Project Summary/Abstract Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and has a dismal prognosis. Tumor heterogeneity and immune suppression (via the PD-1/PDL-1 mechanism) are likely reasons why GBMs almost always recur after treatment. Neo-adjuvant anti-PD-1 immunotherapy has recently been shown to have a modest benefit. Radiation therapy promotes a pro-inflammatory tumor microenvironment and can work synergistically with immunotherapy. Therefore, we propose an early phase clinical trial to compare the anti-tumor immune response before and after the novel combination of neoadjuvant anti-PD-1 immunotherapy and stereotactic radiation (SRT). Ten adult subjects with pathologically confirmed WHO Grade IV, recurrent glioblastoma multiforme that are planning to undergo surgical resection will be recruited. All subjects will have pre-treatment biopsy tissue available. Patients will be administered a single dose of anti-PD-1 immunotherapy followed by SRT followed by surgical resection and then continue on anti-PD-1 until disease progression. In our first aim, we propose to measure the changes in the T cell response against GBM following treatment. We hypothesize that the pre-surgical administration of anti-PD-1 combined with SRT will lead to an increase in the anti-tumor immune response. The primary immune biomarkers will be T cell receptor clonality and CD8+ T cell activation. We will perform single-cell RNA sequencing to study the changes in the T cell and macrophage populations after treatment and interrogate their functional phenotypes. In our second aim, we will assess the changes in intra-tumoral heterogeneity following treatment and identify genomic correlates of immune resistance. Our hypothesis is that treatment will reduce the number of distinct GBM clones as assessed by single cell RNA sequencing. We will also compare the expression profiles of cancer pathways between immune responders versus non-responders using differential enrichment analysis. This will allow us to identify the little-known genomic correlates of anti-PD-1 immune resistance in GBM. Knowledge of these correlates can assist in better selection of GBM patients that are likely to benefit from PD-1 blockade as well as inform us on future combinatorial treatment strategies. Successful completion of our specific aims will deepen our understanding of the changes in the immune response and GBM heterogeneity. Encouraging findings from our study would lead us to pursue a phase II RCT of neoadjuvant anti-PD-1 immunotherapy plus SRT versus best available standard of care.