PROJECT SUMMARY Cancer cells undergo metabolic reprogramming in order to meet elevated energy requirements to fuel proliferation, thus resulting in their differential utilization of many essential metabolites compared to normal cells. Recent advancements in the field of cancer metabolic reprogramming demonstrated significant increase in efficiency of standard cancer treatments when combined with cancer metabolic inhibitors. However, tumor metabolic reprogramming remains poorly understood for the majority of cancers. Moreover, many recent reports revealed evidence that the metabolism of cancer cells in vitro can differ significantly from that of in vivo because in vitro models lack complexity of the tumor microenvironment. However, the progress of studying tumor metabolism in vivo is significantly hampered by the lack of efficient tools that allow real-time noninvasive imaging and quantification of metabolite absorption in animal models of cancer which closely reflect human pathologies. Current strategies have significant limitations and mostly rely on MRI, nuclear imaging techniques such as PET/SPECT, and endpoint ex vivo quantification of metabolite absorption (ex. MS). Here, we propose to develop a novel optical imaging platform that has several important advantages over the existing methods, and allows noninvasive evaluation of the uptake of several essential metabolites using highly sensitive and quantifiable bioluminescent imaging. The method is independent of radioactive and/or short-lived isotopes, less costly, and allows longitudinal monitoring of metabolite absorption during disease progression (e.g., cancer development or clinical intervention such as chemotherapy). While the first application of this approach has been already successfully validated by us using glucose as an example (Maric et.al., Nat Methods, 2019), we propose to expand this technology to develop novel probes to study uptake of several amino acids, fatty acids, and nucleosides that all play central role in cancer metabolic reprogramming. We will perform thorough validation of this platform in cells, healthy transgenic mice and murine animal cancer models to assure that the reagents fulfill the requirements for physiological behavior, stability, safety, and robust signal generation both in vitro and in vivo. In addition, we will optimize in vivo delivery routes, vehicles, and concentrations to achieve high signal/background ratios. In summary, the overall goal of this study is to generate a novel optical imaging platform that would become a universal analytical tool for monitoring nutrient uptake in live cells and animal models of disease. While we plan to apply this platform to unravel tumor metabolic reprogramming, the same method could be adapted for studies of several other important human pathologies, in which changes in metabolism are known to play a significant role, such as diabetes, neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), and many others. Therefore, this novel technology is expected to have a strong, enabling, and long-lasting impact on many physiological and pathological investigations in the field of metabolism and will become a valuable tool for drug discovery, applicable to oncology and other metabolic disorders.

PROJECT NARRATIVE Cancer metabolic reprogramming plays an essential role in tumor development resulting in differential utilization of many essential metabolites by cancer cells compared to normal cells and has been recently recognized as a promising therapeutic target for the improved cancer treatments. However, cancer metabolic reprogramming is poorly understood due to the lack of tools that can quantify nutrient uptake in relevant animal models of human cancers. Here, we propose to develop a novel optical imaging platform that would become a universal analytical method for monitoring nutrient uptake in tumors and would help to understand metabolic vulnerabilities of many different cancers with the overall goal to generate novel, effective treatments.