PROJECT SUMMARY/ABSTRACT Despite its overall decreasing occurrence, colorectal cancer (CRC) remains the fourth most common cause of cancer deaths in the US. Unfortunately, epidemiological studies demonstrate an alarming increase in inci- dence in populations below the age of 50, who are not routinely screened. Furthermore, CRC detection is difficult in high-risk groups, including those with a genetic predisposition (e.g. familial adenomatous polyposis), disease traits (e.g. inflammatory bowel disease), or from certain demographics (e.g. Black-Americans). Thus, there is a significant need for the development of innovative solutions for the early detection of CRC and the prevention of the transition from adenoma to CRC. To address this need, our interdisciplinary research team will develop genetically engineered bacteria using synthetic biology approaches to identify early CRC development, monitor and report changes in the adenoma and CRC microenvironment, and prevent cancer progression. To achieve the above objectives, engineered bacteria have to engraft and colonize the hostile luminal environment, sense and distinguish an abnormal environmental signal, compute this signal, and express a reporter or a therapeutic agent. However, appropriate vectors with these features remain lacking, constraining synthetic biology applica- tions for cancer research. Importantly, CRC is highly associated with E. coli, for which we have many synthetic biology tools. Furthermore, our preliminary proof-of-concept studies have revealed that native E. coli can be engineered to perpetually colonize fully conventional (i.e. non-microbiome depleted) hosts and to execute func- tions of interest, e.g., deconjugation of luminal bile acids. Deconjugated bile acid and resultant farnesoid X re- ceptor (FXR) agonism can suppress CRC development, indicating a potential therapeutic use of engineered native bacteria. Building on our strong supportive preliminary results, we will identify native E. coli from healthy, adenoma, and CRC tissues of a genetic model of CRC and engineer them to detect and treat CRC in response to the cancer microenvironment. Furthermore, we will characterize the effects of different tumor environment factors on the colonization and performances of engineered native E. coli in the colon organoid model in an organ-on-chip with the support of mathematical modeling, thereby identifying specific CRC signals for program- ming the responses of engineered native E. coli as CRC reporters and therapeutics. Finally, we will engineer native bacteria to detect and attenuate the progression of CRC by quantitatively reporting the level of CRC- related cysteine proteases and selectively inhibiting their activity. The research described in this proposal will generate new, much-needed synthetic biology vectors that can be developed as biosensors and therapeutics of adenoma and CRC, as well as many other diseases. Furthermore, this project will enrich our fundamental knowledge about the CRC-microbiome relationship and elucidate the roles of cysteine proteases in CRC pro- gression and treatment.

PROJECT NARRATIVE The proposed research is relevant to public health because it represents a novel approach of using engineered bacteria that can colonize the entire gut and express a desired function for long periods of time. The investigators will use this innovative strategy to engineer microorganisms that help prevent colorectal cancer formation. Thus, the proposed research is relevant to the part of NCI's mission to improve health by the development and accel- eration of the synthetic biology approaches to the understanding, diagnosis, and treatment of colorectal cancer in those who are particularly at risk.