Extrahepatic Cells Research Articles

Abstract 1072: Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a fatal primary malignancy with limited treatment options and a dismal 5year survival rate of <10%. Poor prognosis and early recurrence after surgical treatment warrants further molecular characterization of these tumors to evaluate therapeutic sensitivities and identification of biomarkers for CCA. Although several Next Generation Sequencing (NGS) studies have revealed some targetable genomic aberrations, the lack of preclinical and translational models has severely limited the identification of clinically relevant targeted therapies for CCA. So far, very few putative molecular targets/pathways have been identified using patient-derived xenograft (PDX) models, CCA cell derived xenograft models (CDX) and CCA cell lines, only 4 of which have progressed to preclinical testing for therapeutic value. In this study we describe development and comprehensive characterization of patient derived xenograft models of CCA from freshly resected patient tissue. Patient tumor cells from 8 patients were engrafted subcutaneously in highly immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj) mice for PDX development. Two (CCA-1 and CCA-7) of 8 (25 %) engraftments yielded successful PDX model. The PDX avatar and matched patient tumor samples were genomically characterized at the whole exome and transcriptome level to check the fidelity of PDX models. Exome sequencing analysis of PDX tumors demonstrate higher genomic aberrations as compared to their patient counterparts. Preliminary analysis of transcriptomic profiles from CCA TCGA dataset, long term established intra- and extra-hepatic CCA cell lines (Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1 and HuH-28), CCA CDX model from Mz-ChA-1 cells and CCA PDX models demonstrate that CCA PDX models more faithfully recapitulate patient tumors as compared to long term established cell lines. In summary, this proof of concept study demonstrates that PDX avatars of CCA could serve as a good model system for preclinical target discovery and validation for a deadly and neglected disease such as CCA. Citation Format: Sanhita Rath, Sen Peng, Lauren Hartman, George Reid, Harshil Dhruv, Shannon Glaser, Fanyin Meng, Gianfranco Alpini, Scott Celinski. Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1072.

Plasma exosomes exacerbate alcohol- and acetaminophen-induced toxicity via CYP2E1 pathway

Cellular CYP2E1 is well-known to mediate alcohol- (ALC) and acetaminophen- (APAP) induced toxicity in hepatic and extra-hepatic cells. Although exosomes have been gaining importance in understanding mechanism of intra- and inter-cellular communication, the functional role of drug metabolizing cytochrome P450 (CYP) enzymes in human plasma exosomes are yet to be explored. In our previous study, we reported that human plasma-derived exosomes contain substantial level of functional CYP2E1. In the current project, we investigated the potential role of plasma exosomal CYP2E1 in mediating ALC- and APAP-induced toxicity. We treated hepatic and extra-hepatic (monocytic) cells with exosomes ± ALC/APAP. We observed that the plasma exosomes containing CYP2E1 cargo further exacerbate ALC- and APAP-induced toxicity in both hepatic and monocytic cells. Further, both exosomes- and ALC/APAP-induced toxicity was reduced/abolished by a selective inhibitor of CYP2E1 enzyme activity (diallyl ether). However, only ALC-, but not exosome-induced toxicity was reduced/abolished by CYP2E1 siRNA. These findings suggest that ALC/APAP-induced toxicity in the presence of exosomes are mediated, at least in part, by CYP2E1 enzyme. To validate these in vitro findings, we characterized plasma exosomal contents in a binge-drinking animal model and their effect on ALC/APAP-induced toxicity in monocytic cells. Our results showed that ALC exposure caused a significant induction of the plasma exosomal CYP2E1 level in a binge drinking murine model. These exosomes containing increased levels of CYP2E1 caused significant toxicity in monocytic cells compared to exosomes derived from control mice. Overall, our results showed an important role of exosomal CYP2E1 in exacerbating ALC- and APAP-induced toxicity. The study is significant in terms of understanding the role of exosomal CYP2E1 in cell-cell interactions, and their effects on drug-induced toxicity.

Extracellular Vesicles: Intercellular Mediators in Alcohol-Induced Pathologies.

