Pancreatitis Tissues Research Articles

Expression and Prognostic Value of Cripto-1 in Pancreatic Cancer

Objective To investigate the expression of Cripto-1 in pancreatic cancer and to analyze its clinical significance. Methods Cripto-1 expression in normal pancreas,pancreatic cancer and adjacent non-tumor tissues,chronic pancreatitis tissues and other related tissues was evaluated using immunohistochemistry.The association of Cripto-1 expression with the clinicopathological characteristics and the prognostic value of Cripto-1 in patients with pancreatic cancer were analyzed. Results The expression of Cripto-1 was higher in chronic pancreatitis tissues,pancreatic cancer and its metastases than in normal pancreas(P=0.019,P=0.025,and P=0.018,respectively).Cripto-1 overexpression was correlated with poorly differentiated pancreatic cancer.The patients with Cripto-1 upregulation had shorter median survival time(8 months vs.16 months,χ 2=4.787,P=0.029).However,multivariate analysis failed to demonstrate overexpression of Cripto-1 an independent prognostic indicator in patients with pancreatic cancer(HR=1.341,95% CI=0.800-2.248,P=0.266).Conclusions Cripto-1 may play an important role in the development and progression of pancreatic cancer.It can be a potential prognostic biomarker for patients with pancreatic cancer.

Dysregulated SREBP1c/miR-153 signaling induced by hypertriglyceridemia worsens acute pancreatitis and delays tissue repair

Severe acute pancreatitis (AP) is a life-threatening disease with up to 30% mortality. Therefore, prevention of AP aggravation and promotion of pancreatic regeneration are critical during the course and treatment of AP. Hypertriglyceridemia (HTG) is an established aggravating factor for AP that hinders pancreatic regeneration; however, its exact mechanism remains unclear. Using miRNA sequencing and further verification, we found that miRNA-153 (miR-153) was upregulated in the pancreas of HTG animal models and in the plasma of patients with HTG-AP. Increased miR-153 aggravated HTG-AP and delayed pancreatic repair via targeting TRAF3. Furthermore, miR-153 was transcriptionally suppressed by sterol regulatory element-binding transcription factor 1c (SREBP1c), which was suppressed by lipoprotein lipase malfunction-induced HTG. Overexpressing SREBP1c suppressed miR-153 expression, alleviated the severity of AP, and facilitated tissue regeneration in vivo. Finally, therapeutic administration of insulin also protected against HTG-AP via upregulating SREBP1c. Collectively, our results not only provide evidence that HTG leads to the development of more severe AP and hinders pancreatic regeneration via inducing persistent dysregulation of SREBP1c/miR-153 signaling, but also demonstrate that SREBP1c activators, including insulin, might be used to treat HTG-AP in patients.

The Expression Profile and Textural Characteristics of C595-Reactive MUC1 in Pancreatic Ductal Adenocarcinoma for Targeted Radionuclide Therapy.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is a cancer of low survival needing novel treatment approaches such as targeted therapies. If a target is overexpressed on PDAC cells but has minimal expression on normal cells, it is considered a good candidate for targeted therapy. Identifying targets with this expression pattern can help to optimise targeted therapies to be therapeutically effective without compromising on tolerability. The aim of this study was to assess the expression of the MUC1 receptor using the C595 antibody. We performed a series of cell line and tissue studies to identify if the expression of the MUC1 receptor changes between different pancreatic pathologies, including PDAC and normal pancreatic tissue. We found that the MUC1 receptor is both overexpressed and more uniformly expressed in PDAC compared to the other tissue types assessed. This indicates that the MUC1 receptor is a feasible target for targeted therapies of PDAC.Improvements in the prognosis of pancreatic ductal adenocarcinoma (PDAC) rely on the development of effective treatments to target advanced disease. Mucin 1 (MUC1) is a transmembrane glycoprotein which is involved in the metastatic progression of PDAC and is a receptor-of-interest for targeted radionuclide therapy. The aim of this study was to determine the feasibility of MUC1-based targeted radionuclide therapy for PDAC, by evaluating the expression profile of MUC1 in different pancreatic cells and tissues using the C595 antibody. MUC1 expression was evaluated in four PDAC cell lines (PANC-1, BxPC-3, CAPAN-1 and AsPC-1) using flow cytometry and immunocytochemistry. Immunohistochemistry was performed on primary and metastatic PDAC, pancreatitis, pancreatic intra-epithelial neoplasia and normal pancreatic tissue samples to identify potential changes in C595-reactive MUC1 expression across different disease groups. C595-reactive MUC1 expression was found to varying degrees in the cell lines (11.5–93.1%). A pixel analysis of the immunohistochemical staining demonstrated highest MUC1 expression in primary PDAC tissue (mean pixel value of 205.4), followed by other pancreatic cancer types (204.9), pancreatic intra-epithelial neoplasia (203.8), metastatic PDAC (201.5), chronic pancreatitis (198.1) and normal pancreatic tissue (191.4). The increased expression in malignant tissues and reduced expression in benign tissues indicate that C595-reactive MUC1 is a potential target for targeted radionuclide therapy of PDAC.

