ID #831 Intravenous delivery of oncolytic virotherapy reshapes the tumour immune microenvironment in paediatric-type diffuse high-grade glioma mouse models

Abstract

Most paediatric-type diffuse high-grade glioma (PDHGG) subgroups have a cold tumour immune microenvironment (TIME), with few infiltrating lymphocytes, in part contributing to their poor clinical outcomes, especially histone-mutant diffuse midline (DMG-H3K27) and hemispheric glioma (DHG-H3G34). One promising avenue which may ‘heat up’ the TIME and provide novel therapeutic options is the use of oncolytic viruses (OVs), which in addition to directly lysing tumour cells, have been shown to increase immune infiltration in early phase trials. Most brain tumour studies to-date have utilised intra-tumoural injection of virus, whereas systemic delivery would reduce surgical risks, improve treatment for disseminated disease and allow repeat dosing. We sought to determine whether Reovirus, a naturally-occurring OV which crosses the blood-brain barrier, was able to elicit immunogenic changes in PDHGG mouse models via intravenous delivery. Using KPD (DMG-H3K27) and GPAD (DHG-H3G34) syngeneic models in an immunocompetent background, we confirmed a baseline low degree of tumour immunogenicity by a multimodal combination of assays including flow cytometry, immunohistochemistry, scRNA-seq and spatial transcriptomics/proteomics. Confirming sensitivity of cells derived from these tumours to reovirus in vitro, we subjected tumour-bearing mice to three 1x10^8 PFU doses intravenously over five days. In KPD, we observed a significant increase in CD8+ T cells in reovirus versus controls seven days post-treatment by flow cytometry (p = 0.0151), including proliferating effector Ki67+/PD1+/CD8+ T cells. Concurrently, we observed a reduction of both exhausted TIM3+/LAG3+/CD8+ T cells and FoxP3+/CD4+ immunosuppressive Tregs. Additionally, Reovirus increased myeloid cell populations, with neutrophils (LY6G+) and inflammatory monocytes (F4/80-/LY6C+) elevated seven days post-treatment, and dendritic cells (CD11c+) increased at day fourteen. Ongoing work is exploring the spatial organisation of our observed immune response. Intravenous delivery of reovirus therefore reshapes the relatively cold TIME in the DMG-H3K27 model, with potential to improve responsiveness to other immunotherapies and provides a clinically relevant platform for combinatorial studies.