Project Summary It is increasingly clear that bacteria play an important role in human health. While it is natural to focus on how intestinal bacteria affect disease, intriguing findings have elucidated the extent to which bacteria inhabit solid tumors. Microbes have been detected in lung, pancreatic, breast, oral, gallbladder, ovarian, liver, and colorectal cancers. Localization has been ascribed to several mechanisms, including preference for anaerobic or facultative anaerobic bacteria to grow in the hypoxic core of tumors, presence of bacterial nutrients, lack of immune surveil- lance, and leakiness of the often poorly structured vasculature surrounding neoplastic tissue. This tendency for localization to solid tumors suggests that bacteria could be engineered for precise and robust drug production and delivery from within the solid tumor environment. This dovetails with 20 years of progress in synthetic biology, which has tended to focus on microbial engineering. However, information on how the tumor microenvironment affects bacterial growth is largely unknown. The microenvironment will affect bacterial gene expression that ul- timately underlies the functionality of engineered therapies, and it is difficult to imagine a predictive framework for engineered bacterial therapies without a quantitative understanding of how bacteria react to the environment of a growing tumor. We will use a probiotic strain of E. coli with an established safety record to develop a novel class of biosensors to noninvasively investigate bacterial growth in the tumor microenvironment. Initially, we will develop lysis-based biosensors that respond to specific tumor environment targets: hypoxia, pH, glucose, and lactate (Aim 1). We will also engineer an inducible quorum sensing (QS) system that enables external control of bacterial population dynamics, including the ability to eliminate a specific strain whenever desired (Aim 1). Together these strains will allow us to modulate and monitor population dynamics in vivo, enabling both sens- ing of the local environment and maintenance of an external control switch. We will test these strains using an established in vitro organoid model (Aim 2) and in two clinically relevant animal models for solid tumor growth. Additionally, we will use our previously developed dynOMICS technology to screen tumor extract from the two animal models and construct a second suite of biosensors for monitoring the tumor environment (Aim 2). These biosensors will then be tested in the animal models. We will visualize bacterial populations in a colorectal tumor model with bacteria that are engineered to produce luciferase in order to monitor colony dynamics using our es- tablished methods (Aim 3). We will also build on recently reported technology whereby bacteria are modified for use with ultrasound through addition of gas vesicles that permit high resolution imaging of the engineered bac- teria. We will use the ultrasound method to investigate NASH-induced hepatocellular carcinoma (HCC) where a high-fat diet is used to induce HCC at 20 weeks in mice (Aim 4). This project will quantitatively characterize how bacterial strains sense, respond, and grow in the tumors. The results will establish a platform for future exploration of therapies that are produced and delivered by bacteria that grow within solid tumors.

Project Narrative We will develop a platform for bacterial synthetic biology in complex, heterogeneous environments, using tumors as a model. We will develop genetic tools that allow bacteria to sense, respond to, and compensate for the tumor environment, as well as experimental techniques to monitor their dynamics using in vitro organoid models and animal models of hepatocellular carcinoma and colorectal cancer. Given that some bacteria can naturally localize to tumors, this synthetic biology platform will set the stage for engineering bacterial cancer biosensors and therapies.