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice

Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/- and KPC/Cdh11-/- mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/- and KPC/Cdh11-/- mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11+/- and KPC/Cdh11-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Qualitative diagnostic signature for pancreatic ductal adenocarcinoma based on the within-sample relative expression orderings.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of pancreatic cancer, which is one of the most aggressive malignant neoplasms with a 9.3% five-year survival rate. The pathological biopsy is the current golden standard for confirming suspicious lesions of PDAC, but it is not entirely reliable because of the insufficient sampling amount and inaccurate sampling location. Therefore, developing a robust signature to aid the accurate diagnosis of PDAC is critical. Based on the within-sample relative expression orderings of gene pairs, we identified a qualitative signature to discriminate both PDAC and adjacent samples from both chronic pancreatitis and normal samples in the training datasets and validated it in other independent datasets produced by different laboratories with different measuring platforms. A six-gene-pair signature was identified in the training data and validated in eight independent datasets. For surgical samples, 96.63% of 356 PDAC tissues, 100% of 11 pancreatitis tissues of non-cancer patients, and 23 of 24 normal pancreatic tissues were correctly classified. Especially, 59 of 60 cancer-adjacent normal tissues of PDAC patients were correctly identified as PDAC. For biopsy samples, all of 11 PDAC biopsy tissues were correctly classified as PDAC. The signature can distinguish both PDAC and PDAC-adjacent normal tissues from both chronic pancreatitis and normal tissues of non-cancer patients even when the sampling locations are inaccurate, which can aid the diagnosis of PDAC.

Early Diagnosis of Pancreatic Ductal Adenocarcinoma by Combining Relative Expression Orderings With Machine-Learning Method.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer deeply affecting human health. Diagnosing early-stage PDAC is the key point to PDAC patients’ survival. However, the biomarkers for diagnosing early PDAC are inexact in most cases. Therefore, it is highly desirable to identify an effective PDAC diagnostic biomarker. In the current work, we designed a novel computational approach based on within-sample relative expression orderings (REOs). A feature selection technique called minimum redundancy maximum relevance was used to pick out optimal REOs. We then compared the performances of different classification algorithms for discriminating PDAC and its adjacent normal tissues from non−PDAC tissues. The support vector machine algorithm is the best one for identifying early PDAC diagnostic biomarker. At first, a signature composed of nine gene pairs was acquired from microarray gene expression data sets. These gene pairs could produce satisfactory classification accuracy up to 97.53% in fivefold cross-validation. Subsequently, two types of data from diverse platforms, namely, microarray and RNA-Seq, were used to validate this signature. For microarray data, all (100.00%) of 115 PDAC tissues and all (100.00%) of 31 PDAC adjacent normal tissues were correctly recognized as PDAC. In addition, 88.24% of 17 non-PDAC (normal or pancreatitis) tissues were correctly classified. For the RNA-Seq data, all (100.00%) of 177 PDAC tissues and all (100.00%) of 4 PDAC adjacent normal tissues were correctly recognized as PDAC. Validation results demonstrated that the signature had a good cross-platform effect for early detection of PDAC. This work developed a new robust signature that might be a promising biomarker for early PDAC diagnosis.

Qualitative Transcriptional Signature for the Pathological Diagnosis of Pancreatic Cancer.

