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We examined the expression of Lewis antigens, particularly Lewis Y, on the intrahepatic biliary epithelial cells in normal livers and various hepatobiliary diseases with immunohistochemical and immunoelectron microscopic methods. In normal livers, Lewis Y was consistently and generally negative in the bile ductules and small bile ducts, respectively. This antigen was frequently and strongly expressed on these ducts and ductules showing variable pathological changes such as necroinflammation and proliferation in a majority of hepatobiliary diseases independent on their etiology, whereas a majority of normal-appearing bile ducts and ductules in these pathological conditions were negative. Immunoelectron microscopically, gold particles suggesting the presence of Lewis Y were demonstrated on microvilli facing the biliary lumen, lateral cell membranes, secretory granules and Golgi apparatus of abnormal biliary epithelial cells in various hepatobiliary diseases. These data suggest that neoexpression of Lewis Y antigen is a highly sensitive, nonspecific phenotypic change of carbohydrate residues occurring in the abnormal biliary epithelial cells in various hepatobiliary diseases.
Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14-KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.
During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.
Biliary disorders can lead to life-threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue-specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte-markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.
Chimerism after orthotopic liver transplantation (OLT) has largely been investigated in intrahepatic cellular constituents. However, little is known about chimerism in the extrahepatic and large intrahepatic bile ducts. Our aim was to evaluate the presence and extent of chimerism after OLT in the peribiliary glands (PBG) and the luminal epithelium of the large donor bile ducts.
ERα, one of the classical receptors of estrogen, has been found to be abnormally up-regulated in patients with primary biliary cholangitis (PBC), which is an important factor leading to ductopenia. ERα-mediated signaling pathways are involved in proliferation of human intrahepatic biliary epithelial cells (HiBECs) and portal inflammation. Our previous studies have shown that the expression levels of ERα in the liver tissues of PBC patients are positively correlated with the levels of serum pro-inflammatory cytokines. The present study was designed to assess the relationship between abnormal ERα expression in small bile ducts and the progression of PBC. We examined the levels of multiple cytokines and analyzed their relationship with clinical parameters of livers functions in a cohort of 43 PBC patients and 45 healthy controls (HC). The levels of ERα expression and the relation with the levels of cytokines were further assessed. The localization of cytokines and ERα-mediated signaling pathways in liver were examined using immunohistochemistry. The possible underlying mechanisms of these alterations in PBC were explored in vitro. Our results demonstrated that the levels of IL-6, IL-8, and TNF-α were increased in PBC patients, and positively correlated with the serum AKP levels and ERα expression levels. Moreover, the expression of these cytokines were up-regulated in HiBECs that were stimulated with 17β-estradiol and PPT (an ERα agonist) and they also were positive in intrahepatic bile duct of PBC patients. The ERα-mediated expression of pro-inflammatory cytokines was induced by JNK, P38, and STAT3 phosphorylation in HiBECs. In addition, the CD54 expression was increased in HiBECs after ERα activation, which induced peripheral blood monouclear cells (PBMCs) recruitment. In conclusion, the present study highlighted a key role of abnormal ERα expression in inducing an inflammatory phenotype of HiBECs, which was critical in the development of inflammation and damage in small bile duct.
The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.
The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli's disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney.
Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.
Myeloid-related protein 8 (MRP8) and 14 (MRP14) are abundantly expressed in several kinds of benign and malignant tumors. However, little is known about their clinicopathological significance in intrahepatic cholangiocarcinoma (ICC), biliary intraepithelial neoplasia (BilIN), intraductal papillary neoplasm of bile duct (IPNB), or inflammatory hepatic biliary ducts epithelium (IHBD). This study aimed to investigate the diagnostic and prognostic values of MRP8 and MRP14 as new biomarkers for ICC. We examined MRP8 and MRP14 expression levels by immunohistochemistry in IHBD (n = 15), BilIN (BilIN1 = 24, BilIN2 = 9, BilIN3 = 5), IPNB (n = 18) and ICC (n = 416). The differential diagnostic and prognosis values were also evaluated. The results showed that the ratio of tumor-infiltrating MRP8 and MRP14 positive immune cells, relative to biliary epithelial cells, was significantly increased in ICC tissues compared with nonmalignant tissues, including IHBD, BilIN1, BilIN2, BilIN3, and IPNB (P value < 0.05). In addition, over-expression levels of MRP8 and MRP14 were correlated with overall survival (OS) and time to recurrence (TTR) by univariate analysis; MRP8/MRP14 combination was an independent prognostic factor for OS and TTR. MRP8 and MRP14 expression might help to identify the benign bile duct diseases from ICC, as high expression of MRP8 and MRP14 suggests a poor prognosis after surgical resection.
Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer arising from the bile ducts with a need for earlier diagnosis and a greater range of treatment options. KRAS/NRAS mutations are common in ICC tumours and 6-32% of patients also have isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations associated with metabolic changes. This feasibility study investigated sequencing circulating tumour DNA (ctDNA) combined with metabolite profiling of plasma as a method for biomarker discovery in ICC patients. Plasma was collected from four ICC patients receiving radio-embolisation and healthy controls at multiple time points. ctDNA was sequenced using Ampliseq cancer hotspot panel-v2 on Ion Torrent PGM for single nucleotide variants (SNV) detection and with Illumina whole genome sequencing for copy number variants (CNV) and further targeted examination for SNVs. Untargeted analysis of metabolites from patient and control plasma was performed using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS). Metabolite identification was performed using multi-parameter comparisons with analysis of authentic standards, and univariate statistical analysis was performed to identify differences in metabolite abundance between patient and control samples. Recurrent somatic SNVs and CNVs were identified in ctDNA from three out of four patients that included both NRAS and IDH1 mutations linked to ICC. Plasma metabolite analysis revealed biomarker metabolites associated with ICC and in particular 2-hydroxyglutarate (2-HG) levels were elevated in both samples from the only patient showing a variant allele in IDH1. A reduction in the number of CNVs was observed with treatment. This study demonstrates that ctDNA and metabolite levels can be identified and correlated in ICC patient blood samples and differentiated from healthy controls. We conclude that combining genomic and metabolic analysis of plasma offers an effective approach to biomarker identification with potential for disease stratification and early detection studies.
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.
Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.
Carcinomas of the pancreatobiliary system confer an especially unfavorable prognosis. The differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and its subtypes versus liver metastasis of ductal adenocarcinoma of the pancreas (PDAC) is clinically important to allow the best possible therapy. We could previously show that E-cadherin and N-cadherin, transmembrane glycoproteins of adherens junctions, are characteristic features of hepatocytes and cholangiocytes. We therefore analyzed E-cadherin and N-cadherin in the embryonally related epithelia of the bile duct and pancreas, as well as in 312 iCCAs, 513 carcinomas of the extrahepatic bile ducts, 228 gallbladder carcinomas, 131 PDACs, and precursor lesions, with immunohistochemistry combined with image analysis, fluorescence microscopy, and immunoblots. In the physiological liver, N-cadherin colocalizes with E-cadherin in small intrahepatic bile ducts, whereas larger bile ducts and pancreatic ducts are positive for E-cadherin but contain decreasing amounts of N-cadherin. N-cadherin was highly expressed in most iCCAs, whereas in PDACs, N-cadherin was negative or only faintly expressed. E- and N-cadherin expression in tumors of the pancreaticobiliary tract recapitulate their expression in their normal tissue counterparts. N-cadherin is a helpful marker for the differential diagnosis between iCCA and PDAC, with a specificity of 96% and a sensitivity of 67% for small duct iCCAs and 50% for large duct iCCAs.
Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant neoplasm arising from the intrahepatic bile ducts. As a scaffold protein of lipid rafts, flotillin-2 is upregulated in several types of cancer and promotes tumor progression and metastasis. To the best of our knowledge, the present study was the first to detect the upregulation of flotillin-2 in iCCA tissues compared with matched adjacent non-tumor tissues. In addition, immunohistochemistry was used to investigate the expression of flotillin-2 in a microarray consisting of 92 iCCA tissues. A total of 59 samples (64.1%) exhibited high flotillin-2 expression, which was significantly related to lymph node metastasis (P=0.029) and tumor-node-metastasis stage (P=0.016). Further in vitro study demonstrated that knockdown of flotillin-2 inhibited the invasive capability of iCCA cell lines, further supporting the participation of flotillin-2 in cancer invasion and metastasis. Moreover, Kaplan-Meier analysis showed patients with high flotillin-2 expression had worse overall survival outcomes. The multivariate Cox proportional hazards model further revealed that high flotillin-2 expression was an independent indicator (P=0.005) of poor prognosis for patients with iCCA. Collectively, the present study revealed that as a promoter of invasion and an independent marker of poor prognosis, flotillin-2 may serve as a potential target for the development of novel therapeutic agents for iCCA.