Though alcoholic liver injury plays the primary role in direct alcohol-related morbidity, alcohol consumption is also interlinked with many other diseases in extra-hepatic tissues/organs. The mechanism of alcoholic tissue injury is well documented, however the mechanisms that affect extra-hepatic tissues have not yet been well defined. Extracellular vesicles (EVs) such as exosomes and microvesicles, have been identified as key components of alcohol-induced extra-hepatic effects. We have reviewed the recent findings on the potential impact of alcohol-modified EVs/exosomes production and their downstream effects on extra-hepatic tissues. In this review, we discuss the available information on the cross-talk between hepatocytes and immune cells via EV/exosomal cargos (miRNA, mRNA, protein, etc.) in alcoholic liver diseases. We also discuss the effects of alcohol exposure on the contents of EVs/exosomes derived from various extra-hepatic tissues and their associated pathological consequences on recipient cells. Finally, we speculate on other potential EV/exosomal agents that may mediate alcohol-induced tissue damage. Graphical Abstract Alcohol can alter contents of extracellular vesicles (EVs) (e.g. exosomes) such as miRNAs, protein, cytokines, etc. in hepatic and extra-hepatic cells. The transfer of these alcohol modified EVs to nearby or distant cells can play vital role in inflammatory pathways in alcohol induced pathogenesis/comorbidities.

Functional Role of microRNAs in Patient‐Derived Xenograft Models of Human Cholangiocarcinoma

BACKGROUND & AIMSPatient‐derived xenograft (PDX) models are now being widely used in cancer research including liver and biliary carcinogenesis, and have the potential to feedback and improve the therapeutic effects in cancer patients. MicroRNAs (miRNAs) are highly conserved 19–25 nucleotide non‐coding small RNAs which are primarily involved in gene silencing. Our aim was to characterize the functional roles of microRNA regulated protein translocations in patient‐derived cholangiocarcinoma xenograft tumors.METHODSmiRNA expression in human cholangiocarcinoma cancer tissues, patient‐derived cholangiocarcinoma xenograft tissues, human extrahepatic cholangiocarcinoma cancer stem cells (GB‐CSCs)/Mz‐ChA‐1 extrahepatic cholangiocarcinoma cancer cells and their derived xenograft tumor tissues, as well as H69 non‐malignant cholangiocytes was assessed using a hybridization based microarray and was further evaluated by Taqman real‐time PCR analysis. Promoter methylation, matrix metalloproteinase (MMP) 1, 2, 3 and 9 mRNA expression was quantitated by real‐time PCR and western blot analysis.RESULTSWe identified miR‐200 family including miR‐200a, miR‐200b and miR‐200c that are differentially expressed in doxorubicin‐enriched cell fractions in a patient derived mouse xenograft tumor model of extrahepatic cholangiocarcinoma. Members of the miR‐200 family were notably more silenced in GB‐CSCs compared with their parental extrahepatic cholangiocarcinoma cancer cells (GBCs) and Mz‐ChA‐1 cells. Expression of miR‐200 family microRNAs and transmembrane protein MMP‐2 and MMP‐9 were substantially enhanced concomitant with GBCs differentiation and loss of self‐renewal, whereas forced expression of a polycistron construct encoding miR‐200 miRNAs significantly decreased chemoresistance to doxorubicin. Methylation‐specific PCR detected that miR‐200b and miR‐200c promoter was hypermethylated in GB‐CSCs, GBCs as well as Mz‐ChA‐1 cells. Cell migration and invasion were significantly decreased by miR‐200 precursor introduction in GB‐CSCs and Mz‐ChA‐1 cells. Bioinformatics and dual‐luciferase reporter assays identified ZEB1 and ZEB2 as the direct targets of miR‐200 family.CONCLUSIONOur results define a novel oncogenic regulatory mechanism of specific microRNA group in patient derived cholangiocarcinoma xenograft models and suggesting that miR‐200 cluster may be the molecular targets for eradication of human cholangiocarcinoma.Support or Funding InformationTGEN grant from Translational Genomics Research InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation

Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

Metformin Induces Apoptosis and Inhibits Proliferation through the AMP-Activated Protein Kinase and Insulin-like Growth Factor 1 Receptor Pathways in the Bile Duct Cancer Cells.

