Biliary Cells Research Articles

Loss of Wnt secretion by macrophages promote heptobiliary injury following DDC diet

Exposure of mice to a hepatotoxic diet containing 3,5‐diethoxycarbonyl‐1, 4‐dihydrocollidine (DDC) induces biliary damage followed by hepatocyte injury mimicking human cholestatic liver disease. Hepatobiliary injury is repaired through two known phenomena ‐ atypical ductular proliferation that contributes to ductular repair and hepatocyte proliferation that assist in replacing lost hepatocytes. Also, hepatocyte trans differentiation to biliary cells has been reported in this model as a repair mechanism. Using liver‐specific β‐catenin conditional knockout (KO) mice, previously we reported lack of b‐catenin in hepatocytes and cholangiocytes worsens the hepatic injury after DDC treatment. However, the source of Wnt, specifically Wnt secreted by macrophages contributing to DDC‐diet induced hepatobiliary repair has not been studied. In this report, we investigated the role for macrophage‐specific Wntless (Wls), a critical regulator of Wnt secretion, in DDC‐induced hepatobiliary injury and regeneration. We generated macrophage specific Wls knockout mice, which lack the ability of macrophages to secrete Wnts. Mac‐Wls male KO (n=5) and littermate controls (n=5) were fed a DDC diet for 14 days. Mac‐Wls KO mice on the DDC diet showed larger liver weight to body weight ratios than control. Steatosis was observed in both groups. We observed increased serum levels of ALT, ALP, and bilirubin in Mac‐Wls KO mice compared to wild type littermates. Interestingly, Mac‐Wls KO mice after DDC diet had notably more inflammation compared to control. In addition, F4/80 positive macrophages were more abundant in Mac‐Wls KO mice after DDC treatment. Wild type macrophages polarized to M1 showed upregulation of pro‐inflammatory cytokines and iNOS2 expression. Interestingly, M1 polarized macrophages showed reduced Wls expression, while M2 polarization with IL‐4 did not alter Wls expression. These findings suggest a possibility that lack of Wnt secretion by macrophages under inflammatory conditions may lead to increased inflammation by inflammatory cells including macrophages and dendritic cells. Taken together, these results suggest macrophage specific wls‐deficient mice are more prone to injury, inflammation in response to DDC‐induced injury.

PPARγ and Its Ligands: Potential Antitumor Agents in the Digestive System.

Peroxisome proliferator-activated receptor γ (PPARγ) is a versatile member of the ligand-activated nuclear hormone receptor superfamily of transcription factors, with expression in several different cell lines, especially in the digestive system. After being activated by its ligand, PPARγ can suppress the growth of oral, esophageal, gastric, colorectal, liver, biliary, and pancreatic tumor cells, suggesting that PPARγ ligand is a potential anticancer agent in PPARγ-expressing tumors. This review highlights key advances in understanding the effects of PPARγ ligands in the treatment of tumors in the digestive system.

Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic–nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.

Helicobacter Species are Possible Risk Factors of Cholangiocarcinoma.

Several infectious agents are considered to be causes of cancer in human, mainly hepatitis B and C viruses, high-risk human pailloma viruses, Helicobacter pylori, Clonorchis sinensis, and Opisthorchis viverrini. Here we described the evident research and the association between Helicobacter spp. and biliary tract cancer particularly cholangiocarcinoma (CCA). Global epidemiological studies have suggested that Helicobacter spp. are possible risk factors for biliary tract diseases. Molecular studies support a linkage of Helicobacter spp. with CCA development. H. pylori, H. bilis, and H. hepaticus, are found in CCA, but the most common species are H. pylori and H. bilis. The type of CCA are associated with Helicobacter spp. include extrahepatic CCA, and common bile duct cancer. Up to the present, however, the results from different regions, materials and methods, sub-sites of cancer, and controls have not been consistent, thus introducing heterogeneity. Therefore, a comparison between co-Helicobacter spp.-CCA in the countries with low and high incident of CCA is required to settle the question. Furthermore, clarifying variation in the role of Helicobacter species in this CCA, including pathogenesis of CCA through enhanced biliary cell inflammation and proliferation, is necessary.

