Glioblastoma (GBM) is the most aggressive brain tumor with currently no cure. While immunotherapies have shown promising results in treating other cancers, GBM has proven resistant, with limited impact on overall survival. Oncolytic viruses (OVs) are being explored as potential therapeutic strategies for GBM. However, understanding the complex interplay between OV infection and replication kinetics, and the antiviral immune response, represents a significant challenge. Murine models that closely recapitulate the type I interferon (IFN) signalling of human GBM are required to accurately predict therapeutic outcomes. However, type I IFN signalling-mediated antiviral response appears to be more robust in murine GBM tumor cells compared to human cells. This master thesis aims to engineer a murine GBM cell line and employ a pharmacological approach to attenuate type I IFN signalling, thereby more closely mimicking the antiviral response of human GBM cells.
We employed CRISPR-Cas9 technology to generate a partial knock-out (KO) of the type I interferon receptor 1 gene (Ifnar1) in the SB28-GD2 cell line and investigated the impact on cell proliferation and cell surface immune marker expression profiles in vitro. Additionally, we evaluated in vivo tumorigenesis of the Ifnar1 KO cell line in immunocompetent mice. We also explored a pharmacological approach using ruxolitinib, a Janus Kinase (JAK)1/2 inhibitor, to inhibit type I IFN signalling and investigated its influence on OV-induced cell death in vitro. Furthermore, we characterised type I IFN signalling inhibition kinetics at the messenger RNA (mRNA) and protein level.
We demonstrated the in vitro activity and functionality of type I IFN signalling in SB28-GD2 cells, a crucial feature for a suitable cell line to explore OV replication kinetics. Our results indicate that partial Ifnar1 KO is sufficient to attenuate IFNAR1 expression. Additionally, we revealed a decreased cell proliferation rate in vitro in the engineered cells, and in vivo implantation of these cells resulted in prolonged mouse survival. Finally, ruxolitinib-mediated inhibition of type I IFN signalling accelerated OV-induced cell death in vitro, consistent with enhanced OV replication.
Altogether, these data contribute to further development of a murine GBM model that aims to optimise OV therapeutic approaches. In parallel, our data highlights the importance of considering the influence of IFNAR1 axis on tumor growth in vivo. Given the major changes in tumorigenicity in vivo induced by Ifnar1 editing, further investigations of IFNAR1 signalling should be conducted in human GBM.
