TMIC-70. Integrative spatiotemporal proteomic and metabolomic characterization of immune infiltration following intracranial injection of oncolytic immunotherapy in glioblastoma patients

Abstract Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor in adults, characterized by rapid proliferation, an immunologically inert microenvironment, diffuse tumor cell infiltration and therapy resistance. Progress in developing effective glioblastoma treatments is impeded by a lack of mechanistic insight into how GBM cells and the surrounding microenvironment respond to therapeutic interventions. To address this prominent gap, we developed an innovative on-therapy, longitudinal, and multi-regional tissue sampling approach in patients receiving rQNestin34.5v.2, an engineered oncolytic biologic, based on herpes simplex virus 1, to investigate the dynamic evolution of tumor and immune ecosystems during treatment. Using targeted spatial proteomics (CyCIF) alongside spatial metabolomics (MALDI-MSI), we analyzed over 100 samples from six patients, profiling immune, stromal, and malignant cells across both space and time. Our CyCIF data revealed that T and B cells infiltrate and colocalize within rQnestin34.5v.2–positive regions, forming clusters that reflect active immune interactions. These immune-rich zones were inversely correlated with SOX2⁺ and OLIG2⁺ tumor cells. Notably, patients with longer survival showed a progressive increase in CD3⁺ T cells, driven by expanding cytotoxic T cell populations alongside a reduction in CD4⁺ T cells. Despite increased immune infiltration, post-trial tissues displayed an immunosuppressive environment characterized by few cytotoxic T cells and reduced tumor cells. Integrating spatial proteomics and metabolomics, we observed elevated purine metabolites—including ATP, GTP, and xanthine—in regions of heightened immune infiltration. Extracellular ATP and GTP likely facilitate T cell activation and migration through purinergic signaling and GTPase pathways. These metabolic patterns suggest that purine metabolism plays a key role in shaping immunologically active tumor microenvironments following treatment. Together, these findings provide novel mechanistic insights into the evolving tumor-immune landscape during rQnestin34.5v.2 therapy in glioblastoma, highlighting purine metabolism as a potential driver of immune activation within the tumor microenvironment. This integrated spatial approach offers a powerful framework to guide the development of more effective immunotherapeutic strategies for this challenging malignancy.