Project Summary There is a clear imperative to develop potent, cost effective therapeutics to confront the challenge cancer poses to society. Here we address this need by developing synthetically engineered cells effective against a broad range of cancer types with a special emphasis on colorectal cancer (CRC). This cancer type is the second most common cause of cancer death in the US, with more than 50,000 Americans dying every year. Recent research demonstrates the power of genetic engineering to make significant advances towards more efficacious cancer therapy. The introduction of genetically engineered cells, such as chimeric antigen receptor T (CAR T) cells, has shown great promise for treating many types of B cell malignancies, but unfortunately targeting CAR T cells to solid tumors remains challenging. In this project we will use the tools of synthetic biology to make new engineered therapies based on bacterial rather than mammalian cells. Certain bacterial species have demonstrated a useful ability to “home in” and selectively colonize solid tumors without infecting healthy tissue. This tumor targeting property will be exploited in the proposed work to deliver safe, effective therapies directly to the locations where they are needed most: the solid core of tumors. Previously we developed a bacterial therapeutic and tested it in an animal model of metastatic disease. In contrast to other approaches utilizing bacterial cells, this “lysis strain” does not require specialized genetic modifications for the secretion of encoded cargo, it simply releases it into the environment when the cells burst. Initially we will genetically modify the lysis strain to produce a wide range of therapeutics for testing, including toxins (from bacteria, animals and plants), enzymes, antibiotics, and apoptotic peptides. Next we will analyze the tumor microbiome from human samples since we hypothesize that the native bacterial population's composition will provide a unique signature (analogous to a fingerprint) that can be used to divide tumors into distinct subtypes. We expect to use these fingerprints to identify other species with superior suitability for therapeutic delivery in treating CRC. Once identified we will develop two in vitro assays for testing the candidate strains. We will use microfluidic technology to create a high throughput co-culturing system for bacteria and a cancer cell line. In parallel, we will develop a co-culturing system for bacteria and organoids that are generated from the same human tumor samples which had been previously used for strain identification and fingerprinting. Lastly we will test the most promising therapies in an animal model of colorectal cancer to determine efficacy in a pre- clinical model.