Normal Bile Duct Tissues Research Articles

USP28 promotes tumor progression and glycolysis by stabilizing PKM2/Hif1-α in cholangiocarcinoma.

Ubiquitination is one of the important modification of proteins which can be reversed by deubiquitinating enzymes (DUBs). Ubiquitin specific protease 28 (USP28) belongs to the deubiquitinase family, which plays a cancer-promoting function in many types of cancers such as pancreatic cancer and breast cancer. So far, the molecular function and significance of USP 28 in cholangiocarcinoma remain unclear. In this study, we evaluated the expression of USP28 using tissue microarray (TMA), reverse transcription polymerase chain reaction (qRT-PCR), and online databases. We investigated the effect of USP28 on the progression of CCA through in vitro and in vivo functional experiments. In addition, we explored downstream molecular pathways using Western blotting (WB), immunofluorescence (IF), and mass spectrometry techniques. Here, we found that cholangiocarcinoma tissue had higher USP 28 expression than normal bile duct tissue, and that high USP 28 levels were significantly associated with a malignant phenotype and poorer prognosis in cholangiocarcinoma patients. Both in vitro and in vivo, USP28 could mediate the deubiquitination of PKM2, thereby activating the downstream Hif1-α signaling pathway, promoting glycolysis and energy supply, and finally promoting tumor progression. In summary, USP28 activated downstream Hif1-α by reducing the ubiquitination level of PKM2, furthermore, promoting the level of glycolysis in CCA cells for tumor progression.

MBD2 regulates the progression and chemoresistance of cholangiocarcinoma through interaction with WDR5

BackgroundCholangiocarcinoma (CCA) is a highly malignant, rapidly progressing tumor of the bile duct. Owing to its chemoresistance, it always has an extremely poor prognosis. Therefore, detailed elucidation of the mechanisms of chemoresistance and identification of therapeutic targets are still needed.MethodsWe analyzed the expression of MBD2 (Methyl-CpG-binding domain 2) in CCA and normal bile duct tissues using the public database and immunohistochemistry (IHC). The roles of MBD2 in CCA cell proliferation, migration, and chemoresistance ability were validated through CCK-8, plate cloning assay, wound healing assays and xenograft mouse model. In addition, we constructed a primary CCA mouse model to further confirm the effect of MBD2. RNA-seq and co-IP-MS were used to identify the mechanisms by how MBD2 leads to chemoresistance.ResultsMBD2 was upregulated in CCA. It promoted the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 directly interacted with WDR5, bound to the promoter of ABCB1, promoted the trimethylation of H3K4 in this region through KMT2A, and activated the expression of ABCB1. Knocking down WDR5 or KMT2A blocked the transcriptional activation of ABCB1 by MBD2. The molecular inhibitor MM-102 targeted the interaction of WDR5 with KMT2A. MM-102 inhibited the expression of ABCB1 in CCA cells and decreased the chemoresistance of CCA to cisplatin.ConclusionMBD2 promotes the progression and chemoresistance of CCA through interactions with WDR5. MM-102 can effectively block this process and increase the sensitivity of CCA to cisplatin.

The COL7A1/PI3K/AKT axis regulates the progression of cholangiocarcinoma

BackgroundThe role and molecular mechanisms of collagen type VII (COL7A1) in cholangiocarcinoma (CCA) remain unknown. MethodsWe analyzed the expression of COL7A1 in CCA and its relationship with patient prognosis using bioinformatic techniques. Expression levels of COL7A1 in CCA cells and tissues were detected using reverse transcription-quantitative PCR, western blotting, and immunohistochemistry. The effects of COL7A1 expression on the proliferation, migration, and invasion of CCA cells were assessed using CCK-8, colony formation, and Transwell assays. Bioinformatics and luciferase reporter gene assays were performed to examine the binding of KLF4 to COL7A1, and cytological experiments further verified the role of KLF4 in regulating the CCA phenotype through COL7A1. Xenograft mouse models were established to investigate the effects of COL7A1 on CCA tumor growth in vivo. ResultsCCA tissues exhibited higher COL7A1 expression than normal bile duct tissues. There was no significant correlation between high or low COL7A1 expression and the survival time of patients with CCA. COL7A1 knockdown inhibited CCA cell proliferation, migration, and invasion. Furthermore, COL7A1 knockdown suppressed the activation of the PI3K/AKT signaling pathway. KLF4 can bind to COL7A1 and regulate COL7A1 expression, which in turn regulates the PI3K/AKT signaling pathway and impacts the proliferation and metastasis of CCA cells. ConclusionOur findings suggest that KLF4 regulates CCA cell proliferation, migration, and invasion via the COL7A1/PI3K/AKT axis.

