PROJECT SUMMARY Immune checkpoint blockade (ICB) has yielded durable tumor regression and stabilized disease in 10-30% of patients for a range of solid and hematological malignancies. While its promising results have revolutionized cancer care, much work is needed to expand ICBs' benefit to a greater number of cancer patients. Various studies have highlighted the microbiota's impact on the innate and adaptive immunity and its potential role as a modifiable target to improve ICB response rates. Dysbiosis and decreased gut microbial diversity have been linked to poorer outcomes in patients receiving ICB. In two ongoing clinical trials, preliminary results of fecal microbiota transplantation of an ICB-responsive patient's microbiota into an ICB-non-responsive patient exhibit restored clinical response. Additionally, enrichment of specific bacteria has been identified in both mice and human that respond to checkpoint blockade. This unexpected link between the microbiome and cancer holds a promising opportunity to enhance cancer treatment by modifying the patient's microbiota. While there are a growing number of studies on microbiota-ICB interactions, mechanisms through which the microbiota modulates immune responses to cancer and cancer treatment remains unknown. To better understand the microbiota's contribution to immune activity and ICB treatment, I have established a gnotobiotic model of anti- PD-L1 treated melanoma. I have demonstrated and standardized methods to evaluate how a defined microbial community can inhibit B16 melanoma response to anti-PD-L1, and I have begun fractionating non-responder communities to identify and characterize effector species driving response failure. This grant aims to understand the robustness of ICB-microbiota interactions across different cancer types and tumor models, identify and characterize the first bacteria to drive non-response to anti-PD-L1 and explore potential mechanisms of non-response. Aim 1 – Based on preliminary data, I have selected two mice SPF microbiotas and two defined human microbiotas that exhibit contrasting tumor growth response rates to anti- PD-L1 when colonized into germfree, B16 melanoma-bearing mice. I aim to understand the robustness and generalizability of microbiome-immunotherapy findings across tumor types and ICBs by exploring tumor growth differences following checkpoint blockade therapy (anti-PD-L1, anti-PD-1, and anti-CTLA-4). Aim 2 – Based on previous lab findings that at baseline, germ free mice respond to anti-PDL1, we anticipate that there exists one or more effector species in each non-responder community that drives non-response to anti-PD-L1. To strategically elucidate the causative bacterial strains, I will fractionate each NR microbiota into orthogonal sub-communities, colonize germ-free mice, evaluate tumor growth trends, and analyze myeloid and T-cell populations in tumors and draining lymph nodes. By studying these gnotobiotic animals with different clinical respons...