Virotherapy of the Hepatocellular Carcinoma Characterization of Resistance to the Recombinant Vaccinia Virus GLV-0b347 in Murine HCC Cell Lines with Distinct Oncogenic Mutations

Abstract

After recent progress in cancer treatments through the emergence of targeted- and immuno-therapies, hepatocellular cancer (HCC) remains one of the most ill-treatable cancers to date. Late onset symptoms result in diagnosis of HCC in late, progressive, and often disseminated stages. Therefore, even in developed countries with adequate medical resources, HCC has a high mortality rate and new treatment options are urgently needed. The selection of patients for individual and tumor-specific therapies will most likely play an important role in future treatment strategies, which may soon include oncolytic viruses. Oncolytic virotherapy is a promising new treatment option for patients with advanced and disseminated disease. Since the approval of Imlygic® (T-Vec) oncolytic virotherapy has entered clinical practice. Many vaccinia virus-based oncolytic viruses (OVs) are currently undergoing clinical trials. Other virotherapeutic agents based on a variety of viruses are also gaining ground. The aim of this thesis was to investigate the oncolytic effectiveness of the recombinant vaccinia virus GLV-0b347 on murine cell lines of hepatocellular carcinoma (mHCC). For this purpose, six mHCC cell lines were selected that carry specific oncogenic mutations similar to those found in human HCCs. The mutations included activating mutations in proto-oncogenes Akt, Myc and Nras, as well as knockouts of the tumor suppressor genes p53 and p19 Arf. The mHCC cell lines were infected with the recombinant vaccinia virus GLV-0b347. Phase contrast and fluorescence photos of the infected cell layers revealed successful infection and patterns of viral spread in all cell lines. To determine the efficacy of GLV-0b347-mediated oncolysis, the cell mass based SRB viability assay was performed. The results showed a heterogeneous response of the different cell lines, which were then classified into three different sensitivity groups. Subsequently, the oncolytic efficacy in the four most responsive cell lines was investigated using the real-time cell monitoring system xCELLigence RTCA. The continuous measurement confirmed the results of the cell mass-based viability assay. The quantification of viral replication in the four mHCC cell lines showed differences in viral replication efficiency between cell lines of the susceptible and moderately susceptible group. The differences were most evident when comparing the maximum titers within 96 hours post infection. Finally, the expression of a panel of antiviral response genes was analyzed by RT-qPCR to compare a susceptible to a moderately susceptible mHCC cell line. Differences in basal gene expression and gene induction in response to infection with GLV-0b347 were observed. While the susceptible cell line showed higher induction of crucial genes like IFN-β and IRF7, the moderately susceptible showed higher baseline expression of IFN-β, IRF7 and AIM2. The overall findings show that within one cancerous entity oncolytic efficacy of a virus can vary considerably. These differences are caused, among other factors, by the underlying oncogenic mutations of the tumor. Mutations of the innate immune response, especially of the interferon system, probably also play a decisive role. The complex background of oncolytic resistances requires further clarification. Future research should focus on identifying prognostically reliable markers and developing strategies to direct the most effective oncolytic virus to each individual patient.