Potential of Anti-Leukotriene Drugs as New Therapeutic Agents for Inhibiting Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA) is a cancer with a poor prognosis due to difficulties in diagnosis and limited treatment options, highlighting the urgent need for new targeted therapies. In a clinical setting, we found that leukotriene levels in bile were higher than in serum. Immunohistochemical analysis of surgically resected samples also revealed that CysLT receptor 1 (CysLTR1) was more highly expressed in CCA than in normal bile duct tissue, prompting us to investigate leukotriene as a potential therapeutic target in CCA. In vitro studies using CCA cell lines expressing CysLTR1 showed that leukotriene D4, a major ligand of CysLTR1, promoted cell proliferation, with increased phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Additionally, treatment with two clinically available anti-allergic drugs-zileuton, an inhibitor of CysLT formation, and montelukast, a CysLTR1 inhibitor-had inhibitory effects on cell proliferation and migratory capacity, accompanied by the reduced phosphorylation of AKT and ERK1/2. Furthermore, the simultaneous administration of both drugs synergistically enhanced the inhibitory effect on cell proliferation. Our study suggests that use of these drugs may represent a novel approach to treat CCA through drug repositioning.

Human vascularized bile duct-on-a chip: a multi-cellular micro-physiological system for studying cholestatic liver disease

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity of in vitro models that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured in ex vivo systems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

DNA methylation-based classifier differentiates intrahepatic pancreato-biliary tumours.

Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is challenging. Both tumours have similar morphological and immunohistochemical pattern and share multiple driver mutations. We hypothesised that DNA methylation-based machine-learning algorithms may help perform this task. We assembled genome-wide DNA methylation data for iCCA (n=259), PAAD (n=431), and normal bile duct (n=70) from publicly available sources. We split this cohort into a reference (n=399) and a validation set (n=361). Using the reference cohort, we trained three machine learning models to differentiate between these entities. Furthermore, we validated the classifiers on the technical validation set and used an internal cohort (n=72) to test our classifier. On the validation cohort, the neural network, support vector machine, and the random forest classifiers reached accuracies of 97.68%, 95.62%, and 96.5%, respectively. Filtering by anomaly detection and thresholds improved the accuracy to 99.07% (37 samples excluded by filtering), 96.22% (17 samples excluded), and 100% (44 samples excluded) for the neural network, support vector machine and random forest, respectively. Because of best balance between accuracy and number of predictable cases we tested the neural network with applied filters on the in-house cohort, obtaining an accuracy of 95.45%. We developed a classifier that can differentiate between iCCAs, intrahepatic metastases of a PAAD, and normal bile duct tissue with high accuracy. This tool can be used for improving the diagnosis of pancreato-biliary cancers of the liver. This work was supported by Berlin Institute of Health (JCS Program), DKTK Berlin (Young Investigator Grant 2022), German Research Foundation (493697503 and 314905040 - SFB/TRR 209 Liver Cancer B01), and German Cancer Aid (70113922).

AURKB activates EMT through PI3K/AKT signaling axis to promote ICC progression

Intrahepatic cholangiocarcinoma (ICC) is a fatal disease and the molecular mechanism of its progression remains unknown. Aurora Kinase B (AURKB) is a central regulator of chromosome separation and cytokinesis and is abnormally expressed in a variety of cancer cells. This research aimed to explore the effect of AURKB in occurrence and metastasis of ICC. We found that AURKB showed a progressive up-regulation pattern from normal bile duct tissue to ICC with high invasion. Our data showed that AURKB significantly promoted ICC cell proliferation, induced epithelial-mesenchymal transition (EMT), migration and invasion through gain- and loss- of function experiments. In vivo results consistently showed that AURKB up-regulation not only promoted tumor growth, but also promoted tumor metastasis. Importantly, we discovered that AURKB regulates the expressions of EMT-related genes via PI3K/AKT signaling axis. Herein, our results suggest that AURKB induced EMT through the activation of PI3K/AKT signaling pathway is critical to the progression of ICC, which may be a prospective therapeutic treatment for overcoming ICC metastasis and progression.