It is currently difficult for pathologists to diagnose pancreatic cancer (PC) using biopsy specimens because samples may have been from an incorrect site or contain an insufficient amount of tissue. Thus, there is a need to develop a platform-independent molecular classifier that accurately distinguishes benign pancreatic lesions from PC. Here, we developed a robust qualitative messenger RNA signature based on within-sample relative expression orderings (REOs) of genes to discriminate both PC tissues and cancer-adjacent normal tissues from non-PC pancreatitis and healthy pancreatic tissues. A signature comprising 12 gene pairs and 17 genes was built in the training datasets and validated in microarray and RNA-sequencing datasets from biopsy samples and surgically resected samples. Analysis of 1,007 PC tissues and 257 non-tumor samples from nine databases indicated that the geometric mean of sensitivity and specificity was 96.7%, and the area under receiver operating characteristic curve was 0.978 (95% confidence interval, 0.947–0.994). For 20 specimens obtained from endoscopic biopsy, the signature had a diagnostic accuracy of 100%. The REO-based signature described here can aid in the molecular diagnosis of PC and may facilitate objective differentiation between benign and malignant pancreatic lesions.

Fibroblasts from Distinct Pancreatic Pathologies Exhibit Disease-Specific Properties.

Although fibrotic stroma forms an integral component of pancreatic diseases, whether fibroblasts programmed by different types of pancreatic diseases are phenotypically distinct remains unknown. Here, we show that fibroblasts isolated from patients with pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), periampullary tumors, and adjacent normal (NA) tissue (N = 34) have distinct mRNA and miRNA profiles. Compared with NA fibroblasts, PDAC-associated fibroblasts were generally less sensitive to an antifibrotic stimulus (NPPB) and more responsive to positive regulators of activation such as TGFβ1 and WNT. Of the disease-associated fibroblasts examined, PDAC- and CP-derived fibroblasts shared greatest similarity, yet PDAC-associated fibroblasts expressed higher levels of tenascin C (TNC), a finding attributable to miR-137, a novel regulator of TNC. TNC protein and transcript levels were higher in PDAC tissue versus CP tissue and were associated with greater levels of stromal activation, and conditioned media from TNC-depleted PDAC-associated fibroblasts modestly increased both PDAC cell proliferation and PDAC cell migration, indicating that stromal TNC may have inhibitory effects on PDAC cells. Finally, circulating TNC levels were higher in patients with PDAC compared with CP. Our characterization of pancreatic fibroblast programming as disease-specific has consequences for therapeutic targeting and for the manner in which fibroblasts are used in research. SIGNIFICANCE: Primary fibroblasts derived from various types of pancreatic diseases possess and retain distinct molecular and functional characteristics in culture, providing a series of cellular models for treatment development and disease-specific research.

MicroRNA-10a promotes epithelial-to-mesenchymal transition and stemness maintenance of pancreatic cancer stem cells via upregulating the Hippo signaling pathway through WWC2 inhibition.

MicroRNAs (miRNAs)-mediated cancer stem cells (CSCs) have drawn wide attention. This study aimed to probe the role of miR-10a in epithelial-mesenchymal transition (EMT) and stemness maintenance of pancreatic CSCs (PCSCs). Differentially expressed miRs and genes in pancreatic cancer (PC) were predicted via an online database, and the miR-10a and WW and C2 domain containing 2 (WWC2) expression were identified via a comparative study in PC and pancreatitis tissues. PCNCs were isolated and identified, and then the functional roles of miR-10a and WWC2 in proliferation, invasion, migration, self-renewal, colony formation abilities, EMT, and stemness maintenance of PCNCs were determined. The effects of miR-10a on tumor growth in vivo were studied by performing a xenograft tumor in nude mice. Consequently, miR-10a was highly expressed while WWC2 was lowly expressed in PC tissues. miR-10a could target WWC2 expression. miR-10a inhibition reduced EMT and stemness maintenance of PCSCs via enhancing WWC2 expression. The in vitro results were reproduced in in vivo studies. miR-10a promoted EMT and stemness maintenance of PCSCs via activating the Hippo signaling pathway. Our study provided evidence that miR-10a inhibition reduced EMT and stemness maintenance of PCSCs via upregulating WWC2 expression and inhibiting the Hippo signaling pathway.

SST gene hypermethylation acts as a pan-cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood-based diagnosis.

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell‐free (cf) DNA isolated from plasma samples of PDAC patients and cancer‐free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan‐cancer biomarker detectable in tumor tissue, and liquid biopsy samples.