Biliary tree stem cells are involved in the pathogenesis of primary sclerosing cholangitis

Biliary tree stem cells (BTSCs) are multipotent stem cells located in peribiliary glands (PBGs) of extrahepatic and large intrahepatic bile ducts (1). Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct injury are poorly understood (2). Our aims are to study the role of BTSC in the pathogenesis of biliary fibrosis in PSC. Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n=6), liver explants from patients with PSC (n=11), and primary biliary cirrhosis (n=6). Specimens were processed for histology, immunohistochemistry and immunofluorescence. In PSC samples, progressive hyperplasia and mucinous metaplasia of PBGs were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. PBG hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of PBGs was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, PBGs and myofibroblasts displayed enhanced expression of Hedgehog pathway components. PBGs in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy. The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma. This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009.

Establishment of various biliary tract carcinoma cell lines and xenograft models for appropriate preclinical studies.

We recently reported several driver genes of biliary tract carcinoma (BTC) that are known to play important roles in oncogenesis and disease progression. Although the need for developing novel therapeutic strategies is increasing, there are very few BTC cell lines and xenograft models currently available for conducting preclinical studies. Using a total of 88 surgical BTC specimens and 536 immunodeficient mice, 28 xenograft models and 13 new BTC cell lines, including subtypes, were established. Some of our cell lines were found to be resistant to gemcitabine, which is currently the first choice of treatment, thereby allowing highly practical preclinical studies to be conducted. Using the aforementioned cell lines and xenograft models and a clinical pathological database of patients undergoing BTC resection, we can establish a preclinical study system and appropriate parameters for drug efficacy studies to explore new biomarkers for practical applications in the future studies.

The hepatic, biliary, and pancreatic network of stem/progenitor cell niches in humans: A new reference frame for disease and regeneration.

Stem/progenitors for liver, biliary tree, and pancreas exist at early stages of development in the definitive ventral endoderm forming the foregut. In humans, they persist postnatally as part of a network, with evidence supporting their contributions to hepatic and pancreatic organogenesis throughout life. Multiple stem cell niches persist in specific anatomical locations within the human biliary tree and pancreatic ducts. In liver and pancreas, replication of mature parenchymal cells ensures the physiological turnover and the restoration of parenchyma after minor injuries. Although actively debated, multiple observations indicate that stem/progenitor cells contribute to repair pervasive, chronic injuries. The most primitive of the stem/progenitor cells, biliary tree stem cells, are found in peribiliary glands within extrahepatic and large intrahepatic bile ducts. Biliary tree stem cells are comprised of multiple subpopulations with traits suggestive of maturational lineage stages and yet capable of self-replication and multipotent differentiation, being able to differentiate to mature liver cells (hepatocytes, cholangiocytes) and mature pancreatic cells (including functional islet endocrine cells). Hepatic stem cells are located within canals of Hering and bile ductules and are capable of differentiating to hepatocyte and cholangiocyte lineages. The existence, phenotype, and anatomical location of stem/progenitors in the adult pancreas are actively debated. Ongoing studies suggest that pancreatic stem cells reside within the biliary tree, primarily the hepatopancreatic common duct, and are rare in the pancreas proper. Pancreatic ducts and pancreatic duct glands harbor committed pancreatic progenitors. The hepatic, biliary, and pancreatic network of stem/progenitor cell niches should be considered as a framework for understanding liver and pancreatic regeneration after extensive or chronic injuries and for the study of human chronic diseases affecting these organs. (Hepatology 2016;64:277-286).

Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48–72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells.