Effect of FXR agonist GW4064 in the treatment of hilar cholangiocarcinoma in rats

The study objective was to observe the treatment effect of the farnesoid X receptor (FXR) agonist GW4064 in a rat model of hilar cholangiocarcinoma to explore a new therapeutic target for gene therapy for hilar cholangiocarcinoma. Eighty male Wistar rats were randomly divided into four groups (treatment group, model group, control group and sham operation group, 20 rats in each group). The four groups were fed a standard diet. The treatment group and the model group were injected with a suspension of cholangiocarcinoma QBC939 cells into the hilar bile duct with a microsyringe, the control group was injected with normal saline, and the sham operation group was not injected with anything. A modified tail suspension test (TST) was used to evaluate the vitality of the rats. At 4 weeks, one rat in the treatment group and model group was euthanized, and the changes in the hilar bile duct were recorded. The procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma occurred in the treatment group and model group. Then, the treatment group was injected with GW4064 intraperitoneally at a dose of 50 mg/kg/day. One week after injection, the rats in the four groups were euthanized. Pathological examination confirmed that tumours had formed, and hilar bile duct tissues were taken from the four groups. FXR, Bsep, Ntcp and NF-κB expression in the hilar bile duct was detected by real-time polymerase chain reaction (RT–PCR) and immunohistochemistry. After three weeks, the rats in the treatment group and model group ate less, and their weight was significantly reduced. Six weeks later, hilar cholangiocarcinoma was detected in the treatment group and model group. After treatment with GW4064, the ratios of FXR/GAPDH mRNA, Bsep/GAPDH mRNA, Ntcp/GAPDH mRNA and NF-κBp65/GAPDH mRNA were significantly different among the four groups. Under a light microscope, FXR protein reacted with anti-FXR antibody, Bsep protein reacted with anti-Bsep antibody, Ntcp protein reacted with anti-Ntcp antibody and NF-κBp65 protein reacted with anti-NF-κBp65 antibody, and they showed granular expression. Every pathological section included 4,800 cells, and there were different numbers of positive cells in each group. FXR expression in the hilar cholangiocarcinoma of rats was significantly lower than that in normal hilar bile duct tissues. GW4064 increased the expression of FXR in tumour tissues. These findings suggest that FXR may be a new therapeutic target and that GW4064 may be helpful in the treatment of hilar cholangiocarcinoma.

FXR expression in rats of hilar cholangiocarcinoma

The study objective was to detect the expression of farnesoid X receptor (FXR) in a rat model of hilar cholangiocarcinoma to provide a new therapeutic target for gene therapy in hilar cholangiocarcinoma. Sixty male Wistar rats (weighing 190 ± 8 g) were randomly divided into three groups (experimental group, control group and sham operation group, 20 rats in each group). The three groups were fed a standard diet. The QBC939 cell suspension of cholangiocarcinoma was injected into the hilar bile duct in the experimental group with a microsyringe. The control group was injected with normal saline, and the sham operation group was not injected with any drugs. A modified tail suspension test (TST) was used to evaluate the mental state and physical activity of rats every day. At 5 weeks, one rat in the experimental group was euthanized, and the changes in the hilar bile duct were recorded. The procedure was repeated at one and half months. After one and half months, hilar cholangiocarcinoma only occurred in the experimental group. Pathological examination confirmed the formation of tumours, and hilar bile duct tissues were taken from the three groups. FXR expression in the hilar bile duct was detected by real-time polymerase chain reaction (RT–PCR) and immunohistochemistry. After two weeks, the rats in the experimental group ate less, and their weight was significantly reduced. One and half months later, hilar cholangiocarcinoma was detected in 16 rats in the experimental group. The levels of alanine aminotransferase and aspartate transaminase in the experimental group were higher than those in the other two groups. The ratio of FXR/GAPDH mRNA was significantly different among the hilar cholangiocarcinoma, control and sham operation groups. Under the light microscope, FXR protein reacted with anti-FXR antibody and showed granular expression. Every pathological section included 4800 cells. A total of 1856 positive cells were in the experimental group, 3279 positive cells were in the control group, and 3371 positive cells were in the sham operation group. FXR expression in the hilar cholangiocarcinoma of rats was significantly lower than that in normal hilar bile duct tissues, suggesting that drugs targeting FXR may be a new strategy for the treatment of hilar cholangiocarcinoma.

