Role of Gut-Immune Interactions in Aging-Associated Bladder Cancer - ABSTRACT Bladder cancer (BCa) is predominantly a disease of aging with the risk of developing invasive disease peaking in the 8th decade of life. Elderly patients have higher rates of co-morbidities and are thus often not suitable candidates for standard of care surgery and/or chemotherapy or immunotherapy. Moreover, unlike other cancers such as breast and colon, BCa in the elderly is more aggressive and associated with poorer outcomes. Aging is also associated with both increased baseline levels of tissue inflammation and major differences in the gut microbiome. Aging-associated changes in the microbiome and inflammation have been linked to cancer incidence and progression, and poorer outcomes with immunotherapy and chemotherapy in multiple cancers. In spite of the numerous studies describing these changes, mechanistic studies attempting to understand how these changes impact BCa are comparatively absent. To investigate this phenomenon in a translational setting, we exposed young (8 weeks at start) and older mice (78 weeks at start) to the model BCa carcinogen N-Butyl- N-(4-hydroxybutyl) nitrosamine (BBN). We found older animals had increased baseline levels of inflammation and developed larger, more aggressive tumors after 16 weeks of BBN. Older animals also had more inflamed tumors as we found higher levels of recruited neutrophils and macrophages. Innate immune cells, particularly neutrophils, have been associated with a pro-tumorigenic phenotype in colon, liver, and other cancers and thus we proposed altered inflammation may be a cause of the larger tumors. As aging results in major changes in microbial diversity, we also evaluated 16S sequencing of the fecal microbiome to determine if these animals underwent similar changes. While the microbiomes were similar at the start of the study, we found major divergences between the groups over time, with significant differences in ß-diversity between young or old animals with cancer. This has led to the hypothesis that gut dysfunction drives neutrophil recruitment to enhance BCa formation in aged animals. In Specific Aim 1, I will assess gut dysfunction and microbial divergence over time in young and old animals. In Specific Aim 2, I will use fecal microbiota transplantation to determine if switching microbiota constituency between young and old animals during tumor formation can affect BCa tumorigenesis. In Specific Aim 3, I will use pharmacological and genetic inhibition of specific aspects of neutrophil biology to evaluate the role of neutrophils in BCa development in young and old mice. Completion of these aims will provide the first murine model for aging related increases in BCa. Moreover, this research sets the stage for future R01 studies aimed at detailed mechanistic studies understanding the signaling mechanisms that mediate these observations as well as studies using combination therapy with immunotherapy as a novel treatment for BCa. Dedicated time to perform these studies will serve as the critical preliminary data to propel the applicant, Dr. Ben Woolbright, towards an independent career.
