R. Semeraro

Cholangiocarcinoma: Update and future perspectives

Cholangiocarcinoma is commonly considered a rare cancer. However, if we consider the hepato-biliary system a single entity, cancers of the gallbladder, intra-hepatic and extra-hepatic biliary tree altogether represent approximately 30% of the total with incidence rates close to that of hepatocellular carcinoma, which is the third most common cause of cancer-related death worldwide. In addition, cholangiocarcinoma is characterized by a very poor prognosis and virtually no response to chemotherapeutics; radical surgery, the only effective treatment, is not frequently applicable because late diagnosis. Biomarkers for screening programs and for follow-up of categories at risk are under investigation, however, currently none of the proposed markers has reached clinical application. For all these considerations, cancers of the biliary tree system should merit much more scientific attention also because a progressive increase in incidence and mortality for these cancers has been reported worldwide. This manuscript deals with the most recent advances in the epidemiology, biology and clinical presentation of cholangiocarcinoma.

Biliary tree stem/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo‐alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co‐expressing liver and pancreatic markers (e.g. PDX1+/SOX17+) are EpCAM+/−, and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis.

Intra-hepatic and extra-hepatic cholangiocarcinoma: New insight into epidemiology and risk factors

Cholangiocarcinoma (CCA) is a malignant tumour that arises from biliary epithelium at any portion of the biliary tree. CCA is currently classified as intra-hepatic or extra-hepatic CCA (EH-CCA). Recent evidences suggest that intra-hepatic CCA (IH-CCA) and EH-CCA are biologically different cancers, giving further support to a number of recent epidemiological studies showing large differences in terms of incidence, mortality and risk factors. The purpose of this manuscript is to review recent literature dealing with the descriptive epidemiology and risk factors of CCA with a special effort to compare IH- with EH-CCA.

Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder

BACKGROUND & AIMS Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored. METHODS Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis. RESULTS The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice. CONCLUSIONS Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

Multipotent stem/progenitor cells in the human foetal biliary tree

BACKGROUND & AIMS Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. METHODS Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. RESULTS Foetal biliary tree cells proliferated clonogenically for more than 1 month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. CONCLUSIONS Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo. These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life.

An oestrogen receptor β-selective agonist exerts anti-neoplastic effects in experimental intrahepatic cholangiocarcinoma

BACKGROUND Cholangiocarcinoma cells over-express oestrogen receptor-β, which displays anti-proliferative and pro-apoptotic effects. AIM To evaluate the effects of a newly developed and highly selective oestrogen receptor-β agonist (KB9520) on experimental intrahepatic cholangiocarcinoma. METHODS In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-β silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-α and oestrogen receptor-β negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-α) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested. RESULTS In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-α and β (HepG2) and in cells expressing only oestrogen receptor-α (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-β expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis. CONCLUSIONS KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-β, suggesting that oestrogen receptor-β selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma.

Prostate Apoptosis Response-4 Is Expressed in Normal Cholangiocytes, Is Down-Regulated in Human Cholangiocarcinoma, and Promotes Apoptosis of Neoplastic Cholangiocytes When Induced Pharmacologically

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

Sensitivity of human intrahepatic cholangiocarcinoma subtypes to chemotherapeutics and molecular targeted agents: A study on primary cell cultures