A2M is a potential core gene in intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (ICC) is a type of malignant tumor ranking the second in the incidence of primary liver cancer following hepatocellular carcinoma. Both the morbidity and mortality have been increasing in recent years. Small duct type of ICC has potential therapeutic targets. But overall, the prognosis of patients with ICC is usually very poor.MethodsTo search latent therapeutic targets for ICC, we programmatically selected the five most suitable microarray datasets. Then, we made an analysis of these microarray datasets (GSE26566, GSE31370, GSE32958, GSE45001 and GSE76311) collected from the Gene Expression Omnibus (GEO) database. The GEO2R tool was effective to find out differentially expressed genes (DEGs) between ICC and normal tissue. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were executed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.8. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze protein–protein interaction of these DEGs and protein–protein interaction of these DEGs was modified by Cytoscape3.8.2. Survival analysis was performed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool.ResultsA total of 28 upregulated DEGs and 118 downregulated DEGs were screened out. Then twenty hub genes were selected according to the connectivity degree. The survival analysis results showed that A2M was closely related to the pathogenesis and prognosis of ICC and was a potential therapeutic target for ICC.ConclusionsAccording to our study, low A2M expression in ICC compared to normal bile duct tissue was an adverse prognostic factor in ICC patients. The value of A2M in the treatment of ICC needs to be further studied.

NTCP Change in Rats of Hilar Cholangiocarcinoma and Therapeutic Significance

Background: The study aims to detect the expression of Na+/taurocholate cotransporter polypeptide in hilar cholangiocarcinoma of rat model, to provide a new therapeutic target for gene therapy of hilar cholangiocarcinoma. Methods: 60 male Wistar rats (weighing 190 ± 8 g) were randomly divided into 3 groups (experimental group, control group, and sham operation group; 20 rats in each group). The 3 groups were fed with standard diet. The QBC939 cell suspension of cholangiocarcinoma was injected into the hilar bile duct in the experimental group with a micro syringe. The control group was injected with normal saline, and the sham operation group was not injected with any drugs. Comprehensive behavior score and Basso Beattie Bresnahan were used to evaluate the mental state and exercise of rats every day. At 5 weeks, one rat in the experimental group was killed, and the changes in hilar bile duct were recorded. The procedure was repeated at one and half months. After one and half months, hilar cholangiocarcinoma only occurred in the experimental group. Pathological examination confirmed the formation of tumor, and hilar bile duct tissues were taken from the 3 groups. Na+/taurocholate cotransporter polypeptide expression in hilar bile duct was detected by real-time polymerase chain reaction and immunohistochemistry. Results: After 2 weeks, the rats in experimental group ate less, and their weight was significantly reduced compared with the other 2 groups. One and half months later, hilar cholangiocarcinoma was detected in 16 rats in the experimental group. The levels of alanine aminotransferase and aspartate transaminase in the experimental group were higher than those in the other 2 groups. The ratio of Na+/taurocholate cotransporter polypeptide/GAPDH mRNA in hilar cholangiocarcinoma, control group, and sham operation group was significantly different. Under the light microscope, Na+/taurocholate cotransporter polypeptide protein reacted with anti-Na+/taurocholate cotransporter polypeptide antibody and showed granular expression. Every pathological section included 4800 cells. 3823 positive cells were in the experimental group, 1765 positive cells were in the control group, and 1823 positive cells were in the sham operation group. Conclusions: Na+/taurocholate cotransporter polypeptide expression in hilar cholangiocarcinoma of rats was significantly higher than normal hilar bile duct tissues, suggesting that drugs targeting Na+/taurocholate cotransporter polypeptide may be a new strategy for the treatment of hilar cholangiocarcinoma.

MiR-155-5p suppresses SOX1 to promote proliferation of cholangiocarcinoma via RAF/MEK/ERK pathway

BackgroundAccumulating evidence has demonstrated the close relation of SOX1 with tumorigenesis and tumor progression. Upregulation of SOX1 was recently shown to suppress growth of human cancers. However, the expression and role of SOX1 in cholangiocarcinoma (CCA) is not well characterized.MethodsExpression levels of SOX1 in CCA tissues and normal bile duct tissues were examined using public GEO database. Western blot and immunohistochemistry were used to confirm the expression levels. Cell proliferation assay (CCK-8) and colony formation assay were performed to assess proliferation of CCA cells. A mouse model of subcutaneous transplantable tumors was used to evaluated proliferation of CCA in vivo. The putative regulating factor of SOX1 were determined using Targetscan and dual-luciferase reporter assay.ResultsSOX1 was downregulated in CCA tissues. Overexpression of SOX1 significantly inhibited cell proliferation in vitro and suppressed tumor growth in vivo. miR-155-5p directly targeted the 3′-untranslated region (3′UTR) of SOX1 and inhibited expression of SOX1, resulting in the activation of RAF, MEK and ERK phosphorylation, and thus CCA proliferation. However, restoration of SOX1 expression in miR-155-5p overexpressing cell lines decreased the phosphorylation level of RAF, MEK and ERK, as well as the proliferation of CCA cells.ConclusionMiR-155-5p decreased the expression of SOX1 by binding to its 3′UTR, which activated the RAF/MEK/ERK signaling pathway and promoted CCA progression.

