Simple SummaryNovel therapeutic approaches combining oncolytic virus (OV) therapy and radiotherapy are being investigated to improve treatment for glioblastoma (GBM). However, following radiotherapy cellular senescence is induced in a portion of GBM cells and it is unknown how the radiation-induced senescent state may impact the therapeutic potential of OVs. The aim of this study was to evaluate oncolytic properties of a vaccinia virus (VACV) mutant, ∆F4LΔJ2R, as well as wild-type VACV in radiation-induced senescent GBM cells. We find that both viruses exhibited attenuated phenotypes towards GBM cells under irradiated senescence-enriched conditions relative to non-irradiated controls, suggesting senescence-associated antiviral activity and underscoring the important considerations for treatment strategies combining VACV-based OVs with radiotherapy.AbstractGlioblastoma (GBM) is a malignant brain cancer refractory to the current standard of care, prompting an extensive search for novel strategies to improve outcomes. One approach under investigation is oncolytic virus (OV) therapy in combination with radiotherapy. In addition to the direct cytocidal effects of radiotherapy, radiation induces cellular senescence in GBM cells. Senescent cells cease proliferation but remain viable and are implicated in promoting tumor progression. The interaction of viruses with senescent cells is nuanced; some viruses exploit the senescent state to their benefit, while others are hampered, indicating senescence-associated antiviral activity. It is unknown how radiation-induced cellular senescence may impact the oncolytic properties of OVs based on the vaccinia virus (VACV) that are used in combination with radiotherapy. To better understand this, we induced cellular senescence by treating GBM cells with radiation, and then evaluated the growth kinetics, infectivity, and cytotoxicity of an oncolytic VACV, ∆F4LΔJ2R, as well as wild-type VACV in irradiated senescence-enriched and non-irradiated human GBM cell lines. Our results show that both viruses display attenuated oncolytic activities in irradiated senescence-enriched GBM cell populations compared to non-irradiated controls. These findings indicate that radiation-induced cellular senescence is associated with antiviral activity and highlight important considerations for the combination of VACV-based oncolytic therapies with senescence-inducing agents such as radiotherapy.