Background/Aims: Metformin has been found to have antineoplastic activity in some cancer cells. This study was performed to determine whether metformin inhibits the proliferation of bile duct cancer cells by inducing apoptosis and its effects on the expression of gene-related proteins involved in cancer growth.Methods: Human extrahepatic bile duct cancer cells (SNU-245 and SNU-1196) were cultured. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to determine the effect of metformin on the cell proliferation. Apoptosis was measured by a cell death detection enzyme-linked immunosorbent assay and a caspase-3 activity assay. Expression levels of various proteins, with or without specific small interfering ribonucleic acid-induced gene disruption, were measured by Western blot analysis. The migratory activity of the cancer cells was evaluated by wound healing assay.Results: Metformin suppressed cell proliferation in bile duct cancer cells by inducing apoptosis. Metformin inhibited mammalian target of rapamycin (mTOR) by activation of tuberous sclerosis complex 2 (TSC-2) through phosphorylation of adenosine monophosphate-activated protein kinase at threonine-172 (AMPKThr172). Hyperglycemia impaired metformin-induced AMPKThr172 activation and enhanced phosphorylation of AMPK at serine-485 (AMPKSer485). Metformin blocked the inhibitory effect of insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor substrate 1 (IRS-1) pathway on TSC-2, and hyperglycemia impaired metformin-induced inhibition of IGF-1R/IRS-1 pathway and modulated the invasiveness of bile duct cancer cells; however, this effect was impaired by hyperglycemia.Conclusions: Metformin has antineoplastic effects in bile duct cancer, and hyperglycemic environment interrupts the effect of metformin. In addition, AMPK and IGF-1R play a key role in the proliferation of bile duct cancer cells.

Existence of Occult Hepatitis C virus Infection in Local Haemodialysis Setting

Introduction: Among chronic hepatitis C patients, the viral agent is well known to replicate in the hepatocytes and extrahepatic cells. Previous studies have demonstrated a new form of hepatitis C called 'Occult HCV Infection' (OCI). It is defined as the presence of HCV RNA in the hepatocytes but undetectable HCV RNA in the serum or plasma. The exact natural history, epidemiology, pathogenesis and clinical importance of OCI are unknown. Several studies have been carried out worldwide, but none from Malaysia. This study investigated OCI among local patients undergoing routine hemodialysis at a referral hospital in Pahang, Malaysia. Materials and Methods: Blood was collected from both anti-HCV positive (n=10) and anti-HCV negative (n=18) patients. Inclusion criteria for the seronegative patients included elevated liver enzymes. Results: 10 chronic hepatitis C patients were recruited in this study. All had viral RNA detected in both serum and peripheral blood mononuclear cells (PBMC). Viral quasispecies was common in this group of patients. Meanwhile, out of 18 seronegative patients, 3 patients (16%) showed active viral replication in PBMC. Conclusion: This study proved the existence of OCI in local haemodialysis setting. Further investigation is needed to get the actual prevalence of OCI.

Hedgehog Signaling Modulates Interleukin-33-Dependent Extrahepatic Bile Duct Cell Proliferation in Mice.

Hedgehog (HH) signaling participates in hepatobiliary repair after injury and is activated in patients with cholangiopathies. Cholangiopathies are associated with bile duct (BD) hyperplasia, including expansion of peribiliary glands, the niche for biliary progenitor cells. The inflammation‐associated cytokine interleukin (IL)‐33 is also up‐regulated in cholangiopathies, including cholangiocarcinoma. We hypothesized that HH signaling synergizes with IL‐33 in acute inflammation‐induced BD hyperplasia. We measured extrahepatic BD (EHBD) thickness and cell proliferation with and without an IL‐33 challenge in wild‐type mice, mice overexpressing Sonic HH (pCMV‐Shh), and mice with loss of the HH pathway effector glioma‐associated oncogene 1 (Gli1lacZ/lacZ). LacZ reporter mice were used to map the expression of HH effector genes in mouse EHBDs. An EHBD organoid (BDO) system was developed to study biliary progenitor cells in vitro. EHBDs from the HH overexpressing pCMV‐Shh mice showed increased epithelial cell proliferation and hyperplasia when challenged with IL‐33. In Gli1lacZ/lacZ mice, we observed a decreased proliferative response to IL‐33 and decreased expression of Il6. The HH ligands Shh and Indian HH (Ihh) were expressed in epithelial cells, whereas the transcriptional effectors Gli1, Gli2, and Gli3 and the HH receptor Patched1 (Ptch1) were expressed in stromal cells, as assessed by in situ hybridization and lacZ reporter mice. Although BDO cells lacked canonical HH signaling, they expressed the IL‐33 receptor suppression of tumorigenicity 2. Accordingly, IL‐33 treatment directly induced BDO cell proliferation in a nuclear factor κB‐dependent manner. Conclusion: HH ligand overexpression enhances EHBD epithelial cell proliferation induced by IL‐33. This proproliferative synergism of HH and IL‐33 involves crosstalk between HH ligand‐producing epithelial cells and HH‐responding stromal cells.

Squamous Cell Carcinoma of the Extrahepatic Common Hepatic Duct.