Upregulation of Stress-Induced Protein Kinase CK1 Delta is associated with a Poor Prognosis for patients with Hepatocellular Carcinoma.

Objective: This study was designed to analyze the expression of CSNK1D in hepatocellular carcinoma (HCC) and investigate the relationship between the expression of CSNK1D and the prognosis of HCC patients. Methods: The CSNK1D and alpha-fetoprotein (AFP) expression levels in patients with HCC and their corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA) and sorted with a Perl program. CSNK1D and AFP expression differences in liver tissue and liver cancer were compared and analyzed, based on the online database human cancer metastasis database, the relationships between the expression levels of CSNK1D and AFP and the proliferation and metastases of HCC were explored. The immunohistochemical data obtained from the Human Protein Atlas Database further verified the differences in the expression levels of CSNK1D and AFP in liver tissues and liver cancer tissues. Through Kaplan-Meier survival analysis, the effects of CSNK1D and AFP expression levels on the prognosis of patients with HCC were investigated, and the influences of and patients' gender, age and grades of cancer cells, tumor size, the status of lymph node metastasis, distant metastasis, and tumor stage on the expression of CSNK1D were analyzed with R language. The influence of differential expressions of CSNK1D on survival time was compared and the prognostic factors influencing the survival of HCC patients were statistically explored by univariate analysis and multivariate analysis. The potential influencing mechanism of CSNK1D on the prognosis of HCC patients was explored by Gene Set Enrichment Analysis (GSEA) enrichment. Results: The expression level of CSNK1D and AFP in cancer foci was significantly higher than that in normal tissues, However, in the same patient, the expression levels of AFP in paracarcinoma tissues and cancer tissues showed no significant difference. The expression level of CSNK1D in HCC with distant metastases was higher than that in those without metastasis, but the expression level of AFP in metastatic HCC was lower than that in those HCC without metastases. In immunohistochemical tests, CSNK1D was moderately positive in normal liver tissues, slightly positive in normal bile duct tissues, and highly positive in HCC. AFP was slightly positive in normal liver tissues and negative in HCC, but it was not detected in normal intrahepatic bile duct tissue. Survival analysis results suggested that the higher expression level of CSNK1D corresponded to the shorter survival period, whereas the expression level of AFP showed no significant influence on survival time. The expression level of CSNK1D was not correlated with gender, age, the status of lymph node metastasis status, or distant metastasis of patients. The main factors influencing the expression level of CSNK1D included tumor size, cancer cell grade, and tumor stage. The expression levels of CSNK1D in T2 and T3 were higher than that in T1. The expression levels of CSNK1D in G3 and G4 were higher than that in G1. The expression levels of CSNK1D in Stage II and Stage III were higher than that in Stage I. Univariate analysis suggested that tumor size, cell grade, distant metastasis, clinical stage, and CSNK1D expression level were the prognostic factors influencing the survival of patients. Multivariate analysis suggested that CSNK1D expression level was an independent factor influencing the prognosis of HCC patients. GSEA enrichment analysis indicated that CSNK1D mainly affected the prognosis of HCC patients through cell cycle, WNT signaling pathway, amino acid degradation metabolism, and other pathways. Conclusion: CSNK1D is an independent influencing factor for the prognosis of HCC patients and has the potential to be developed as a potential therapeutic target for HCC, and better than AFP in predicting the prognosis of HCC.

Abstract 624: Clinical evaluation of the MasSpec Pen technology for rapid diagnosis and margin assessment in pancreatic cancer surgery

