Experimental Model of Biliary Tract Cancers: Subcutaneous Xenograft of Human Cell Lines in Immunodeficient Nude Mice.

The ectopic xenograft mouse model of cancer is a commonly employed tool for in vivo investigations, particularly for studying cell tumorigenicity and testing the efficacy and tolerability of systemic or local anti-cancer therapies. The model displays advantageous features with an easy-access to visualize and monitor tumor growth in real-time with a caliper. Although the tumor development occurs in an ectopic location, the histology of the tumor resembles that of human cancer upon pathological examination. This suggests that when human malignant cells are transplanted into immunocompromised mice, they can educate and attract murine cells from the surrounding environment to recapitulate a tumor structure. The experimental protocol for ectopic xenograft models is straightforward, making them reproducible, cost-effective, and conductive to shorter experimental durations. Here, we detail the utilization of ectopic xenograft models in studying biliary tract cancers (BTC), which involves subcutaneously grafting human BTC cell lines originating from different biliary tree locations onto immunocompromised nude mice.

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

Hepatitis B virus X protein enhances the development of liver fibrosis and the expression of genes associated with epithelial-mesenchymal transitions and tumor progenitor cells.

The hepatitis B virus X protein (HBx) has pleiotropic biological effects, which underlies its potential role in cell transformation. However, its involvement in hepatic fibrosis remains unclear. In this study, we wanted to clarify, in vivo, the role of HBx protein in the development of liver fibrosis. Mice transgenic for the full-length HBx (FL-HBx) were used. To create liver fibrosis, FL-HBx transgenic and control mice were chronically exposed to carbon tetrachloride (CCl4). Modulation of the expression of proteins involved in matrix remodeling, hepatic metabolism and epithelial-mesenchymal transition (EMT) were investigated. In transgenic mice, FL-HBx expression potentiates CCl4-induced liver fibrosis with increased expression of proteins involved in matrix remodeling (Collagen1a, α-Sma, PdgfR-ß, MMP-13). In FL-HBx transgenic mice, an increase in EMT was observed with a higher transcription of two inflammatory cytokines (TNF-α and TGF-ß) and a decrease of glutamine synthetase expression level. This was associated with a sustained cell cycle and hepatocyte polyploidy alteration consistent with p38 and ERK1/2 overactivation, increase of PLK1 transcription, accumulation of SQSTM1/p62 protein and increase expression of Beclin-1. This correlates with a higher expression of tumor progenitor cell markers (AFP, Ly6D and EpCam), indicating a higher risk of progression from fibrosis to hepatocellular carcinoma (HCC) in the presence of FL-HBx protein. In conclusion, our results show that FL-HBx protein enhances the development of liver fibrosis and contributes to the progression of liver disease from chronic hepatitis to HCC.

Alteration of splicing factors' expression during liver disease progression: impact on hepatocellular carcinoma outcome.

PURPOSE: Trans-acting splicing factors (SF) shape the eukaryotic transcriptome by regulating alternative splicing (AS). This process is recurrently modulated in liver cancer suggesting its direct contribution to the course of liver disease. The aim of our study was to investigate the relationship between the regulation of SFs expression and liver damage. METHODS: The expression profile of 10 liver-specific SF and the AS events of 7 genes associated with liver disorders was assessed by western-blotting in 6 murine models representing different stages of liver damage, from inflammation to hepatocellular carcinoma (HCC). Relevant SFs (PSF, SRSF3, and SRSF6) and target genes (INSR, SRSF3, and SLK) modulated in mice were investigated in a cohort of 179 HCC patients. RESULTS: Each murine model of liver disease was characterized by a unique SF expression profile. Changes in the SF profile did not affect AS events of the selected genes despite the presence of corresponding splicing sites. In human HCC expression of SFs, including the tumor-suppressor SRSF3, and AS regulation of genes studied were frequently upregulated in tumor versus non-tumor tissues. Risk of tumor recurrence positively correlated with AS isoform of the INSR gene. In contrast, increased levels of SFs expression correlated with an extended overall survival of patients. CONCLUSIONS: Dysregulation of SF expression is an early event occurring during liver injury and not just at the stage of HCC. Besides impacting on AS regulation, overexpression of SF may contribute to preserving hepatocyte homeostasis during liver pathogenesis.

Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.

Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-α-stimulated signaling pathways and limits the extent of liver inflammation.

Hepatitis B splicing-regulated protein (HBSP) of the hepatitis B virus (HBV) was uncovered a few years ago but its function remains unknown. HBSP expression occurs from a spliced viral transcript that increases during the course of liver disease. This study aimed at characterizing the impact of HBSP on cellular signaling pathways in vitro and on liver pathogenesis in transgenic (Tg) mice. By RT-qPCR array, NF-κB-inducible genes appeared modulated in HepG2 cells transduced with a HBSP-encoding lentivirus. Using luciferase and Western blot assays, we observed a decreased activation of the NF-κB pathway in HBSP-expressing cells following TNF-α treatment, as illustrated by lower levels of phosphorylated IκB-α. Meanwhile, the level of phosphorylated JNK increased together with the sensitivity to apoptosis. The contrasting effects on JNK and IκB-α activation upon TNF-α stimulation matched with a modulated maturation of TGF-ß-activated kinase 1 (TAK1) kinase, assessed by 2-dimensional SDS-PAGE. Inhibition of the NF-κB pathway by HBSP was confirmed in the liver of HBSP Tg mice and associated with a significant decrease of chemically induced chronic liver inflammation, as assessed by immunohistochemistry. In conclusion, HBSP contributes to limit hepatic inflammation during chronic liver disease and may favor HBV persistence by evading immune response.

Regulation of interleukin-6 in head and neck squamous cell carcinoma is related to papillomavirus infection.

The prevalence of head and neck squamous cell carcinoma (HNSCC) related to human papillomavirus (HPV) is increasing, unlike tobacco- and alcohol-associated cancers. To gain a clearer understanding of the molecular mechanisms implicated in HNSCC, depending on the presence or not of a viral sequence, we investigated the expression of proteins detected in the tumor regions of HNSCC patients. Twenty-two untreated HNSCC patients were selected according to the presence of HPV-16. For six patients, tumor and controlateral healthy tissues were tested for viral detection before quantitative proteomic analysis. After confirmation by Western blot, proteins were connected into a network, leading to investigate interleukin-6 (IL-6) by immunocytochemistry and ELISA. 41 ± 5% of proteins quantified by proteomics were differentially expressed in tumor compared with healthy regions. Among them, 36 proteins were retained as modulated in HPV-16 positive or negative tumors, including cytokeratins, tubulins, annexin A1, and serpin B1. Network analysis suggested a central role of IL-6, confirmed by overexpression of IL-6 in tumor tissues as in sera of HPV-negative HNSCC compared with HPV-16-positive tumors. This modulation may contribute to the survival and proliferation of cancer cells, although it was not related to tumor stage or to the level of HPV-16 DNA.

Detection and genotyping of human papillomavirus by real-time PCR assay.

BACKGROUND: Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients. OBJECTIVE: Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples. STUDY DESIGN: Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity. RESULTS: Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10(3)cells using in-house HPV (6, 11 and 16) viral load assays. CONCLUSION: The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.