We report a rare case of hilar squamous cell carcinoma. A 62-year-old Korean woman complaining of nausea was referred to our hospital. Her biliary computed tomography revealed a 28 mm-sized protruding solid mass in the proximal common bile duct. The patient underwent left hemihepatectomy with S1 segmentectomy and segmental excision of the common bile duct. Microscopically, the tumor was a moderately differentiated squamous cell carcinoma of the extrahepatic bile duct, without any component of adenocarcinoma or metaplastic portion in the biliary epithelium. Immunohistochemically, the tumor was positive for cytokeratin (CK) 5/6, CK19, p40, and p63. Squamous cell carcinoma of the extrahepatic bile duct is rare. To date, only 24 cases of biliary squamous cell carcinomas have been reported. Here, we provide a clinicopathologic review of previously reported extrahepatic bile duct squamous cell carcinomas.

Silencing of Kangai 1 C-terminal interacting tetraspanin suppresses progression of cholangiocarcinoma

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

Extrahepatic cancers and chronic HCV infection.

Infectious agents, such as HCV, account for ∼15% of human cancers. HCV infects not only hepatocytes but also extrahepatic cells. Chronic HCV infection can induce chronic inflammation with qualitative and quantitative alterations of the immune repertoire and tissue microenvironment, which could induce various neoplasias. Epidemiological studies and meta-analyses suggest an increased rate of extrahepatic cancers in patients with chronic HCV infection along with a higher risk of intrahepatic cholangiocarcinoma, pancreatic cancer and non-Hodgkin lymphoma (NHL), highlighting the need to screen for HCV infection in patients with these cancers. Development of B cell NHL has been associated with HCV infection, with a relative risk of ∼1.5. Direct transformation related to the presence of the virus and chronic antigenic stimulation are the two major non-exclusive mechanisms involved in HCV-related lymphomagenesis. HCV infection alters survival of patients with lymphoma, and sustained virologic response (SVR) substantially improves prognosis. Antiviral treatments might induce remission of indolent lymphoma when SVR is achieved even without chemotherapy, emphasizing the role of HCV in lymphomagenesis in this context. However, studies are needed to provide prospective evidence of a causal relationship between chronic HCV infection and other extrahepatic cancers and to determine whether the risk of extrahepatic cancers is reduced with SVR. In this Review, we report on recent studies analysing the risk of extrahepatic cancers associated with chronic HCV infection. Although there is no doubt regarding the direct and indirect causality between HCV and NHL, an increased risk of other cancers is less clear, with the exception of cholangiocarcinoma.

Liver Regeneration Supported by Muse Cells.

Cellular compensation from extrahepatic resources is expected to improve the prognosis of liver diseases. Currently, liver dysfunction is treated by a variety of modalities including drugs, cytokines, vascular interventions, energy devices, surgery, and liver transplantation; however, in recent years there have been few significant advancements in treatment efficacy. A next-generation therapeutic strategy for liver disease, cellular compensatory therapy (i.e., cell therapy), is now being considered for clinical practice. Liver dysfunction is attributed to a lack of sufficient functional cells. However, processes involved in recovery of liver function are not fully elucidated, which has complicated the interpretation of treatment effects at the cellular level. Our genotyping study of living donor liver transplantation revealed that a variety of graft liver tissues contained the donor genotype, indicating that extrahepatic cells had differentiated into liver component cells during liver regeneration. Multilineage-differentiating stress-enduring (Muse) cells appear to be a strong candidate for extrahepatic resources that can contribute to liver regeneration. Muse cells are defined as stage-specific embryonic antigen 3-expressing cells that contribute to tissue regeneration and have the potential to differentiate into three germ layers. The significant advantage of Muse cells over other "pluripotent cells" is that Muse cells are present in bone marrow/blood as well as a variety of connective tissues, which provides safety and ethical advantages for clinical applications. Here, we review current therapeutic topics in liver diseases and discuss the potential for cell therapy using Muse cells based on our recent studies of Muse cell administration in a mouse model of physical partial hepatectomy.

Revisiting the liver: from development to regeneration - what we ought to know!

