PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is one of the deadliest forms of cancer in the United States and other industrialized nations. It is clear becoming evident that the initiation and development of colonic tumors can be influenced by the composition of the microorganisms that colonize the gastrointestinal tract (GI microbiota). The composition of the GI microbiota has been linked to beneficial effects on the host, by protecting against cancer, as well as harmful influences where tumor growth is promoted. The composition and function of the microbiota, in turn, can also be altered by colonic tumors. Therefore, understanding how GI microorganisms interact with host tissue is essential to improve our mechanistic understanding of CRC. The extreme complexity of the mammalian GI microbiota, composed of many hundreds of species and subspecies, however, poses a significant challenge in identifying specific bacterial metabolic pathways that are important in host-microbe interactions. As an alternative, we propose to use an underutilized gnotobiotic mouse model to determine how tumor growth influences the composition and activity of the gut microbial community. The Altered Schaedler Flora (ASF) represents a gnotobiotic animal model where mice are colonized with only 8 known bacterial species. The ASF model was originally developed to standardize mouse colonies for use by the National Cancer Institute, but now represents a potentially powerful system to study bacteria-host interactions. We hypothesize that tumor growth leads to significant changes in the structure of the GI microbiota (spatial localization) as well as functional changes (gene expression) as the bacteria respond to the altered physiological activity of colonic tumors. Changes in the location of the ASF community will be measured by new in situ RNA hybridization technology. Also, global transcriptional analysis using deep DNA sequencing technology (RNA-seq) will be used to identify genes whose expression is altered in the diseased colon. In combination, these approaches will allow us to identify specific genes that are important for colonization of the tumor microenvironment, The results from these studies will also be directly applicable to understand host-microbe dynamics in more conventional mouse models, as well as humans do to the similarity in major taxonomic classes of bacteria. These studies will significantly improve our understanding of how tumor growth influences the gut microbiota and will also contribute to our long-term goal of developing new interventions for prevention of CRC. This effort is consistent with the mission of the NCI to support research and training with respect to the cause, prevention and treatment of cancer. This proposed research is also in line with the intent of the R03 funding mechanism as it will allow us to develop the ASF animal model for colorectal cancer studies. Translational outcomes of these efforts will include targeted alteration of the GI microbiome for health benefits, as well as identifying new biomarkers for the early detection of cancer.