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and is up-regulated in biliary tract cancer (BTC), contributing to aggressive clinical features. In this study we investigated the cytotoxic effects of PTC-209, a recently developed inhibitor of BMI1, in BTC cells. PTC-209 reduced overall viability in BTC cell lines in a dose-dependent fashion (0.04 - 20 µM). Treatment with PTC-209 led to slightly enhanced caspase activity and stop of cell proliferation. Cell cycle analysis revealed that PTC-209 caused cell cycle arrest at the G1/S checkpoint. A comprehensive investigation of expression changes of cell cycle-related genes showed that PTC-209 caused significant down-regulation of cell cycle-promoting genes as well as of genes that contribute to DNA synthesis initiation and DNA repair, respectively. This was accompanied by significantly elevated mRNA levels of cell cycle inhibitors. In addition, PTC-209 reduced sphere formation and, in a cell line-dependent manner, aldehyde dehydrogease-1 positive cells. We conclude that PTC-209 might be a promising drug for future in vitro and in vivo studies in BTC.

The effects of buthionine sulfoximine on the proliferation and apoptosis of biliary tract cancer cells induced by cisplatin and gemcitabine.

Patients with biliary tract cancer (BTC) have a poor prognosis. Advanced BTC patients have been treated with cisplatin in combination with gemcitabine, however, the treatment has had little impact on survival rates, and more effective treatments are urgently required for this disease. Previous studies discovered that buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, was able to enhance the cytotoxic effect of various drugs in cancer cells. Phase I studies demonstrated that continuous-infusion of BSO was relatively non-toxic and resulted in the depletion of tumor GSH. However, the synergistic effect of BSO and cisplatin in BTC cells remains unknown, and no reports are available regarding sensitization to gemcitabine by BSO. In the present study, the effect of BSO in combination with cisplatin or gemcitabine in the treatment of BTC cells was examined in vitro. Cytotoxic effects were measured using an MTT assay, Annexin V assay and fluorescence-activated cell sorting analysis. Antiapoptotic protein expression levels were examined using western blot analysis. The results revealed that a sub-toxic concentration of BSO was capable of significantly enhancing cisplatin-induced apoptosis in BTC cells. The mechanisms of BSO's effect on BTC cells may be attributable to the reduction of GSH levels and downregulation of the expression of antiapoptotic proteins (Bcl-2, Bcl-xL and Mcl-1). Furthermore, BSO enhanced the antiproliferative effect of gemcitabine. In conclusion, the present data are the first results to indicate that BSO may sensitize BTC cells to standard first-line chemotherapeutic agents (cisplatin and gemcitabine). Combining BSO with cisplatin and gemcitabine is a promising therapeutic strategy for the treatment of BTC.

PKCα regulates TMEM16A-mediated Cl⁻ secretion in human biliary cells.

TMEM16A is a newly identified Ca(2+)-activated Cl(-) channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing intracellular Ca(2+) concentration ([Ca(2+)]i), the regulatory pathways have not been elucidated. Protein kinase C (PKC) contributes to ATP-mediated secretion in BECs, although its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl(-) transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca(2+)-activated Cl(-) currents. Currents demonstrated outward rectification and reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl(-) currents with biophysical properties identical to TMEM16A in control cells but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca(2+)-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.

TRAIL in Combination with Subtoxic 5-FU Effectively Inhibit Cell Proliferation and Induce Apoptosis in Cholangiocarcinoma Cells.

In the past decade, the incidence and mortality rates of cholangiocarcinoma (CCA) have been increasing worldwide. The relatively low responsiveness of CCA to conventional chemotherapy leads to poor overall survival. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has emerged as the most promising anti-cancer therapeutic agent since it is able to selectively induce apoptosis of tumor cells but not normal cells. In this study, we aimed to investigate the therapeutic effect of TRAIL in CCA cell lines (M213, M214 and KKU100) compared with the immortal biliary cell line, MMNK1, either alone or in combination with a subtoxic dose of 5-fluorouracil (5-FU). We found that recombinant human TRAIL (rhTRAIL) was a potential agent which significantly inhibited cell proliferation and mediated caspase activities (caspases 8, 9 and 3/7) and apoptosis of CCA cells. The combined treatment of rhTRAIL and 5-FU effectively enhanced inhibition of CCA cell growth with a smaller effect on MMNK1. Our finding suggests TRAIL to be a novel anti-cancer therapeutic agent and advantage of its combination with a conventional chemotherapeutic drug for effective treatment of CCA.

