Teresa W. Fan

In Vivo and In Vitro Metabolomic Analysis of Anaerobic Rice Coleoptiles Revealed Unexpected Pathways

The ability of the rice (Oryza sativa L.) seedling to tolerate extended hypoxia during submergence is largely attributed to the biochemical adaptation of its coleoptile. Rice coleoptiles are capable of sustaining ATP production and cytoplasmic pH, unlike flood-sensitive organs, such as maize shoots. Fermentation reactions leading to the production of ethanol, alanine, succinate, and γ-aminobutyrate (GAB) are active in both types of tissues and thus may not account for the difference in tolerance. We have shown previously that rice coleoptiles undergo nitrate reduction and metabolism, which is efficient in alleviating cytoplasmic acidosis and regenerating NAD. Here, we employed 13C-2-acetate tracer methods with in vivo 13C NMR measurement, including in vivo isotopomer analysis, to probe the tricarboxylic acid (TCA) cycle and interacting pathways in rice coleoptiles during anaerobiosis. We found that the TCA cycle underwent multiple turns based on the metabolic scrambling of 13C label patterns in glutamine and malate. The in vivo kinetics of the 13C label incorporation into glutamic acid, glutamine, and GAB supports a separate pool of glutamate that was derived from the glutamate dehydrogenase reaction and subsequently decarboxylated to yield GAB. Both reactions consume additional H+ and/or NADH. Moreover, the higher rate of 13C enrichment at C-3 than C-2 of malate suggests the contribution of the glyoxylate cycle to malate synthesis, which could replenish the TCA cycle carbons diverted to GAB, glutamate, and glutamine synthesis. All of the above reactions contribute to the maintenance of glycolysis for energy production.

Abstract 2501: Assessing metabolic activity of patient-derived models of non-small cell lung cancer using stable isotope resolved metabolomics (SIRM)

Stable Isotope-Resolved Metabolomics (SIRM) has been a useful tool in interrogating metabolic reprogramming in cancer. We have recently published several protocols using SIRM in patient-derived models of non-small cell lung cancer (NSCLC)1. In this case study, we compared central metabolic activity in three different model systems, all derived from the same patient. Slices from the human tumor (Warburg slices 2,3), slices from an NSG mouse bearing the human tumor 4, and the patient-derived cell culture were incubated in [U-13C]-glucose or [U-13C,15N]-glutamine for 24 hours. Samples were then harvested and analyzed by gas-chromatography-mass spectrometry and NMR. All three models displayed similar levels of fractional enrichment after 24 hours, making metabolic comparison possible, despite their very different 3D architectures. Glucose metabolism in particular, showed consistent labeling patterns of metabolites in glycolysis and the Krebs cycle. Glutamine metabolism, however, was distinct in the cell culture, with a pattern that indicated upregulation of anaplerotic glutaminolysis. Activation of glutaminolysis may be an important transformative step in the establishment of a viable cell culture. We are also in the process of validating the present finding by comparing multiple patient-derived models of NSCLC and tracking the evolution of their metabolic activity. 1 Lane, A. N., Higashi, R. M. & Fan, T. W.-M. Preclinical models for interrogating drug action in human cancers using Stable Isotope Resolved Metabolomics (SIRM) Metabolomics 12, 1-15 (2016). 2 Fan, T. W.-M. et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small cell lung cancer study. CSH Molec. Case Studies J. 2, a00083, (2016). 3 Fan, T. W.-M., Lane, A. N. & Higashi, R. M. Stable Isotope Resolved Metabolomics Studies in ex vivo Tissue Slices. Bio-protocol 6, e1730 (2016). 4 Lane, A. N., Yan, J. & Fan, T. W.-M. 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts. Bio-protocol 5, e1650 (2015). Citation Format: Connor J. Kinslow, Jinlian Tan, Yihua Cai, Jun Yan, Richard M. Higashi, Andrew N. Lane, Teresa W. Fan. Assessing metabolic activity of patient-derived models of non-small cell lung cancer using stable isotope resolved metabolomics (SIRM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2017-2501

Abstract SY02-02: Exploring the lung cancer metabolome, in vivo and ex vivo, for individualized medicine

2502: Liquid diet introduction of tracers into mice for stable isotope-resolved metabolomics (SIRM) investigations