Abstract Precise removal of pancreatic ductal adenocarcinoma (PDAC) with microscopically negative margins, commonly assessed by frozen section analysis, is associated with longer disease-free survival. However, histologic complexities and tissue-processing artifacts can render frozen section analysis of PDAC margins a challenging and time-consuming task, with reported accuracies dependent on the skillset and subspecialty of the pathologist on call. We developed the MasSpec Pen, a handheld device coupled to a mass spectrometer, for rapid (~15 s) and nondestructive molecular analysis and diagnosis of tissues. The MasSpec Pen supplies a discrete water droplet onto a tissue's surface, allowing diagnostic metabolites and lipids to be extracted into the droplet and then transmitted into a mass spectrometer for analysis. Here, we evaluate the performance of the MasSpec Pen for intraoperative diagnosis of PDAC in human pancreatic and bile duct margins. Pancreatic and bile duct tissue samples (N=157) were obtained from the Cooperative Human Tissue Network and Baylor College of Medicine and stored at -80°C prior to analysis. A Q Exactive mass spectrometer (Thermo Scientific) coupled to the MasSpec Pen was used for analysis of thawed samples in the negative ion mode. Tissues were then cryo-sectioned, H&E stained, and blindly evaluated by a pathologist. Based on the distinct molecular profiles acquired, we generated two statistical classifiers using lasso penalized logistic regression for distinguishing PDAC from healthy pancreas and bile duct tissue based on a sparse set of molecular features indicative of disease state. For distinguishing normal pancreas from PDAC, an overall accuracy of 91.5%, sensitivity of 95.5%, and specificity of 89.7% was achieved for training, validation, and test sets. Classification results for discriminating normal bile duct from PDAC had an overall accuracy of 95%, sensitivity of 92%, and specificity of 100% in training and validation. We have begun clinical testing of the MasSpec Pen in human surgeries following its successful translation to an operating room at Texas Medical Center. To date, the MasSpec Pen has been used to analyze in vivo and fresh ex vivo tissue in 19 pancreatic surgeries. When predicting on 64 intraoperative analyses using classification models built on banked data, 93.8% agreement with final postoperative pathology reports was achieved. While further validation studies are needed, our results show that the MasSpec Pen can distinguish PDAC from normal pancreas and bile duct tissues with high accuracy and is compatible for in vivo use, suggesting this technology may be valuable for near real-time margin evaluation during pancreatic oncologic surgeries. Citation Format: Mary E. King, Jialing Zhang, John Q. Lin, Kyana Y. Garza, Rachel J. DeHoog, Clara L. Feider, Alena V. Bensussan, Marta Sans, Anna Krieger, Sunil Badal, Michael F. Keating, Sadhna Dhingra, Wendong Yu, George Van Buren, Christopher Pirko, Kirtan Brahmbhatt, William E. Fisher, James W. Suliburk, Livia S. Eberlin. Clinical evaluation of the MasSpec Pen technology for rapid diagnosis and margin assessment in pancreatic cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 624.

Promoter hypermethylation of early B cell factor 1 (EBF1) is associated with cholangiocarcinoma progression.

DNA hypermethylation in a promoter region causes gene silencing via epigenetic changes. We have previously reported that early B cell factor 1 (EBF1) was down-regulated in cholangiocarcinoma (CCA) tissues and related to tumor progression. Thus, we hypothesized that the DNA hypermethylation of EBF1 promoter would suppress EBF1 expression in CCA and induce its progression. In this study, the DNA methylation status of EBF1 and mRNA expression levels were analyzed in CCA and normal bile duct (NBD) tissues using a publicly available database of genome-wide association data. The results showed that the DNA methylation of EBF1 promoter region was significantly increased in CCA tissues compared with those of NBD. The degree of methylation was negatively correlated with EBF1 mRNA expression levels. Using methylation-specific PCR technique, the DNA methylation rates of EBF1 promoter region were investigated in CCA tissues (n=72). CCA patients with high methylation rates of EBF1 promoter region in the tumor tissues (54/72) had a poor prognosis. Higher methylation rates of EBF1 promoter region have shown in all CCA cell lines than that of an immortal cholangiocyte cell line (MMNK1). Upon treatment with the DNA methyltransferase inhibitor 5-Aza-dC, increased EBF1 expression levels and reduced DNA methylation rates were observed in CCA cells. Moreover, restoration of EBF1 expression in CCA cells led to inhibition of cell growth, migration and invasion. In addition, RNA sequencing analysis suggested that EBF1 is involved in suppression of numerous pathways in cancer. Taken together, DNA hypermethylation in the EBF1 promoter region suppresses EBF1 expression and induces CCA progression with aggressive clinical outcomes.