Expression of Toll-like receptor (TLR) 2 and TLR4 in the livers of mice infected by Clonorchis sinensis.

Clonorchis sinensis is one of the most important foodborne pathogens in humans, and can cause biliary diseases such as gallstones, cholecystitis, cholangitis, and cholangiocarcinoma. Toll-like receptors (TLRs) as sensors are crucial to initiating both innate and adaptive immune defenses against pathogens. However, little is known about the hepatic expression of TLRs of hosts induced by C. sinensis infection. In the present study, the expression and distribution of TLR2 and TLR4 were investigated in a mouse model of clonorchiasis on days 28, 56, 84, and 112 post-infection (PI) using real-time quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemically staining, respectively. The levels of cytokines that are mediated by TLR2 and TLR4 were also evaluated using a cytometric bead array. Results showed that the transcripts of TLR2 and TLR4 were upregulated on day 28 PI in C. sinensis-infected mice compared with non-infected ones (p < 0.01). In addition, their proteins were strongly immunohistochemically positive in the cytoplasm and membrane of endothelial cells, fibroblasts, and biliary epithelium cells of C. sinensis-infected mice. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were increased with activation of TLR2 and TLR4. The expression of TLR2 and TLR4 is upregulated against C. sinensis infection, which suggests that TLR2 and TLR4 might be involved in immune responses during C. sinensis infection.

Natural killer cells regulate T cell immune responses in primary biliary cirrhosis.

The hallmark of primary biliary cirrhosis (PBC) is the presence of autoreactive T- and B-cell responses that target biliary epithelial cells (BECs). Biliary cell cytotoxicity is dependent upon initiation of innate immune responses followed by chronic adaptive, as well as bystander, mechanisms. Critical to these mechanisms are interactions between natural killer (NK) cells and BECs. We have taken advantage of the ability to isolate relatively pure viable preparations of liver-derived NK cells, BECs, and endothelial cells, and studied interactions between NK cells and BECs and focused on the mechanisms that activate autoreactive T cells, their dependence on interferon (IFN)-γ, and expression of BEC major histocompatibility complex (MHC) class I and II molecules. Here we show that at a high NK/BEC ratio, NK cells are cytotoxic for autologous BECs, but are not dependent on autoantigen, yet still activate autoreactive CD4(+) T cells in the presence of antigen presenting cells. In contrast, at a low NK/BEC ratio, BECs are not lysed, but IFN-γ production is induced, which facilitates expression of MHC class I and II molecules on BEC and protects them from lysis upon subsequent exposure to autoreactive NK cells. Furthermore, IFN-γ secreted from NK cells after exposure to autologous BECs is essential for this protective function and enables autoreactive CD4(+) T cells to become cytopathic. NK cell-mediated innate immune responses are likely critical at the initial stage of PBC, but also facilitate and maintain the chronic cytopathic effect of autoantigen-specific T cells, essential for progression of disease.

Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies.

Biliary intraductal metastasis from advanced gastric cancer: radiologic and histologic characteristics, and clinical outcomes of percutaneous metallic stent placement.

