Abstract SY02-02: Exploring the lung cancer metabolome, in vivo and ex vivo, for individualized medicine
Authors: Fan Teresa W. , Sun Ramon , Warmoes Marc , Sun Qiushi , Song Huan , Mahan Angela , Martin Jeremiah , Higashi RICHARD M. , Lane Andrew N. 
Published: July 2017

Abstract
Lung cancer is a leading cause of cancer death worldwide. This disease is of serious concern in Kentucky, which leads the nation in both lung cancer incidence and mortality. The past decade of research in cancer metabolism reveals the untapped value of exploring human metabolome for the discovery of novel therapeutic and diagnostic biomarkers for human cancers and other diseases. To better understand metabolic reprogramming in individual tumors of lung cancer patients, we have developed stable isotope tracers (e.g., [13C6]-glucose) coupled with NMR and MS-based stable isotope-resolved metabolomics (SIRM) analysis directly in patient and in patient-derived ex vivo and in vivo models. In the in vivo patient study, we mapped differential metabolic network between paired cancerous (CA) and noncancerous (NC) tissues procured from non–small cell lung cancer (NSCLC) patients infused with [13C6]-glucose and snap-frozen immediately after resection (1). In particular, we uncovered elevated anaplerotic pyruvate carboxylase (PC) activity in CA versus paired NC tissues. Proliferating cancer cells require active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. Pyruvate carboxylation represents one of the two major anaplerotic pathways that replenish the Krebs cycle intermediates; the other involves glutaminolysis initiated by glutaminase (GLS). We also found that PC but not GLS protein was overexpressed (median 8-10 fold; n=86) in CA tissues relative to paired NC tissues and that PC expression was functionally important to NSCLC cell growth both in vitro and in vivo (1). We also utilize the “Warburg slice” concept to systematically define metabolic distinctions between thinly sliced paired CA versus NC lung tissues freshly resected from individual NSCLC patients and cultured in stable isotope tracers. This ex vivo tissue slice culture system is excellently suited for delineating reprogrammed metabolic pathways in CA tissues without systemic interferences (2-4). These lung tissue slices were metabolically viable for up to 72 hr, while maintaining their 3D architecture and microenvironment (4). We found that the reprogrammed metabolic network in the cultured human tissue slices recapitulated that in vivo (1). These advantages make the ex vivo human tissue slice systems a unique preclinical model for exploring human target tissue metabolism and how it underlies the response to anticancer agents such as chemopreventive Se compounds, enzyme inhibitors (5), and the immune modular β-glucan (4). We found that selenite blocked PC anaplerosis and elicited massive necrosis in CA but not in NC lung tissues. CA lung tissue slices also responded to β-glucan with perturbed metabolic activity and histopathologic changes, which were consistent with polarization towards M1-type macrophages. We were intrigued to find that the metabolism and histopathology of these CA lung tissue slices from different patients responded distinctly to these anticancer agents, which could be translated into individual patients' response to drugs. Furthermore, to delineate systemic and microenvironmental influences on cancer metabolism, we compared and found that the ex vivo tissue slice cultures incorporated to a higher extent 13C from [13C6]-glucose into glycolytic, PPP, and purine nucleotide products than the corresponding mouse PDX in vivo, illustrating the high metabolic viability of the ex vivo tissue slice cultures. These new developments in preclinical models and mechanistic metabolic interrogations promise to provide rigorous prediction for individual patients' response to therapeutics while revealing new and exciting targets for the next generation of personalized therapeutics. Supported by 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, 3R01ES022191-04S1, 3R01CA118434-02S1, 1U24DK097215-01A1, P30CA177558; KLCRP, and the KY Challenge for Excellence.
References
1. Sellers K, Fox MP, Bousamra M II, Slone SP, Higashi RM, Mille, DM, et al. Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J Clin Invest 2015;125:687-98.
2. Fan TW, Lane AN, Higashi RM. (2016) Stable isotope resolved metabolomics Studies in ex vivo tissue slices. Bio-protocol 2016;6:e1730.
3. Lane AN, Higashi RM, Fan TWM. (2016) Preclinical models for interrogating drug action in human cancers using Stable Isotope Resolved Metabolomics (SIRM). Metabolomics 2016;12:1-15.
4. Fan TW, Warmoes MO, Sun Q, Song H, Turchan-Cholewo J, Martin JT, et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator beta-glucan in a two-case ex vivo non-small-cell lung cancer study. Cold Spring Harb Mol Case Stud 2016;2:a000893.
5. Xie H, Hanai JI, Ren JG, Kats L, Burgess K, Bhargava P, et al. (2014) Targeting lactate dehydrogenase-A inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells. Cell Metab 2014;19:1-15.
Citation Format: Teresa W. Fan, Ramon Sun, Marc Warmoes, Qiushi Sun, Huan Song, Angela Mahan, Jeremiah Martin, RICHARD M. Higashi, Andrew N. Lane. Exploring the lung cancer metabolome, in vivo and ex vivo, for individualized medicine [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 SY02-02. doi:10.1158/1538-7445.AM2017-SY02-02