“Through-Stent-Enterography”: first experience with a novel technique intended to improve safety in endosonographic-guided gastroenterostomy (with video)

Background and Aims Endosonographic-guided gastroenterostomy (EUS-GE) is a novel technique to manage symptoms of gastric outlet obstruction. Major challenges are the high mobility of intestinal loops and the transient loss of endosonographic visibility during the puncture. This can lead to stent-misdeployment, which can be associated with potentially fatal adverse events. By injecting contrast medium through the guidewire-channel of the lumen apposing metal stent (LAMS) application system under fluoroscopic guidance, a positive enterogram can confirm the position of the stent inside the intestinal lumen before its deployment. The aim of this study was to describe this novel technique and to assess its feasibility. Methods The data of 39 consecutive patients undergoing EUS-GE with “through-stent-enterography” between July 2020 and March 2022 were retrospectively collected and analyzed. Primary endpoint was to assess the technical success. Secondary endpoints were to assess adverse events, rate of reinterventions and clinical success. Results Technical success was achieved in all cases (n=39). In two cases a second puncture was required to place the stent successfully. In one case, misdeployment could be avoided after a negative enterogram. In the other case, misdeployment occurred despite a positive enterogram a reintervention was needed. Clinical success was achieved in 92.3% (n=36). No major adverse events or mortalities were encountered. Conclusions “Through-stent-enterography” after the puncture to confirm the correct position of the stent in the small bowel is a novel and simple technique, which can potentially reduce the risk of misdepolyment of the stent

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Metabolic Activation of Intrahepatic CD8+ T Cells and NKT Cells Causes Nonalcoholic Steatohepatitis and Liver Cancer via Cross-Talk with Hepatocytes

SummaryHepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8+ T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice.

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment

Modeling human liver cancer heterogeneity: virally induced transgenic models and mouse genetic models of chronic liver inflammation.

In addition to being the most common primary liver cancer, hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death in humans. Treatment options are limited for this chemoresistant cancer, with liver transplantation and surgical intervention in early stages being the most successful treatments. Drug development over the past 15 years has focused on generating mouse models that mimic the human pathology for HCC. This has enabled the laboratory testing of potentially new human therapeutics. Described in this unit are the classification of HCC and an overview of hepatitis virus-related transgenic and genetically engineered mouse models (GEMMs) that are employed for elucidating the mechanism(s) responsible for the development of HCC, with particular emphasis on genetic, dietary, and environmental factors. © 2014 by John Wiley & Sons, Inc

Characterization of the function of the transactivating NF-κB subunit RelA/p65 in liver regeneration after partial hepatectomy in mice

Um die Rolle der transaktiviernen NF-κB Untereinheit RelA/p65 in der Leberregeneration nach partieller Hepatektomie (PHx) zu untersuchen, wurden zwei verschiedene konditionale Mausmodelle benützt. RelA/p65 wurde entweder Hepatozyten-spezifisch (RelaF/FAlbCre-), oder in Hepatozyten und Nicht-Parenchymzellen (RelaF/FMxCre-Mäuse) genetisch inaktiviert. Erstaunlicherweise zeigte sich sowohl in RelaF/FAlbCre- als auch in RelaF/FMxCre-Mäusen eine erfolgreiche Leberregeneration ohne vermehrte Leberschädigung nach PHx. In RelaF/FAlbCre-Mäusen wurde ein beschleunigter Zellzykluseintritt nach PHx beobachtet. Dies hatte jedoch keine signifikante Auswirkung auf die Regeneration der Lebermasse. Somit ist die transaktivierende NF-κB Untereinheit RelA/p65 nicht essentiell für eine erfolgreiche Leberregeneration nach PHx.Two different conditional mouse models were used in order to determine the role of the transactivating NF-κB subunit RelA/p65 in liver regeneration after partial hepatectomy (PHx). RelA/p65 was either genetically inactivated specifically in hepatocytes (RelaF/FAlbCre-) or in hepatocytes and in non-parenchymal cells (RelaF/FMxCre-mice). Surprisingly, both RelaF/FAlbCre- and RelaF/FMxCre-mice showed effective liver regeneration without increased liver damage after PHx. In RelaF/FAlbCre-mice an accelerated cell-cycle entry was found after PHx. However, this had no significant effect on liver mass regeneration. Thus, the transactivating NF-κB subunit RelA/p65 is not essential for successful liver regeneration after PHx

The direct and indirect roles of HBV in liver cancer: prospective markers for HCC screening and potential therapeutic targets.

Chronic hepatitis B virus (HBV) infection remains the number one risk factor for hepatocellular carcinoma (HCC), accounting for more than 600 000 deaths/year. Despite highly effective antiviral treatment options, chronic hepatitis B (CHB), subsequent end-stage liver disease and HCC development remain a major challenge worldwide. In CHB, liver damage is mainly caused by the influx of immune cells and destruction of infected hepatocytes, causing necro-inflammation. Treatment with nucleoside/nucleotide analogues can effectively suppress HBV replication in patients with CHB and thus decrease the risk for HCC development. Nevertheless, the risk of HCC in treated patients showing sufficient suppression of HBV DNA replication is significantly higher than in patients with inactive CHB, regardless of the presence of baseline liver cirrhosis, suggesting direct, long-lasting, predisposing effects of HBV. Direct oncogenic effects of HBV include integration in the host genome, leading to deletions, cis/trans-activation, translocations, the production of fusion transcripts and generalized genomic instability, as well as pleiotropic effects of viral transcripts (HBsAg and HBx). Analysis of these viral factors in active surveillance may allow early identification of high-risk patients, and their integration into a molecular classification of HCC subtypes might help in the development of novel therapeutic approaches

Oncogenic potential of hepatitis B virus encoded proteins.

Due to the limited treatment options hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death, and hepatitis B virus (HBV) infection is the major risk factor for development of HCC worldwide. HCC is typically preceded by chronic inflammation, but may also develop in the absence of liver disease on the basis of HBV infection and even when virus replication is controlled by antivirals. In this situation, HBV antigen expression persists and direct oncogenic effects of HBV are integration of the viral DNA into the host genome as well as direct effects of viral proteins. These factors have to be taken into account in order to personalize HCC surveillance in CHB and unravel novel therapeutic approaches