MiR-375 Inhibits Autophagy and Further Promotes Inflammation and Apoptosis of Acinar Cells by Targeting ATG7.

MicroRNAs have been considered to be closely related with the development of severe acute pancreatitis (SAP), and microRNA-375 (miR-375) was believed to be a marker of SAP. We aim to investigate the role of miR-375 in regulating SP. Cerulein and lipopolysaccharide were used to establish the models of SAP. AR42J cell line was chosen for study in vitro. Flow cytometry was applied for assessing apoptosis. The contents of inflammatory factors were detected with related enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction assays. Hematoxylin and eosin staining was applied to observe the pathological changes of pancreatic tissues. Immunohistochemistry analysis was conducted for investigating the expression of light chain 3. The level of miR-375 in pancreatitis tissues and cell lines was upregulated. Overexpression of miR-375 promoted inflammation and the apoptosis of acinar cells through inhibiting autophagy. The binding site between miR-375 and ATG7 was identified, and miR-375 could directly regulate the ATG7. microRNA-375 suppressed autophagy and promoted inflammation and the apoptosis of acinar cells via targeting ATG7. We proved that miR-375 could inhibit autophagy and promote inflammation and the apoptosis of acinar cells through regulating ATG7. This study first proves that miR-375 modulates the development of SAP through targeting ATG7.

Harmonic Motion Imaging of Pancreatic Tumor Stiffness Indicates Disease State and Treatment Response.

Pancreatic ductal adenocarcinoma (PDA) is a common, deadly cancer that is challenging both to diagnose and to manage. Its hallmark is an expansive, desmoplastic stroma characterized by high mechanical stiffness. In this study, we sought to leverage this feature of PDA for two purposes: differential diagnosis and monitoring of response to treatment. Harmonic motion imaging (HMI) is a functional ultrasound technique that yields a quantitative relative measurement of stiffness suitable for comparisons between individuals and over time. We used HMI to quantify pancreatic stiffness in mouse models of pancreatitis and PDA as well as in a series of freshly resected human pancreatic cancer specimens. In mice, we learned that stiffness increased during progression from preneoplasia to adenocarcinoma and also effectively distinguished PDA from several forms of pancreatitis. In human specimens, the distinction of tumors versus adjacent pancreatitis or normal pancreas tissue was even more stark. Moreover, in both mice and humans, stiffness increased in proportion to tumor size, indicating that tuning of mechanical stiffness is an ongoing process during tumor progression. Finally, using a brca2-mutant mouse model of PDA that is sensitive to cisplatin, we found that tissue stiffness decreases when tumors respond successfully to chemotherapy. Consistent with this observation, we found that tumor tissues from patients who had undergone neoadjuvant therapy were less stiff than those of untreated patients. These findings support further development of HMI for clinical applications in disease staging and treatment response assessment in PDA.

IAP inhibitor, Embelin increases VCAM-1 levels on the endothelium, producing lymphocytic infiltration and antitumor immunity

ABSTRACT There is an increasing unmet need for successful immunotherapeutic interventions. Lymphocyte extravasation via tumor tissue endothelial cells (TECs) is required for lymphocyte infiltration into tumor sites. This study aimed to investigate the clinical significance of dysfunctional TECs in pancreatic ductal adenocarcinoma (PDAC) and identify chemical compounds that boost tumor-infiltrating lymphocyte (TIL) numbers. We performed immunohistochemical detection and clinicopathological analysis of VCAM-1 on TECs, which is essential for lymphocyte trafficking. We characterized the gene expression profiles of TECs from fresh PDAC tissues. We isolated compounds that upregulated VCAM-1 and E-selectin expression in TECs and examined their biological activities. Compared to endothelial cells from chronic pancreatitis tissue, TECs showed significantly lower VCAM-1 and E-selectin expression and significant weaknesses in lymphocyte adhesion and transmigration, resulting in decreased T cell infiltration around vessels. Patients with a relatively high percentage of VCAM-1+ vessels among all vessels in PDAC tissue had an improved prognosis. A bioinformatics survey demonstrated that TNFR1 signaling was involved in abnormal VCAM-1 and E-selectin expression in TECs. We screened compounds affecting TNFR1 signaling, and the IAP inhibitor, Embelin, induced these molecules on TECs and enhanced T cell adhesion to TECs and transmigration. Furthermore, in vivo, Embelin enhanced tumor-infiltrating T cell numbers, leading to an antitumor immune response. Embelin accelerates TIL infiltration and the antitumor immune response by recovering VCAM-1 expression in TECs. Our strategy may be a therapeutic approach for accelerating the immunotherapeutic response in immune-quiescent tumors, leading to clinical trials’ success.

