ABSTRACT Recently, our lab developed a novel immune therapeutic approach delivering TT as a neoantigen surrogate selectively into tumors by Listeria infection in vivo, which attracts pre-existing TT-specific memory T cells (generated earlier in life during childhood vaccinations) now killing the highly immunogenic tumor cells. This has been published last year in Science Transl Med. Our ultimate goal is to test this promising therapy in patients with advanced pancreatic cancer. Although Listeria has been tested for 15 years in cancer patients this was always through intravenous (iv) injections. In previous studies we found that Listeria needs to be injected intraperitoneally (ip), in order to reach the tumor (Listeria is immediately eliminated in the blood through immune clearance). However, ip injections of Listeria has never been done before in patients. Therefore, we developed a tool that visualizes Listeria in vivo by positron emission tomography (PET) scan, i.e. we uploaded Listeria-TT with 18F-fluorodeoxyglucose (FDG). FDG is a glucose analogue, transported into the cell through glucose transporters (GLUTs), and then phosphorylated by hexokinases (HXKs) to FDG-6-phosphate, which become stored in the cell, resulting in a positive FDG signal measurable by PETscan. This methodology is normally used to visualize tumors in vivo. However, here we don’t want FDG signal in tumors but in the Listeria, to monitor Listeria in vivo. It is known that Listeria transports glucose through via PEP (phosphoenolpyruvate) and PTS (phosphotransferase system). Starvation of Listeria-TT bacteria in saline, followed by incubation of the Listeria- TT with FDG (up to 5 mCi) in vitro, and a washing step to eliminate the free FDG (supernatant), resulted in highly radioactive Listeria-TT without killing the Listeria bacteria. More importantly, Listeria-TT-FDG was visible in tumors and metastases by PET/CT without background in normal tissues within 1hr after ip injection. Our results suggest that further improvement is possible (higher doses of FDG, different incubation times, and monitoring). This allows us to monitor the distribution of Listeria in vivo which will answer several questions such as whether Listeria accumulates in tumors and metastases only (like in mice), at what time points do we see the highest number of Listeria in correlation with tumor size, what is the sensitivity of the methodology. In preclinical studies we found that Listeria is only visible in tumors (immune suppressive, allowing the Listeria to survive) and metastases and not normal tissues (lacks immune suppression, Listeria will be immediately eliminated), raising the question can we use Listeria-TT-FDG to determine the success of therapy or diagnosis? Thus, the main goal of this proposal is to finetune the Listeria-TT-FDG for human application. To reach our goal the spec aims are as follow. Aim 1: Characterizing Listeria-TT-FDG in vitro. Aim 2: Testing Listeria-TT-FDG in different mouse m...