Tumor-specific metabolic alterations have been increasingly recognized as a driving force of tumor growth. Glutamine (Gln) is the most abundant amino acid in the blood. Glucose and Gln are two major nutrients utilized by cancers to meet the demand for energy production and biosynthesis. Gln is metabolized through the glutaminolysis pathway, which is highly active in many aggressive forms of human cancers, including triple-negative breast cancer (TNBC), pancreatic and lung cancer, lymphoma and glioblastoma. The first and rate-limiting step of glutaminolysis is catalyzed by mitochondrial glutaminase (GLS) that converts glutamine to glutamate. Drugs targeting key steps of glutaminolysis pathway are being developed, and promising inhibitors of GLS have advanced to early phase clinical trials. However, there are no current clinically feasible methods to estimate the level of cancer glutamine metabolism, nor is there a non-invasive marker that can report the pharmacodynamic (PD) effect of GLS inhibitors. These unmet needs provide strong motivation to develop imaging-based methods to measure cellular glutaminolysis in order to direct GLS-targeted therapy, the focus of this proposal. To interrogate in vivo glutamine metabolism in cancer, our Center has designed and developed two positron emission tomography (PET) imaging probes: L-[5-11C]Glutamine ([11C]Gln) and (2S,4R)-4[18F]Glutamine ([18F]Fluoroglutamine or [18F]4F-Gln ). This project aims to develop and validate quantitative analysis tools for [11C]Gln and [18F]4F-Gln PET using human breast cancer xenografts. Based on our preliminary studies, we have designed studies to test the central hypothesis that the distribution volume of [18F]4F-Gln is a marker of tumor glutamine pool that is an indirect measure of glutamine metabolism, while flux through GLS measured by [11C]Gln PET is an authentic marker of tumor GLS activity. The utility of these markers to report the pharmacodynamic effect of novel glutaminase inhibitor will be developed and validated using breast cancer xenograft models based on cell lines of different breast cancer subtype with varied degrees of dependence on glutamine. The outcome of the proposed project will fulfill the unmet clinical needs for quantitative, non-invasive tools capable of predicting and measuring response to glutamine-targeted therapies. These tools will be ready for clinical trials upon completion of this project to play a critical role in development and deployment of such therapies in patients.