Isotope-Tracer Methods to Study Cancer by Mass Spectrometry-Based Metabolomics in Adult Zebrafish

The zebrafish offers attractive benefits for studying metabolism, as it possesses all of the organs that are needed for metabolic control in humans. Accordingly, zebrafish models have emerged for prevalent metabolic diseases such as obesity, diabetes, and cancer. The metabolism of adult zebrafish, however, has not been extensively studied by using metabolomics or isotope-tracer analysis. In part, this is because its small size complicates analysis of biofluids/tissues from individual animals. The work described herein contains two parts. First, liquid chromatography/mass spectrometry (LC/MS)-based metabolomic methods to maximize metabolite coverage when handling small amounts of biological material were optimized, and a workflow to perform isotope-tracer analysis in adult zebrafish is established. Second, having this platform in place, a zebrafish model of cancer was evaluated. Using animals bearing human oncogenic BRAFV600E-driven melanoma, it was determined that diseased animals exhibit an alanine cycle between tumor and liver. Specifically, tumors excrete glucose-derived alanine as a substrate for hepatic gluconeogenesis, which maintains glucose homeostasis in spite of pathological glucose consumption by tumors. In addition to alanine serving as a gluconeogenic substrate, the data suggest that it transfers the nitrogen burden of the tumor to the liver, leading to an increase in ammonia release. By using a BRAFV600E-specific inhibitor to suppress tumor metabolism, I observed rapid attenuation of the alanine cycling phenotype as well as pathological ammonia excretion. Interestingly, I determined that zebrafish tumors place liver-bound nitrogen onto alanine as a consequence of activated branched-chain amino acid catabolism, which I found to be conserved in melanoma patients. Pharmacologically inhibiting alanine production significantly reduced melanoma propensity to utilize branched-chain amino acids as fuel, resulting in ~50% tumor remission within 10 days of drug treatment. In sum, I establish the adult zebrafish as a valuable model for probing metabolic diseases and shed light on a potential novel therapeutic target for melanoma