Correlation between Expression Differences of Epithelial-Mesenchymal Transition (EMT) in Cholangiocarcinoma Tissue

Background: By studying the expression of epithelial-mesenchymal transition regulators in cholangiocarcinoma and intrahepatic duct stones, the correlation between the expression of epithelial-mesenchymal transition regulators and cholangiocarcinoma was revealed. Objective: The objective is to investigate the correlation between the expression of epithelial-mesenchymal transition (EC) regulatory factors and cholangiocarcinoma in patients with intrahepatic duct stones and cholangiocarcinoma, to investigate the relationship between clinicopathological features and prognosis, and to observe the expression of molecular markers of epithelial-mesenchymal transition (EMT) in intrahepatic duct stones and bile duct carcinoma. Methods: Twenty cases of primary cholangiocarcinoma, 20 cases of intrahepatic cholangiolithiasis complicated with cholangiocarcinoma, and 20 cases of intrahepatic cholangiolithiasis specimens were collected from the Fourth People’s Hospital and the friendly medical unit of Haikou. Immunohistochemistry was used to detect the expression differences of EMT-related molecular markers Twisit1, Twisit2, E-cadherin, N-cadherin, and Vimentin in paraffin sections of normal intrahepatic bile duct tissues and patients with intrahepatic duct stones and cholangiocarcinoma. Results: Immunohistochemical staining revealed epithelial-mesenchymal transition (EMT) in intrahepatic cholangiocarcinoma tissue, intrahepatic cholangiolithiasis with cholangiocarcinoma, intrahepatic cholangiolithiasis with normal intrahepatic cholangiolithiasis, such as Sit1, Twisit2, E-cadherin, N-cadherin, and Vimentin proteins were different. The expression of E-cadherin was decreased in cholangiocarcinoma tissue and intrahepatic cholangiolithiocarcinoma combined with cholangiocarcinoma (P 0.05). There was no difference in the expression of intrahepatic bile duct stones and EMT (P > 0.05). Conclusion: The expression of E-cadherin, the molecular marker of EMT, was down-regulated, while the expression of N-cadherin and Vimentin was up-regulated. Age, gender, depth of tumor invasion, degree of tumor differentiation and lymph node metastasis were correlated with the expression of EMT in intrahepatic cholangiocarcinoma.

Transcription factor HIF1α promotes proliferation, migration, and invasion of cholangiocarcinoma via long noncoding RNA H19/microRNA-612/Bcl-2 axis

Cholangiocarcinoma, which is the most common invasive malignant tumor of the biliary tract, has poor prognosis. There is evidence suggesting that hypoxia-inducible factor 1α (HIF1α) plays an important role in cholangiocarcinoma. Also, microRNA-612 (miR-612) is another key regulator of cholangiocarcinoma. In this study, we investigate the scantly documented interaction of HIF1α and miR-612 in cholangiocarcinoma. We first undertook microarray-based cholangiocarcinoma gene expression profiles to screen out the differentially expressed long noncoding RNAs (lncRNAs) and genes. We used reverse transcription quantitative polymerase chain reaction to detect the expression of HIF1α in normal bile duct and cholangiocarcinoma tissues, and in corresponding cells lines. Cell counting kit 8, scratch, and Transwell assays were used to detect the proliferation, migration and invasion of cholangiocarcinoma cells. Cell cycle distribution and apoptosis were detected by flow cytometry. ChIP, dual luciferase reporter gene assay, RNA pull-down, and RNA immunoprecipitation were used to verify relationship between HIF1α and lncRNA H19, and lncRNA H19 and miR-612. We also monitored tumor formation in nude mice to verify the effect of HIF1α on cholangiocarcinoma. HIF1α expression was elevated in cholangiocarcinoma tissues and cells. Silencing HIF1α reduced proliferation, migration, and invasion of cholangiocarcinoma cells. HIF1α transcriptionally activated the expression of lncRNA H19. Overexpression of miR-612 could rescue the proliferation, migration and invasion of cholangiocarcinoma cells caused by lncRNA H19 overexpression. Taken together, HIF1α activated lncRNA H19-mediated miR-612/Bcl-2 pathway to promote cholangiocarcinoma, suggesting a promising therapeutic target for cholangiocarcinoma.

Long non-coding RNA myocardial infarction associated transcript promotes the proliferation of cholangiocarcinoma cells by targeting miR-551b-3p/CCND1 axis.

