P08.10.B EVOV VACCINE-LIKE PARTICLES RESTORE ANTI-TUMOR IMMUNITY AND SUPPRESS GLIOBLASTOMA PROGRESSION IN PRECLINICAL MODELS

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Abstract BACKGROUND Glioblastoma (GBM) remains a challenge for immunotherapy due to profound immune evasion and a highly suppressive tumor microenvironment. We developed EVOVs—extracellular vesicles derived from glioma stem-like cells (GSCs) infected with oncolytic HSV-1—serving as vaccine-like immune activators. We show that EVOVs reprogram immune-tumor interactions and restore anti-tumor immunity across integrated preclinical models. MATERIAL AND METHODS Patient-derived GSCs (n=6) were infected ex vivo with OV (MOI 0.1 or mock-treated), and EVOVs and EVs were collected after 12h. Proteomic profiling, nanoparticle tracking analysis, and electron microscopy assessed vesicle quality and cargo. Human PBMCs (n=7) were treated with EVs or EVOVs, followed by flow cytometry, cytokine assays, GSC spheroid co-cultures, and single-cell RNA sequencing (scRNA-seq). For co-cultures, multiplex immunofluorescence imaging was performed using the Rarecyte Orion™ platform. In vivo efficacy was evaluated in a humanized NSG mouse model (n=16) using flow cytometry and spatial transcriptomics (10x Genomics Visium). RESULTS EVOVs incorporated pathogen-associated molecular patterns from OV without carrying infectious particles. Proteomic profiling showed enrichment in immune-related pathways and enhanced signatures of NK, NKT, CD4⁺, and CD8⁺ T cells. Notably, EVOV stimulation induced a robust activation of PBMCs, significantly increasing the secretion of IFN-γ, IL-12p70, and IP-10 (p<0.05). In co-cultures, EVOV-primed PBMCs enhanced GSC killing, confirmed by Orion™ multiplex imaging showing increased infiltration of CD8⁺ GrB⁺ effector T cells and reduced PD-L1⁺ and CD11c⁺ cells. Single-cell RNA-seq revealed upregulated interferon signaling and MHC-I antigen presentation in GSCs, and activation of IFNα/γ programs in immune cells.In humanized NSG mice, EVOV treatment led to a significant reduction in exhausted PD1⁺ CD8⁺ T cells and a notable increase in activated CD4⁺ T cells in the peripheral blood. Importantly, 10x Genomics Visium analysis confirmed a significant immune infiltration into tumors and a remarkable decrease in PD1⁺ and LAG-3⁺ inhibitory markers. CONCLUSION EVOVs reshape the glioblastoma immune landscape, reversing tumor-induced suppression and restoring anti-tumor immunity. This strategy offers a powerful new avenue for immunotherapy in GBM and holds promise for extending its benefits to other therapy-resistant malignancies.

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