Abstract
AIMS: Glioblastoma (GBM) is the most aggressive primary brain tumor, necessitating innovative therapeutic ap- proaches. Although oncolytic herpes simplex viruses (oHSV) are promising therapies, their efficacy as monotherapies is limited. In this context, modulating autophagy—a key cellular recycling process that plays a complex role in cancer, influencing survival, immune response, tumor progression, and the tumor microen- vironment (TME)—holds promise. In addition, anti-diabetic drug metformin induces autophagy and exhibits anti-tumoral properties. However, autophagy’s impact on virotherapy and immunotherapy remains unclear. This study investigates how autophagy modulation affects immunotherapy efficacy and the TME in GBM, focus- ing on oHSV and metformin. METHODS: We assessed the ability of different oHSVs, with their genome having the Beclin Binding Domain (BBD) deletion or not, to modulate autophagy, hence hijacking beclin – a key protein of the autophagy pathway – and putatively modulating autophagy. Western blot analysis evaluated autophagy markers (LC3, P62) in murine glioblastoma stem-like cells (mGSCs: 005, NF53) post-infection. Additionally, we evaluated the effect of metformin on mGSC proliferation, sphere formation, and survival. We also analyzed the impact of metformin on survival and im- mune recruitment in orthotopic mGSC models. RESULTS: All tested oHSVs (Δ68H-6, Δ68HR-6, G47Δ, MG18L) unexpectedly induced autophagy – regardless of the BBD status and were cytotoxic to mGSCs. Metformin reduced cell proliferation, suppressed sphere formation, and induced autophagy and AMPK activation in a dose- and time-dependent manner. In vivo, metformin improved survival in NF53-implanted mice, but flow cytometry showed no significant changes in GBM-infiltrating immune cells when used as monotherapy. CONCLUSION: Our findings demonstrate that oHSV and metformin can induce mGSC cell death and autophagy. This study also highlights the complexity of autophagy modulation and underlines the need to better understand how this intricate process is regulated in the context of oncolytic virotherapy. Future work will explore oHSV-metformin synergy to improve immunotherapy for GBM.