Accumulating reports have demonstrated that long non-coding RNAs (lncRNAs) play critical roles in the occurrence and metastasis of cholangiocarcinoma (CCA). LncRNA myocardial infarction associated transcript (MIAT) has been widely reported in hepatocellular carcinoma, pancreatic cancer and colorectal cancer, but the relationship between MIAT and CCA progression has not yet been investigated. In the present study, we found that the expression of MIAT in CCA tissues was prominently higher than that in normal bile duct tissues. Moreover, TCGA-CHOL data in the GEPIA platform further revealed the upregulated expression of MIAT in CCA tissues. Additionally, quantitative real-time PCR results showed that MIAT expression was increased in CCA cell lines compared to the human intrahepatic biliary epithelial cell line. Functionally, MIAT knockdown significantly inhibited cell proliferation and induced G0/G1 phase arrest as well as apoptosis in HuCCT-1 and QBC939 cells. Conversely, ectopic expression of MIAT obviously facilitated the proliferation, cell cycle progression and apoptosis resistance of RBE cells. Mechanistically, MIAT directly interacted with miR-551b-3p and inversely modulated miR-551-3p level in CCA cells. Furthermore, MIAT knockdown reduced the expression of cyclin D1 (CCND1), which was rescued by miR-551b-3p silencing in HuCCT-1 cells. Importantly, CCND1 restoration partially reversed MIAT knockdown-induced proliferation inhibition, G0/G1 phase arrest and apoptosis in HuCCT-1 cells. In conclusion, MIAT was frequently overexpressed in CCA. MIAT contributed to the growth of CCA cells by targeting miR-551b-3p/CCND1 axis.

New staining method using methionyl-tRNA synthetase 1 antibody for brushing cytology of bile duct cancer

Identifying malignant biliary strictures using endobiliary brushing cytology specimens is important for treatment decision-making and prognosis prediction. The sensitivity of brushing cytology specimens based on Papanicolaou (Pap) staining is low, which hampers accurate diagnosis of indeterminate strictures. Here, we assessed the diagnostic value of immunohistochemical (IHC) and immunofluorescence (IF) staining for methionyl-tRNA synthetase 1 (MARS1). Endobiliary brushing cytology specimens were obtained during ERCP from 80 patients with an extrahepatic biliary stricture. Pap and MARS1 IF staining were performed on liquid-based cytology slides derived from these specimens. Sections of bile duct adenocarcinoma and normal bile duct tissue were obtained from 45 patients who underwent surgery for malignant biliary stricture, and MARS1 levels were evaluated by IHC staining. MARS1 IF staining was applied to brushing cytology specimens, and the results showed strong signals in malignant biliary structures but not in the negative for malignancy specimens. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 70.4%, 96.2%, 97.4%, 56.8%, and 78.8%, respectively, for conventional Pap staining and 98.1%, 96.1%, 98.1%, 96.2%, and 97.5%, respectively, for MARS1 IF (P< .0001). When IHC staining was used, MARS1 was detected in 45 bile duct adenocarcinoma sections but not in 15 normal bile duct sections. Moreover, MARS1 mRNA and protein levels were significantly higher in bile duct adenocarcinoma sections according to polymerase chain reaction and Western blot, respectively. The high sensitivity and accuracy of MARS1 IF staining enabled detection of malignancy in patients with indeterminate biliary stricture. Further prospective studies are needed to validate our findings. (Clinical trial registration number: KCT 0003285.).

CD147 augmented monocarboxylate transporter-1/4 expression through modulation of the Akt-FoxO3-NF-κB pathway promotes cholangiocarcinoma migration and invasion.

Cholangiocarcinoma (CCA) is an aggressive type of cancer. The major obstacles for treatment are its late presentation and the occurrence metastases. Targeting the metastatic process may serve as a treatment option. CD147 is a membrane protein that promotes CCA metastasis. High lactate levels in CCA are predicted to result from lactate dehydrogenase A expression and sensitivity to monocarboxylate transporter (MCT) inhibitors. An involvement of CD147 in MCT maturation has been reported, but the exact role of MCT in CCA is not clear. Here, we aimed to assess the mechanism of CD147-promoted CCA progression through MCT regulation. The expression levels of CD147 and MCT-1/4 in human CCA tissues were determined by immunohistochemistry. Two CD147 knockout (CD147 KO) CCAcell (KKU-213) clones were established using the CRISPR/Cas9 system. Cell migration and invasion were determined using a Boyden chamber assay. Temporal protein levels were modified by siRNA, specific inhibitors and/or activators. The expression of target proteins was determined using Western blot analyses. CD147 and MCT-1/4 were found to be overexpressed in CCA tissues compared to normal bile duct tissues. In addition, we found that CD147 knockdown significantly alleviated CCA cell migration and invasion, concomitant with decreased pAkt, pFoxO3, pNF-κB (pp65) and MCT-1/4 levels. Conversely, we found that FoxO3 knockdown led to recovered migration/invasion abilities and increased pp65 and MCT-1/4 expression levels. The involvement of Akt in the regulation of MCT-1/4 expression through CD147 was established by inhibition and activation of Akt phosphorylation. Our data indicate that CD147 promotes the malignant progression of CCA cells by activating the Akt-FoxO3-NF-κB-MCT-1/4 axis. As such, CD147 may serve as a possible target for advanced CCA treatment.