Intrahepatic cholangiocarcinoma (iCCA) arises along the peripheral bile ducts and is often accompanied by a tumor microenvironment (TME) high in extracellular matrices (ECMs). In this study, we aimed to evaluate whether an ECM-rich TME favors iCCA progression. We identified ITGA2, which encodes collagen-binding integrin α2, to be differentially-expressed in iCCA tumors compared with adjacent normal tissues. Elevated ITGA2 is also positively-correlated with its ligand, collagen type I. Increased ITGA2 expression and its role in collagen type I binding was validated in vitro using four iCCA cell lines, compared with a non-cancerous, cholangiocyte cell line. Robust interaction of iCCA cells with collagen type I was abolished by either ITGA2 depletion or integrin α2β1-selective inhibitor treatment. In a phenotypic study, collagen type I significantly enhances clonogenic growth of HuCCA-1 and HuCCT-1 cells by three and sixfold, respectively. Inhibition of integrin α2 expression or its activity significantly blocks collagen type I-induced colony growth in both cell lines. Taken together, our data provide mechanistic evidence that collagen type I promotes growth of iCCA colonies through integrin α2 suggesting that the collagen type I-integrin α2 axis could be a promising target for cancer prevention and a therapeutic opportunity for this cancer.
Mutations in IDH1/2 and the epigenetic silencing of TET2 occur in leukaemia or glioma in a mutually exclusive manner. Although intrahepatic cholangiocarcinoma (iCCA) may harbour IDH1/2 mutations, the contribution of TET2 to carcinogenesis remains unknown. In the present study, the expression and promoter methylation of TET2 were investigated in iCCA. The expression of TET2 was assessed in 52 cases of iCCA (small-duct type, n = 33; large-duct type, n = 19) by quantitative PCR, immunohistochemistry (IHC) and a sequencing-based methylation assay, and its relationships with clinicopathological features and alterations in cancer-related genes (e.g., KRAS and IDH1) were investigated. In contrast to non-neoplastic bile ducts, which were negative for TET2 on IHC, 42 cases (81%) of iCCA showed the nuclear overexpression of TET2. Based on IHC scores (area × intensity), these cases were classified as TET2-high (n = 25) and TET2-low (n = 27). The histological type, tumour size, lymph node metastasis and frequency of mutations in cancer-related genes did not significantly differ between the two groups. Overall and recurrence-free survival were significantly worse in patients with TET2-high iCCA than in those with TET2-low iCCA. A multivariate analysis identified the high expression of TET2 as an independent prognostic factor (HR = 2.94; p = 0.007). The degree of methylation at two promoter CpG sites was significantly less in TET2-high iCCA than in TET2-low iCCA or non-cancer tissue. In conclusion, in contrast to other IDH-related neoplasms, TET2 overexpression is common in iCCA of both subtypes, and its high expression, potentially induced by promoter hypomethylation, is an independent poor prognostic factor.
Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.
Background: Intrahepatic cholangiocarcinoma (ICC) is a high malignant tumor arising from the bile ducts in the liver with a poor prognosis. As current molecular targeted therapies and systemic chemotherapies had limited success in ICC, novel therapeutic targets are needed. In this study, we attempted to investigate the expression and the role of the intermediate conductance calcium-activated potassium channel (KCa3.1) in ICC. Methods: The expression levels of KCa3.1 channel were measured in 81 resected ICC tumor specimens and the clinicopathological significance of these levels were determined. KCa3.1 channel inhibitor and siRNA were used to study the role of KCa3.1 in proliferation, migration, and invasion of ICC cell lines. The effect of KCa3.1 channel blockade on tumor growth in vivo was also studied using xenograft model in nude mice. Results: The protein expression of KCa3.1 channel was upregulated in ICC tissues and was correlated with age, lymph node metastasis and TNM stage. And high KCa3.1 expression indicated a worse prognosis in ICC patients. Blocking KCa3.1 channel with a specific inhibitor TRAM-34 reduced the proliferation and invasion of ICC cells. Knockdown of KCa3.1 could achieve the same effects through decreasing NF-κB activation. Further in vivo studies demonstrated that KCa3.1 channel blockade suppressed ICC tumor growth. Conclusions: Our observations suggested KCa3.1 might be a promising novel therapeutic target in intrahepatic cholangiocarcinoma.
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