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Abstract Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-β leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.

β-catenin signaling is a major oncogenic pathway in hepatocellular carcinoma (HCC). Since β-catenin phosphorylation by glycogen synthase kinase 3β (GSK3β) and casein kinase 1ε (CK1ε) results in its degradation, mutations affecting these phosphorylation sites cause β-catenin stabilization. However, the relevance of missense mutations in non-phosphorylation sites in exon 3 remains unclear. The current study explores significance of such mutations in addition to addressing the clinical and biological implications of β-catenin activation in human HCC.Gene alteration in exon3 of CTNNB1, gene expression of β-catenin targets such as glutamate synthetase (GS), axin2, lect2 and regucalcin (RGN), and protein expression of β-catenin were examined in 125 human HCC tissues.Sixteen patients (12.8%) showed conventional missense mutations affecting codons 33, 37, 41, and 45. Fifteen additional patients (12.0%) had other missense mutations in codon 32, 34, and 35. Induction of exon3 mutation caused described β-catenin target gene upregulation in HCC cell line. Interestingly, conventional and non-phosphorylation site mutations were equally associated with upregulation of β-catenin target genes. Nuclear localization of β-catenin was associated with poor overall survival (p = 0.0461). Of these patients with nuclear β-catenin localization, loss of described β-catenin target gene upregulation showed significant poorer overall survival than others (p = 0.0001).This study suggests that both conventional and other missense mutations in exon 3 of CTNNB1 lead to β-catenin activation in human HCC. Additionally, the mechanism of nuclear β-catenin localization without upregulation of described β-catenin target genes might be of clinical importance depending on distinct mechanism.

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p