Bacterial biosensors for the prevention of gastrointestinal cancers - Project Summary One-third of all cancer related deaths world-wide result from gastrointestinal (GI) cancers, including colorectal (CRC) and pancreatic cancer (PC). Soberingly, 50,000 Americans die every year from CRC alone, despite the majority of these being curable if detected early. Current screening for CRC saves lives but is limited by cost, patient non-compliance, chance of serious complications and operator dependent sensitivity. There is no population screening for PC and so patients are often diagnosed late, at which point effective treatment options are very limited. It is essential to develop more convenient, affordable and effective techniques to diagnose and prevent GI carcinogenesis. The goal of this project is to develop whole-cell, living biosensors capable of detecting tumors within the gastrointestinal system and responding with a diagnostic output. In proof- of-concept work described here in our progress report, we developed a living bacterial biosensor that can detect engineered target DNA naturally released in situ from orthotopic tumors in the mouse colon. We also developed a DNA-sensing circuit to detect and report on arbitrary target DNA sequences, with the ability to distinguish single-base mutations, including important oncogenic variants such as KRAS G12D. This work will be published in Science, an indicator of the excitement and promise engendered by our fundamental research advance, as the first demonstration of rationally engineered bacteria capable of detecting and analyzing DNA of interest. We foresee the application of our system wherever DNA defines illness, sampling is challenging and diagnostic (and therapeutic) outputs are best delivered at the time and place of disease detection. In this project, we will improve upon this technology and progress towards clinical trials. In Aim 1, we will identify naturally competent, endogenous gut microbes. A. baylyi is an excellent DNA biosensor chassis for in vitro applications, but it is not an anaerobe, and we hypothesize sensing in the gut would benefit from using a native resident of the gut tumor microbiome. In Aim 2, we will improve DNA-sensing circuits for cancer mutations, by optimizing the genetic circuitry for the bacterial host, reducing background mutation rates, and adding multiplex detection of additional hotspot mutations to expand detection to common PC associated DNA variants. In Aim 3, we will deliver a diagnostic, urine-detectable output upon sensing tumor DNA or blood. In Aim 4, we will systematically characterize the performance of our newly optimised biosensors in the complex environment of the GI tract in vivo, using orthotopic mouse models of CRC and PC that recapitulate human disease. The result of this project will be whole-cell, living biosensors capable of detecting GI cancers in situ and responding with a diagnostic output. This will in the future provide valuable, non-invasive diagnostics to enable screening for PC, and that are far less onerous for the patient than colonoscopy to detect CRC. The motivation is to increase participation in screening, catch GI cancers in earlier stages, decrease healthcare costs and improve patient outcomes.