The liver is structurally and functionally heterogeneous and complex, and it accomplishes crucial functions for the organism. Its most remarkable potential is its capacity to regenerate after injury in order to maintain whole body homeostasis and guarantee the survival of the individual. Under normal conditions, liver regeneration (LR) is attributed to adult hepatocytes, the main cells in the liver which are able to proliferate in response to different stimuli or injuries. Nevertheless, when liver injury is severe and/or hepatocytes are prevented from proliferation, liver stem/progenitor cells (LS/PCs) participate directing LR to maintain liver mass and functions. Different mechanisms have been shown to guide this second line of LR, such as intrahepatic and extrahepatic liver progenitor cells, as well as transdifferentiation processes between hepatocytes and other liver cells. For this reason, many efforts have been made to elucidate the specific molecular mechanisms which orchestrate this process; this in turn would improve the prognosis and treatment of liver diseases. In this review, we revisit the fascinating process of LR, also with a short overview about liver development, the process from which arises the concept of LS/PCs participating in LR, and very important nowadays when considering cell therapy and tissue bioengineering for the treatment of patients suffering from liver disease.

Specific packaging and circulation of cytochromes P450, especially 2E1 isozyme, in human plasma exosomes and their implications in cellular communications

Cytochrome P450 (CYP) enzymes metabolize the majority of xenobiotics and are mainly found in hepatic and some extra-hepatic cells. However, their presence and functional role in exosomes, small extracellular vesicles that are secreted from various cells into extracellular fluids including plasma, is unknown. In this study, we analyzed the expression and biological activity of CYP enzymes in human plasma exosomes. First, we optimized isolation of plasma exosomes and characterized them for their physical properties and quality. The results showed that the purity of exosomes (<200 nm) improved upon prior filtration of plasma using a 0.22 micron filter. We then analyzed the relative level of exosomal CYP mRNAs, proteins, and enzyme activity. The results showed that the relative level of CYP enzymes in exosomes is higher than in plasma, suggesting their specific packaging in exosomes. Of the seven CYP enzymes tested, the mRNA of CYP1B1, CYP2A6, CYP2E1, and CYP3A4 were detectable in exosomes. Interestingly, the relative level of CYP2E1 mRNA was >500-fold higher than the other CYPs. The results from the Western blot showed detectable levels of CYP1A1, CYP1B1, CYP2A6, CYP2E1, and CYP3A4. Our results also demonstrated that exosomal CYP2E1 and CYP3A4 show appreciable activity relative to their respective positive controls (CYP-induced baculosomes). Our results also showed that CYP2E1 is expressed relatively higher in plasma exosomes than hepatic and monocytic cells and exosomes derived from these cells. In conclusion, this is the first evidence of the specific packaging and circulation of CYP enzymes, especially CYP2E1, in human plasma exosomes. The findings have biological and clinical significance in terms of their implications in cellular communications and potential use of plasma exosomal CYPs as biomarkers.

In vitro detection of cholangiocarcinoma cells using a fluorescent protein-expressing oncolytic herpes virus.

Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.

The stellate cell system (vitamin A-storing cell system)

Past, present, and future research into hepatic stellate cells (HSCs, also called vitaminA-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells) are summarized and discussed in this review. Kupffer discovered black-stained cells in the liver using the gold chloride method and named them stellate cells (Sternzellen in German) in 1876. Wake rediscovered the cells in 1971 using the same gold chloride method and various modern histological techniques including electron microscopy. Between their discovery and rediscovery, HSCs disappeared from the research history. Their identification, the establishment of cell isolation and culture methods, and the development of cellular and molecular biological techniques promoted HSC research after their rediscovery. In mammals, HSCs exist in the space between liver parenchymal cells (PCs) or hepatocytes and liver sinusoidal endothelial cells (LSECs) of the hepatic lobule, and store 50-80% of all vitaminA in the body as retinyl ester in lipid droplets in the cytoplasm. SCs also exist in extrahepatic organs such as pancreas, lung, and kidney. Hepatic (HSCs) and extrahepatic stellate cells (EHSCs) form the stellate cell (SC) system or SC family; the main storage site of vitaminA in the body is HSCs in the liver. In pathological conditions such as liver fibrosis, HSCs lose vitaminA, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. The morphology of these cells also changes from the star-shaped HSCs to that of fibroblasts or myofibroblasts.

Spectrum of Histopathological Findings in Liver Biopsy

The significance of abnormal liver function tests in the absence of diagnostic serology is unclear. The aim of this prospective study is to report liver biopsy findings in a large group of patients with unexplained abnormal liver biochemistry and correlate them with the clinical features to assess the severity of these diseases. Percutaneous liver biopsy is a relatively safe and accurate method of diagnosing liver disease and should be considered in such cases. A total of 63 liver biopsies were studied for a period of 18 months from june 2012 to december 2013, of which, 34.92% (n=22) hepatic tumors, 20.63.%(13) glycogen storage disorders, 4.7% (n=03) cirrhosis, 4.7% (n=03) fatty liver and 4.7% (n=3) viral hepatitis, 4.7%(3) Non alcoholic steato hepatitis were seen. Further, 01.58% case (n=1) each of extra hepatic biliary atresia, amyloidosis, miliary tuberculosis, liver abscess, and liver cell dysplasia were also observed. Liver biopsy was non specific in 6.34% (n=4) and inadequate in 11.11% (n=7) cases. The role of histopathological examination of liver biopsy is highlighted in this paper.