We investigated the sensitivity of intrahepatic cholangiocarcinoma (IHCCA) subtypes to chemotherapeutics and molecular targeted agents. Primary cultures of mucin- and mixed-IHCCA were prepared from surgical specimens (N. 18 IHCCA patients) and evaluated for cell proliferation (MTS assay) and apoptosis (Caspase 3) after incubation (72 hours) with increasing concentrations of different drugs. In vivo, subcutaneous human tumor xenografts were evaluated. Primary cultures of mucin- and mixed-IHCCA were characterized by a different pattern of expression of cancer stem cell markers, and by a different drug sensitivity. Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-IHCCA while Cisplatin or Abraxane were more effective against mucin-IHCCA, where Abraxane also enhances apoptosis. 5-Fluoracil showed a slight inhibitory effect on cell proliferation that was more significant in mixed- than mucin-IHCCA primary cultures and, induced apoptosis only in mucin-IHCCA. Among Hg inhibitors, LY2940680 and Vismodegib showed slight effects on proliferation of both IHCCA subtypes. The tyrosine kinase inhibitors, Imatinib Mesylate and Sorafenib showed significant inhibitory effects on proliferation of both mucin- and mixed-IHCCA. The MEK 1/2 inhibitor, Selumetinib, inhibited proliferation of only mucin-IHCCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-IHCCA. The c-erbB2 blocking antibody was more active against mixed-IHCCA while, the Wnt inhibitor, LGK974, similarly inhibited proliferation of mucin- and mixed-IHCCA. Either mucin- or mixed-IHCCA showed high sensitivity to nanomolar concentrations of the dual PI3-kinase/mTOR inhibitor, NVP-BEZ235. In vivo, in subcutaneous xenografts, either NVP-BEZ235 or Abraxane, blocked tumor growth. In conclusion, mucin- and mixed-IHCCA are characterized by a different drug sensitivity. Cisplatin, Abraxane and the MEK 1/2 inhibitor, Selumetinib were more active against mucin-IHCCA while, Gemcitabine, Gemcitabine-Cisplatin combination, the c-erbB2 blocking antibody and bestatin worked better against mixed-IHCCA. Remarkably, we identified a dual PI3-kinase/mTOR inhibitor that both in vitro and in vivo, exerts dramatic antiproliferative effects against both mucin- and mixed-IHCCA.

Polycystins play a key role in the modulation of cholangiocyte proliferation

BACKGROUND Polycystin-1 and -2 (PC-1 and PC-2) are critical components of primary cilia, which act as mechanosensors and drive cell response to injury. PC-1 activation involves the cleavage/processing of PC-1 cytoplasmic tail, driven by regulated intramembrane proteolysis or ubiquitine/proteasome, translocation in the nucleus and activation of transcription factors. Mutations of PC-1 or PC-2 occur in polycystic liver where cholangiocyte proliferation is enhanced. AIM We evaluated the involvement of PC-1 and PC-2 in modulating cholangiocyte proliferation. METHODS We investigated rat cholangiocytes induced to proliferate by 17beta-oestradiol. Proliferation was evaluated by PCNA immunoblotting or [(3)H]-thymidine incorporation into DNA. PC-1 silencing was performed by siRNA, while inhibition of regulated intramembrane proteolysis or proteasome by gamma-secretase inhibitor, leupeptin or MG115. RESULTS Cholangiocyte proliferation was associated with decreased PC-1 and PC-2 expression, which was inversely correlated with enhanced PCNA. The selective silencing of PC-1 induced activation of cholangiocyte proliferation in association with decreased PC-1 expression. Two different regulated intramembrane proteolysis inhibitors, gamma-secretase-inhibitor and leupeptin, and the proteasome inhibitor, MG115, abolished the 17beta-oestradiol proliferative effect. CONCLUSIONS PC-1 and PC-2 play a major role as modulators of cholangiocyte proliferation suggesting that primary cilia may act as sensors of cell injury driving, when activated, a proliferative cholangiocyte response to trigger the reparative processes.

The fetal liver as cell source for the regenerative medicine of liver and pancreas.

Patients affected by liver diseases and diabetes mellitus are in need for sources of new cells to enable a better transition into clinic programs of cell therapy and regenerative medicine. In this setting, fetal liver is becoming the most promising and available source of cells. Fetal liver displays unique characteristics given the possibility to isolate cell populations with a wide spectrum of endodermal differentiation and, the co-existence of endodermal and mesenchymal-derived cells. Thus, the fetal liver is a unique and highly available cell source contemporarily candidate for the regenerative medicine of both liver and pancreas. The purpose of this review is to revise the recent literature on the different stem cells populations isolable from fetal liver and candidate to cell therapy of liver diseases and diabetes and to discuss advantages and limitation with respect to other cell sources.