Critical Role of TFEB-Mediated Lysosomal Biogenesis in Alcohol-Induced Pancreatitis in Mice and Humans.

Background & AimsAlcohol abuse is the major cause of experimental and human pancreatitis but the molecular mechanisms remain largely unknown. We investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in the pathogenesis of alcoholic pancreatitis.MethodsUsing a chronic plus acute alcohol binge (referred to as Gao-binge) mouse model, we analyzed pancreas injury, autophagic flux, zymogen granule removal, TFEB nuclear translocation and lysosomal biogenesis in GFP-LC3 transgenic mice, acinar cell-specific Atg5 knockout (KO) and TFEB KO mice as well as their matched wild type mice.ResultsWe found that Gao-binge alcohol induced typical features of pancreatitis in mice with increased serum amylase and lipase activities, pancreatic edema, infiltration of inflammatory cells, accumulation of zymogen granules (ZGs) and expression of inflammatory cytokines. While Gao-binge alcohol increased the number of autophagosomes, it also concurrently inhibited TFEB nuclear translocation and TFEB-mediated lysosomal biogenesis resulting in insufficient autophagy. Acinar cell-specific Atg5 KO and acinar cell-specific TFEB KO mice developed severe inflammatory and fibrotic pancreatitis in both Gao-binge alcohol and control diet-fed mice. In contrast, TFEB overexpression inhibited alcohol-induced pancreatic edema, accumulation of zymogen granules and serum amylase and lipase activities. In line with our findings in mice, decreased LAMP1 and TFEB nuclear staining were also observed in human alcoholic pancreatitis tissues.Conclusionsour results indicate that TFEB plays a critical role in maintaining pancreatic acinar cell homeostasis. Impairment of TFEB-mediated lysosomal biogenesis by alcohol may lead to insufficient autophagy and promote alcohol-induced pancreatitis.

Abstract A42: Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4

Abstract Pancreatic ductal adenomcarcinoma (PDAC) is one of the most lethal forms of cancer with a 5-year survival rate of 8%. To determine the composition of different cell types in PDAC, we performed single-cell RNA-sequencing (scRNA-seq) analysis of two resected patient tumors. Nine distinct cell types were identified: (1) ductal carcinoma cells expressing mutant KRAS, (2) normal pancreatic islet cells, (3) B-lymphocytes, (4) macrophages, (5) mast cells, (6) nerve cells, (7-8) two types of cancer-associated fibroblasts (CAFs), and (9) a previously unidentified epithelial type with distinctly high expression of OLFM4 and CRISP3. Histologic analysis of human PDAC and chronic pancreatitis (CP) tissue sections confirmed the presence of this distinct cell type and furthermore demonstrated that it corresponded to inflamed/injured ducts and early pancreatic intraepithelial neoplasms (PanINs) but that actual PDACs did not express OLFM4. CRISP3 was not always expressed in this new cell type, but the expression of OLFM4 was always detected in these histologic structures. This distinct pancreatic epithelial cell type was also observed in mouse models for chronic pancreatitis and in mutant-KRAS induced PanINs. We hypothesize that inflammation-induced OLFM4 expression helps drive PanIN formation, and that its expression could be exploited for early PDAC detection. We are currently performing functional studies using organoids to address the role that OLFM4 might play in generation of this distinct cell type. Citation Format: Manisha Rao, Alice Nemajerova, Jinyu Li, Mei Gao, Ki Oh, Richard Moffit, Joseph Kim, George Georgakis, Aaron Sasson, Agnieszka Bialkowkska, Scott Powers. Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A42.