To investigate the radiologic and histological characteristics of biliary intraductal metastasis of advanced gastric cancer and the clinical outcomes of percutaneous, metallic stent placement. We retrospectively assessed 24 patients with obstructive jaundice related to biliary intraductal metastasis of gastric cancers who underwent PTBD and subsequent metallic stent placement between 2003 and 2012. Intraductal metastases appeared as uniform, concentric, linear (n = 17) or band-like (n = 7), enhanced wall thickening on CT, and 20 patients (83.3%) had cystic ductal lesions. On pathology specimens, malignant cells scattered in the submucosal layer caused a desmoplastic reaction. The technical and clinical success rate of stent placement was achieved in all 24 patients. The median survival time was 203days. Stent occlusion was observed in four patients with 49-278 days following stent placement. The median stent patency time was 156days. The radiologic and histological characteristics of biliary intraductal metastasis of advanced gastric cancer consist of uniform, linear or band-like, enhanced biliary wall thickening and malignant cells scattered in the submucosal layer, together with the desmoplastic reaction without any disruption of the epithelial layer. Uncovered metallic stent placement was also a safe and effective method of palliative treatment in these patients. • The CT findings of intraductal metastasis were linear/band-like, enhanced biliary wall thickening. • The histological finding was malignant cells scattered in the submucosal layer. • It showed a desmoplastic reaction without any disruption of the epithelial layer. • Uncovered metallic stent placement was a safe and effective palliative treatment.

Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression

ObjectivePrevious studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to...

Adult Human Biliary Tree Stem Cells Differentiate to β-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide.

Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Abstract 3354: Cancer-associated or cirrhosis fibroblasts recovered from hepatocellular carcinoma promote macrophages and cancer cells to progressive phenotype

Abstract Background/Aim: Tumor microenvironment plays supportive roles for cancer cells of proliferating, invading and spreading systemically. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) are deemed to be essential constituents forming cancer-surrounding niche. However, the cross-talks between CAFs and TAMs in the settings of hepatocellular carcinoma (HCC) has yet to be deciphered. The lack of a reliable system simulating in vivo-microenvironment has been one of the obstacles hampering investigation of CAFs and TAMs. By using the newly-established model consisting of fibroblasts directly recovered from the disease liver, we aimed to clarify the mechanisms of CAFs modulating the macrophage differentiation and malignant potential of cancer cells in order to search for therapeutic targets. Methods: We enrolled three patients who underwent the resection of HCC and two who did living donor liver transplantation. From the resected or the explanted livers, fibroblasts from cancer and its adjacent tissues were separated by tissue digestion. We examined their ontogeny by the expression of fibroblast-specific markers. For the functional analyses, we examined invasiveness of HuH7 cells on matrigel and migratory response of monocytes in the presence of conditioned media (CM) from the fibroblasts. We assessed the phenotypes/function of macrophages after the differentiation from monocytes in the presence of M-CSF and the CM. We comprehensively assayed cytokine/chemokine in the CM to identify functionally-relevant factors. Results): After the culture, we established three types of fibroblasts, CAFs, cirrhotic liver fibroblasts (LCFs) and non-cancerous fibroblasts (NFs). All of these were positive for vimentin, SMA, PDGFRa and FAP (&amp;gt;99%), confirming that they are ontologically fibroblasts instead of being HCC, hepatocytes or biliary cells. The invasiveness of HuH7 were higher in the presence of the CAF- or LCF-CM compared to those with NF-CM. Similar superiority was observed with the CAF- or LCF-CM in the migratory ability of monocytes. In the process of macrophage differentiation, the CAF- or LCF-CM were capable of polarizing macrophages into TAM/M2 subtypes, as evidenced by their higher production of IL-10 but lesser IL-12. In the CAF- or LCM-CM, the levels of IL6, IL-8, CCL2 and GRO were higher than those in the NF-CM. Conclusion: The CAFs as well as LCFs directly recovered from the cirrhotic liver are functionally competent of driving TAM/M2 differentiation and providing malignant potential to cancer cells, thereby tuning the microenvironment favoring HCC development. Citation Format: Yohei Mano, Tatsuya Kanto, Hirotaka Shoji, Schiyo Yoshio, Masaya Sugiyama, Yosuke Osawa, Kiminori Kimura, Ken Shirabe, Yoshihiko Maehara, Masashi Mizokami. Cancer-associated or cirrhosis fibroblasts recovered from hepatocellular carcinoma promote macrophages and cancer cells to progressive phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2015-3354