**3. Factors Affecting OV Therapy in GBM**

Several oncolytic virotherapy clinical trials have shown impressive and durable responses in a subset of patients, indicating that OVs might be a very promising therapeutic tool for treating GBM. The establishment of an efficient viral infection, lysis of tumor cells, viral spreading and anti-tumor immune activation, all depends on multiple factors (Figure 1). It is therefore conceivable that we may improve OV efficacy if we take these factors into account when selecting patients for treatment.

**Figure 1.** OV restriction mechanisms of GBM tumors. Infiltration of NK cells and tumor-associated macrophages (TAMs) at the tumor site, activation status of autophagy, expression of viral entry molecules and viral sensors (e.g., cGAS-STING) that lead to constitutive active type I interferon pathways, all could hamper the OV replication and oncolysis. Furthermore, cathepsin B expression and expression of specific proteins that drive specific tumor replication (e.g., nestin) could deter-**Figure 1.** OV restriction mechanisms of GBM tumors. Infiltration of NK cells and tumor-associated macrophages (TAMs) at the tumor site, activation status of autophagy, expression of viral entry molecules and viral sensors (e.g., cGAS-STING) that lead to constitutive active type I interferon pathways, all could hamper the OV replication and oncolysis. Furthermore, cathepsin B expression and expression of specific proteins that drive specific tumor replication (e.g., nestin) could determine the OV efficacy. Created with BioRender.com.

### mine the OV efficacy. Created with BioRender.com. *3.1. Viral Entry Molecule Expression*

*3.1. Viral Entry Molecule Expression* Tumor cell infection and oncolysis are a prerequisite for mounting an inflammatory response in the tumor microenvironment and ultimately generating an anti-tumor immune response. This depends on the cell entry possibilities for the virus. As GBM cells are not the natural host cells for entry of most viruses, low levels or even lack of receptor molecules on these cells can form the first obstacle to virotherapy. It has been shown that tremendous inter-tumoral variability exists in expression levels of specific adenovirus and reovirus entry molecules on patient-derived GBM cells [55,56]. As a result, various retargeting strategies have been applied to overcome such limitations, including EGFR and integrin retargeting of OVs [57]. Therefore, OV efficacy could potentially be enhanced by stratification of patients based on expression of specific viral receptor molecules in their tumors. Tumor cell infection and oncolysis are a prerequisite for mounting an inflammatory response in the tumor microenvironment and ultimately generating an anti-tumor immune response. This depends on the cell entry possibilities for the virus. As GBM cells are not the natural host cells for entry of most viruses, low levels or even lack of receptor molecules on these cells can form the first obstacle to virotherapy. It has been shown that tremendous inter-tumoral variability exists in expression levels of specific adenovirus and reovirus entry molecules on patient-derived GBM cells [55,56]. As a result, various retargeting strategies have been applied to overcome such limitations, including EGFR and integrin retargeting of OVs [57]. Therefore, OV efficacy could potentially be enhanced by stratification of patients based on expression of specific viral receptor molecules in their tumors.
