IMMU-67. On-Treatment Spatiotemporal Profiling of Glioblastoma During Oncolytic ImmunoTherapy

Abstract Glioblastoma (GBM) is the most common and deadliest primary brain tumor, with no effective therapies and a longstanding record of failed phase 3 clinical trials—often without mechanistic insight into why promising approaches fall short. To address this critical gap, we implemented a novel on-therapy, longitudinal, and multi-regional tissue sampling strategy in patients treated with rQNestin34.5v2, an engineered oncolytic biologic based on herpes simplex virus 1, to explore how tumor and immune ecosystems evolve during treatment. Using high-resolution spatial transcriptomics (Xenium) and targeted spatial proteomics (CyCIF), we profiled over 80 spatially and temporally distinct samples from six patients, capturing over half a million immune, stromal, and malignant cells. This unique dataset enabled unprecedented resolution of the dynamic interactions between tumor and immune compartments in situ over the course of therapy. Our preliminary analysis revealed discrete, virus-associated lymphoid-like structures that formed early after treatment initiation. These structures were enriched in T cells, B cells, and myeloid cells, and were associated with localized reductions in tumor cell density. Over time, we observed a shift from an initially inflamed microenvironment to a more immunosuppressive state. By the time of radiographic progression, these lymphoid structures had largely dissipated—underscoring the importance of on-treatment sampling in capturing transient, treatment-responsive immune architectures. While these findings are preliminary, they suggest a transient immune structure linked to early therapeutic response, whose collapse may contribute to treatment resistance. Our study illustrates how integrative, on-therapy spatial profiling can begin to reveal mechanisms of response and resistance in GBM, providing a framework for refining future immunotherapeutic strategies in brain tumors and other treatment-refractory cancers.