Fibromodulin is upregulated by oxidative stress through the MAPK/AP-1 pathway to promote pancreatic stellate cell activation

Background/ObjectivesFibromodulin (FMOD) expression in chronic pancreatitis (CP) tissues and its effect on PSC was unknown. Our aim was to investigate the role of FMOD in regulating PSC profibrogenic phenotype and the molecular mechanism of CP. MethodsRat CP models were induced by dibutyltin dichloride. Pancreatic fibrosis was evaluated by Sirius Red staining. The expression of FMOD and α-SMA was measured, the correlation between FMOD expression and fibrosis was investigated in CP models and CP patients. The effects of FMOD on PSCs were examined by CCK-8 and migration assays. We investigated the mechanisms underlying FMOD expression using MND and a MAPK pathway inhibitor. Luciferase reporter and chromatin immunoprecipitation assays were used to investigate the effects of AP-1 on FMOD expression. ResultsSirius Red staining revealed high collagen deposition in model rats. Higher expression of FMOD and α-SMA was observed in fibrotic tissues, and the expression of FMOD was correlated with that of α-SMA and the areas of Sirius Red staining. Upregulation of FMOD increased the expression of collagen I and α-SMA and the proliferation and migration of PSCs. MND induced FMOD and α-SMA expression, and knockdown of FMOD abated α-SMA expression. ERK and JNK inhibitors attenuated FMOD expression as induced by MND. AP-1 upregulated the expression of FMOD. AP-1 binds to the FMOD promoter and transcriptionally regulates FMOD expression. ConclusionFMOD levels are upregulated in fibrosis tissues in CP and it is a critical downstream mediator of oxidative stress. FMOD induces PSC activation and maintains the fibrosis phenotype of PSCs.

P53 Activated by ER Stress Suppresses Caerulein-Induced Acute Pancreatitis Progression in Humanized PRSS1 Transgenic Mice by Inducing Acinar Cell Apoptosis

Background: Acute pancreatitis is a severe pancreatic disorder that remains associated with high mortality due to a lack of effective drugs and management strategies. This study aimed to investigate the molecular pathogenic mechanisms of acute pancreatitis involving p53 and endoplasmic reticulum (ER) stress pathways. Methods: Expression of PRSS1 and p53 in human acute pancreatitis tissues was detected by immunohistochemistry and western blotting. Acute pancreatitis was induced with caerulein in humanized PRSS1 transgenic mice, and its severity was verified by histological imaging, evaluation of edema, and serum amylase and trypsin activity assays. A transferase mediated d-UTP nick end-labeling assay was performed to evaluate acinar cell apoptosis associated with acute pancreatitis. The expression of ER stress genes was assessed by quantitative RT-PCR (qRT-PCR) and western blotting. Results: PRSS1 and p53 were highly expressed in human acute pancreatitis tissues. Expression of human PRSS1 in caerulein-treated mice induced significant acinar cell apoptosis and acute pancreatitis progression. p53 knockout significantly aggravated acute pancreatitis progression in humanized PRSS1 transgenic mice. The ER stress pathway was activated by PRSS1 and mediated the progression of acute pancreatitis in mouse pancreatic tissues. Application of a p53 inhibitor promoted caerulein-induced acute pancreatitis in PRSS1 transgenic mice, while a p53 activator ameliorated the progression of acute pancreatitis. Conclusion: P53, which was activated by the ER stress pathway, suppresses the progression of acute pancreatitis in mice expressing PRSS1 by inducing acinar cell apoptosis. Funding: The article is supported by Scientific Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (CX2018N012) & Clinical Research Program of Nanfang Hospital, Southern Medical University (2018CR046) & Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2016PY011). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: All experimental procedures performed with mice were approved by the Institutional Animal Care and Use Committee of Southern Medical University.

Reduction of intrapancreatic neural density in cancer tissue predicts poorer outcome in pancreatic ductal carcinoma.

