Human Microbiota Influence the Response to Anti-PD-1 in a Pre-clinical Model of Glioblastoma Kory J. Dees,1 Hyunmin Koo,2 J. Fraser Humphreys,3 Joseph A. Hakim,4 David K. Crossman,2 Michael R. Crowley,2 L. Burton Nabors,3 Etty N. Benveniste,1 Casey D. Morrow,1 and Braden C. McFarland1 1Department of Cell, Developmental and Integrative Biology, 2Department of Genetics, 3Department of Neurology, and 4School of Medicine, University of Alabama at Birmingham, Birmingham, AL The composition of the gut microbiome has been shown to determine responsiveness or resistance to immune checkpoint inhibitors (anti-PD-1) in patients with melanoma and other cancers. Unfortunately, although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has not demonstrated efficacy in humans. Most pre-clinical cancer studies have been done in mouse models using mouse gut microbiomes, but there are significant differences between mouse and human microbial gut compositions. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate five independent humanized mouse lines (HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. Interestingly, we found that the HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls, while HuM1, HuM4, and HuM5 mice are resistant to anti-PD-1. The question still remains of whether the “responsive” microbial communities in HuM2 and HuM3 can be therapeutically exploited and applicable in other tumor models, or if the “resistant” microbial communities in HuM1, HuM4, and HuM5 can be depleted and/or replaced. Future studies will assess responder microbial transplants as a method of enhancing immunotherapy.