Measuring and manipulating metabolic fluxes in the tumor microenvironment Tumors have altered metabolism compared to normal tissues, which suggests that drugging metabolism could kill tumors while sparing healthy tissues. However, tumor metabolism has chiefly been measured in vitro, while recent studies have showed that tumor metabolism in the body is distinct from in vitro systems. Therefore, the field needs approaches to measure tumor metabolic fluxes in vivo. During my postdoctoral fellowship, I developed methods to measure glycolytic and tricarboxylic acid cycle (TCA) flux in vivo using kinetic infusion of isotope-labeled tracers. These approaches revealed that tumors have much lower TCA flux than healthy tissues (5 mouse tumor models examined). Though the tumors had higher glycolytic flux than healthy tissues, the total ATP production rate from glycolysis plus TCA cycle-driven oxidative phosphorylation was significantly lower in tumors than in healthy tissues. Moreover, feeding mice a high-fat ketogenic diet increased tumor TCA flux and slowed tumor growth synergistically when combined with chemotherapy. These findings raise two key questions. First, since tumors in vivo are a mix of cancer cells and other infiltrating cells, what is the metabolism of cancer cells versus immune cells or fibroblasts in tumors? Second, can directly upregulating tumor TCA flux slow tumor growth? I propose first to combine my glycolysis and TCA cycle measuring techniques with immunomagnetic and sorting strategies to measure fluxes in cancer cells, immune cells, and fibroblasts (Aim 1). I will apply this strategy to melanoma, a tumor type infiltrated by CD8 T cells which can help control the tumor, and to pancreatic adenocarcinoma, a tumor type where fibroblasts and myeloid cells can be even more abundant than cancer cells. Next, I will directly upregulate TCA flux in tumor cells by using genetic and pharmacologic approaches: overexpressing the NADH uncoupler protein mito- LbNOX, knockout of the TCA suppressor protein PDK, and inhibition of PDK with dichloroacetate. I will confirm that these strategies increase TCA flux using the method I developed and will test whether increased TCA flux slows tumor growth in primary and metastatic breast tumors. Successful completion of these aims will reveal the metabolism of different cell populations in the tumor microenvironment and will test TCA upregulation as a therapeutic strategy in cancer.