Neural invasion is one of the malignant features contributing to locally advanced and/or metastatic disease progression in patients with pancreatic ductal adenocarcinoma (PDAC). Few studies exist on the distribution and state of nerve fibers in PDAC tissue and their clinicopathological impacts. The aim of the present study was to investigate the clinicopathological characteristics and prognostic value of intrapancreatic neural alterations in patients with PDAC. We retrospectively analyzed 256 patients with PDAC who underwent macroscopic curative surgery. Nerve fibers, immunolabeled with a specific neural marker GAP‐43, were digitally counted and compared among PDAC, chronic pancreatitis (CP) and normal pancreatic tissues. Interlobular nerve fibers were apparently hypertrophic in both CP and PDAC, although intrapancreatic neural density and nerve number decreased characteristically in PDAC. They tended to decrease toward the center of the tumor. Kaplan‐Meier survival analyses revealed a statistically significant correlation between low neural density and shorter overall survival (OS) (P = 0.014), and between high neural invasion and shorter OS (P = 0.017). Neural density (P = 0.04; HR = 1.496; 95% CI 1.018‐2.199) and neural invasion ratio (P = 0.064; HR = 1.439; 95% CI .980‐2.114) were prognostic factors of shorter OS in the multivariate analysis. These findings suggest low intrapancreatic neural density in patients with PDAC as an independent prognosticator, which may represent aggressive tumor behavior. Furthermore, we propose a simple, practical and reproducible method (to measure neural density and the neural invasion ratio during conventional histopathological diagnosis of PDAC), which has been validated using another cohort (n = 81).

Long noncoding RNA myocardial infarction-associated transcript regulated the pancreatic stellate cell activation to promote the fibrosis process of chronic pancreatitis.

Long noncoding RNAs (lncRNAs) play crucial roles in fibrosis process. In our previous RNA-seq study, we found that lncRNA myocardial infarction-associated transcript (MIAT) was differentially expressed in pancreatic tissues of chronic pancreatitis (CP) patients. However, the function of MIAT in CP remains unknown. This study was aimed to investigate the function and underlying mechanism of MIAT in pancreatic fibrosis. The expression levels of MIAT, miR-216a-3p, cyclooxygenase 2 (COX-2), α-smooth muscle actin (α-SMA), and collagen I were estimated by Western blot analysis and qualitative reverse transcription polymerase chain reaction. The relationships between miR-216a-3p,MIAT, and COX-2 were confirmed by luciferase reporter assay. The proliferation of human pancreatic stellate cells (HPaSteCs) was detected by cell counting kit-8 assay. We found that MIAT, along with the levels of fibrosis-related proteins α-SMA and collagen I, as well as COX-2 were upregulated, while miR-216a-3p was downregulated in transforming growth factor (TGF)-β1-stimulated HPaSteCs. Mechanistically, MIAT acted as a molecular sponge for miR-216a-3p. Furthermore, we identified COX-2 as a direct target of miR-126a-3p. Additionally, MIAT overturned the inhibitory effect of miR-216a-3p overexpression and COX-2 knockdown on the activation and proliferation of HPaSteCs. Our study provided mechanistic insights into a critical role for MIAT as a miRNA sponge in CP.

Transgenic expression of cyclooxygenase-2 in pancreatic acinar cells induces chronic pancreatitis.

Replacement of the exocrine parenchyma by fibrous tissue is a main characteristic of chronic pancreatitis. Understanding the mechanisms of pancreatic fibrogenesis is critical for the development of preventive and therapeutic interventions. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin synthesis, is expressed in patients with chronic pancreatitis. However, it is unknown whether COX-2 can cause chronic pancreatitis. To investigate the roles of pancreatic acinar COX-2 in fibrogenesis and the development of chronic pancreatitis, COX-2 was ectopically expressed specifically in pancreatic acinar cells in transgenic mice. Histopathological changes and expression levels of several profibrogenic factors related to chronic pancreatitis were evaluated. COX-2 was expressed in the pancreas of the transgenic mice, as detected by Western blot analysis. Immunohistochemical staining showed COX-2 was specifically expressed in pancreatic acinar cells. COX-2 expression led to progressive changes in the pancreas, including pancreas megaly, persistent inflammation, collagen deposition, and acinar-to-ductal metaplasia. Quantitative RT-PCR and immunostaining showed that profibrogenic factors were upregulated and pancreatic stellate cells were activated in the COX-2 transgenic mice. Expression of COX-2 in pancreatic acinar cells is sufficient to induce chronic pancreatitis. Targeting this pathway may be valuable in the prevention of chronic pancreatitis. NEW & NOTEWORTHY COX-2 expression is observed in pancreatic tissues of human chronic pancreatitis. In this study, we showed that COX-2 expression caused the development of chronic pancreatitis in transgenic mice, supporting the idea that COX-2 inhibition may be an effective preventive and therapeutic strategy.