L1 Cell Adhesion Molecule Promotes Migration and Invasion via JNK Activation in Extrahepatic Cholangiocarcinoma Cells with Activating KRAS Mutation.

Extrahepatic cholangiocarcinoma (ECC), a malignant tumor of biliary origin, has a poor prognosis with limited treatment options. The KRAS oncogene is the most commonly mutated gene in ECC and one of the factors that predicts a poor prognosis and low survival rate. L1 cell adhesion molecule (L1CAM) is expressed in ECC cells and acts as an independent poor prognostic factor in predicting patient survival. In this study we investigate the functional significance of L1CAM in ECC cells with activating KRAS mutation. We selected an ECC cell line, EGI-1, with activating KRAS mutation, and then confirmed its expression of L1CAM by RT-PCR, western blot analysis, and flow cytometry. The suppression of L1CAM expression (using a specific lentivirus-delivered shRNA) significantly decreased the migratory and invasive properties of EGI-1 cells, without altering their proliferation or survival. Analyses of signaling effectors in L1CAM-depleted and control EGI-1 cells indicated that L1CAM suppression decreased the levels of both phosphorylated MKK4 and total MKK4, together with c-Jun N-terminal kinase (JNK) phosphorylation. Further, exposure to a JNK inhibitor (SP600125) decreased migration and invasion of EGI-1 cells. These results suggest that L1CAM promotes cellular migration and invasion via the induction of MKK4 expression, leading to JNK activation. Our study is the first to demonstrate a functional role for L1CAM in ECC carrying the activating KRAS mutation. Given that KRAS is the most commonly mutated oncogene in ECC, L1CAM may serve as an attractive therapeutic target for ECC cells with activating KRAS mutation.

Well-Defined Multivalent Ligands for Hepatocytes Targeting via Asialoglycoprotein Receptor.

Targeted delivery of therapeutic agents to hepatocytes is a particularly attractive strategy for the treatment of hepatocellular carcinoma and other liver diseases. The asialoglycoprotein receptor (ASGP-R) is abundantly expressed on hepatocytes and minimally found on extra-hepatic cells, making it an ideal entry gateway for hepatocyte-targeted therapy. Numerous multivalent ligands have been developed to target ASGP-R, among which well-defined multivalent ligands display especially high binding affinity to the receptor. Recently, several gene delivery systems based on such ligands for ASGP-R showed encouraging clinical results, drawing increasing interest in the scientific community and eventually promoting the improvement of current treatment for liver diseases. Here, we review ASGP-R targeting with a special emphasis on well-defined systems and properties such as the linker's length, hydrophilic-hydrophobic balance of the linker, and the spatial geometry of the scaffold. The present manuscript provides important guidelines for the design of multivalent ligands for ASGP-R.

Novel Silicone-Coated 125I Seeds for the Treatment of Extrahepatic Cholangiocarcinoma.

125I seeds coated with titanium are considered a safe and effective interstitial brachytherapy for tumors, while the cost of 125I seeds is a major problem for the patients implanting lots of seeds. The aim of this paper was to develop a novel silicone coating for 125I seeds with a lower cost. In order to show the radionuclide utilization ratio, the silicone was coated onto the seeds using the electro-spinning method and the radioactivity was evaluated, then the anti-tumor efficacy of silicone 125I seeds was compared with titanium 125I seeds. The seeds were divided into four groups: A (control), B (pure silicone), C (silicone 125I), D (titanium 125I) at 2 Gy or 4 Gy. Their anti-tumour activity and mechanism were assessed in vitro and in vivo using a human extrahepatic cholangiocarcinoma cell line FRH-0201 and tumor-bearing BALB/c nude mice. The silicone 125I seeds showed higher radioactivity; the rate of cell apoptosis in vitro and the histopathology in vivo demonstrated that the silicone 125I seeds shared similar anti-tumor efficacy with the titanium 125I seeds for the treatment of extrahepatic cholangiocarcinoma, while they have a much lower cost.