Single cell analyses to uncover drivers of hepatitis B virus-induced hepatocellular carcinoma
Eloi R. Verrier1,2+, Tom Croonenborghs3,4+, Frank Jühling1,2+*, Laura Heydmann1,2, Charlotte Bach1,2, James Bochicchio3, Patrick Pessaux1,5, Catherine Schuster1,2, Mirjam B. Zeisel1,2, Nathalie Pochet3+, Thomas F. Baumert1,2,5+
1Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques; 2Université de Strasbourg, Strasbourg, France; 3Harvard Medical School, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, United States; 4KU Leuven Technology Campus Geel, AdvISe, Geel, Belgium, 5Pôle Hépato-Digestif, Institut Hospitalo-Universitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France
Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and hepatocellular carcinoma (HCC). To cope with the low level of infection in bulk samples, we analyzed differentially expressed host genes depending on HBV infection levels in cell culture and infected human HCC tissue in single cells. A state-of-the-art cell culture system overexpressing NTCP was infected with purified recombinant HBV. Genome-wide gene expression profiles as well as viral pregenomic RNA of individual cells were measured. A similar strategy was applied to study HBV infected HCC liver tissue. Quantifying viral pregenomic RNA within this single cell analyses allowed to distinguish uninfected from infected cells, and to determine individual infection levels. Correlating viral RNA with host gene expressions revealed candidate driver genes. Performing gene set enrichment analysis based on these correlations indicated various HBV-mediated dysregulated pathways, e.g., viral infection, metabolic, and cancer pathways. An important portion of pathways identified in cell culture could be verified in infected human liver tissue. This demonstrates that single cell analyses of HBV-mediated reprogrammed key host factors driving viral persistence, liver disease and cancerogenesis are a powerful tool to uncover novel targets for prevention and treatment of virus-induced HCC.