. cKras Mod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKras Low , cKras Mod , and cKras High mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.

Pancreatic ductal adenocarcinoma (PDAC) shows poor prognosis and high malignancy due to the presence of cancer-initiating cells (CICs) and characteristics of the tumor microenvironment (TME). Organoids are useful for studying PDAC, and establishing organoids is dependent on stem cell growth factors, including Wnt signaling. Herein, using a conventional organoid culture system, we demonstrated that CD44(+)CD24(+) and CD44(+)CD24(+)EpCAM(+) CICs were enriched >65% in a PDAC patient-derived organoid. CICs expressing CD44 formed lumen structures by gathering into circles. Additionally, organoid-derived CD44(-) cancer cells were capable of organoid re-formation and could be re-programed as CD44-expressing CICs in the organoid culture system. To mimic a TME absent artificial stem cell growth factors, a PDAC organoid with vascular niche was established. CICs in the PDAC tumor organoid were maintained by paracrine effects and direct interactions with endothelial cells. Interestingly, CD44(+) cells in PDAC tumor tissue were detected primarily in the vascular niche. Inhibiting both Wnt and Notch signaling in endothelial cells suppressed organoid formation and the maintenance of CD24(+)CD44(+) CICs. Collectively, our results suggest that PDAC patient-derived organoids maintain CICs by interacting with endothelial cells via Wnt and Notch pathways., (Copyright © 2020. Published by Elsevier B.V.)

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-ras G12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-ras G12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Background: Diagnosis of early chronic pancreatitis is a clinical challenge and hindered by the lack of a gold standard. Endoscopic ultrasound (EUS) and the endoscopic pancreatic function test (ePFT) are the most sensitive morphological and functional methods in this setting. EUS-elastography allows for the quantification (strain ratio) of pancreatic fibrosis, and the dynamic evaluation of the main pancreatic duct compliance provides additional information. We developed a multimodal EUS-based approach for the evaluation of the pancreas by integrating these four methods in a single procedure., Objective: We aim to describe morphological and functional pancreatic abnormalities in patients with clinical suspicion of chronic pancreatitis and inconclusive EUS findings by using the multimodal EUS-based approach., Methods: This was a prospective, cross-sectional, observational study of patients with clinically suspected chronic pancreatitis and indeterminate EUS criteria of the disease. EUS criteria of chronic pancreatitis, quantitative pancreatic elastography, ePFT and compliance of the main pancreatic duct were evaluated in a single procedure., Results: In total, 53 patients with 3-4 EUS criteria of chronic pancreatitis were included (mean age 39.7 years, 29 male). Strain ratio was abnormally high in all patients. Peak bicarbonate concentration was decreased in 43 patients (81.1%) and the main pancreatic duct compliance was reduced in 41 patients (77.3%). Some 34 patients (64.1%) had abnormal results at EUS, elastography, ePFT and compliance of the main pancreatic duct., Conclusions: A multimodal EUS-based test for the morphological and functional evaluation of the pancreas is presented, which allows detecting mild pancreatic abnormalities in patients with suspected early chronic pancreatitis. The presence of abnormal morphological and functional evaluation of the pancreas could support the clinical suspicion of early chronic pancreatitis in the appropriate clinical setting.

Objectives: Chronic pancreatitis is the end stage of a pathologic inflammatory syndrome with multiple etiological factors, including genetic. We hypothesized that some pancreatitis etiology originates in pancreatic acinar or duct cells and requires both injury and compensatory mechanism failure., Methods: One hundred pancreatitis patients were assessed using a DNA sequencing panel for pancreatitis. Cooccurrence of variants within and between genes was measured. Gene coexpression was confirmed via published single-cell RNA sequencing., Results: One hundred and twenty-one variants were identified in 2 or more patients, 15 of which were enriched compared with reference populations. Single cell RNA-sequencing data verified coexpression of GGT1, CFTR, and PRSS1 in duct cells, PRSS1, CPA1, CEL, CTRC, and SPINK1 in acinar cells, and UBR1 in both. Multiple-risk variants with injury/stress effects (CEL, CFTR, CPA1, PRSS1) and impaired cell protection (CTRC, GGT1, SPINK1, UBR1) cooccur within duct cells, acinar cells, or both., Conclusions: Pancreatitis is a complex disorder with genetic interactions across genes and cell types. These findings suggest a new, non-Mendelian genetic risk/etiology paradigm where a combination of nonpathogenic genetic risk variants in groups of susceptibility genes and injury/dysfunction response genes contribute to acquired pancreatic disease.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: The yield of genetic testing of main pancreatic duct (MPD) fluid collected during endoscopic retrograde cholangiopancreatography (ERCP) versus endoscopic ultrasound-guided fine-needle aspiration is unclear., Methods: Consecutive MPD fluid samples obtained by endoscopic ultrasound/ERCP with DNA profiling were reviewed, excluding specimens designated "no amplification." Invasive disease included invasive cancer or malignant cytology., Results: One hundred ten samples from 109 patients who underwent ERCP (n = 32) or endoscopic ultrasound-guided fine-needle aspiration (n = 78) were analyzed (2007-2018). Leading indications were dilated MPD and suspected intraductal papillary mucinous neoplasm. Elevated DNA quantity, KRAS, loss of heterozygosity (LOH), and GNAS mutations occurred in 61.5%, 25.5%, 16.4%, and 8.7% of samples, respectively. Elevated DNA quantity occurred more frequently in ERCP samples (84.4% vs 51.9%, P = 0.002); other mutation yields were similar (P > 0.05). Invasive pathology (P = 0.032) was associated with LOH in the subset of patients who underwent surgery (n = 44). Adverse events occurred more frequently after ERCP (28.1% vs 9.0%, P = 0.016)., Conclusions: Endoscopic MPD fluid sampling may yield genetic data to improve diagnosis and risk stratification. In our surgical cohort, LOH was the sole predictor of invasive pathology. Endoscopic ultrasound-guided fine-needle aspiration of MPD fluid, when possible, is preferred because of superior safety profile.

Feline pancreatitis is a challenge to diagnose and no previously published study has described the CT characteristics of the pancreatic duct (PD) in cats. The current prospective analytical study was performed to identify and describe the CT characteristics of the PD in normal cats and to compare that to those cats with an elevated feline pancreatic lipase immunoreactivity (fPLI). Contrast-enhanced CT was performed in 16 normal cats and 13 cats with an elevated fPLI. Two ACVR-certified radiologists blinded to the fPLI status assessed whether or not the PD could be identified, contrast phase during which the PD was most conspicuous, and PD shape in the body, right and left lobes. A second-year radiology resident blinded to the fPLI status measured maximum PD diameter and PD:parenchyma. The PD was identified in 84 of 87 pancreatic segments, which was most conspicuous in the portal phase in 28 of 29 cats. The PD shape was tubular (48/84), tapered (34/84), or beaded (2/84) with no significant difference (P = 1.0 to .1615) between groups. Mean maximal PD diameters of normal cats were 1.5-1.7 mm, which was significantly larger in the body of the pancreas in cats with an elevated fPLI (2.4 mm, P = .0313). Mean PD:parenchyma was not significantly different between groups (P = .2001 to .949). In conclusion, the feline PD can be consistently identified on CT, for which the portal phase is preferred. Cats with an elevated fPLI are more likely to exhibit dilation of the PD in the body of the pancreas on CT., (© 2020 American College of Veterinary Radiology.)