MSACL 2016 US Abstract

Successes and Challenges of Metabolic Reprogramming Elucidation in Cancers Directly from Human Subjects via Stable Isotope Resolved Metabolomics (SIRM)

Richard Higashi (Presenter)
University of Kentucky

Bio: I have over thirty years of experience in the application and development of MS & NMR methods for probing metabolism, from ecophysiology/biochemistry in field plants, invertebrates, & fish, and more recently focused on cancer metabolism studies directly in humans. I direct a mass spectrometry facility which includes GC/LC-MS, IC-MS, and direct infusion pm ultrahigh resolution FT-MS and ICP-MS. I am currently the lead-PI on the NIH Common Funds Resource Center for Stable Isotope Resolved Metabolomics (RC-SIRM) at UK (one of the 6 NIH metabolomics centers), co-PI on a R01 (PD T. Fan) to develop chemoselective probes for various functional groups using both NMR and MS methods, and I head the Analytical Core of an NCI funded (P01, A. Lane director) clinical-based PPG on lung cancer metabolism. I am also Associate Director of MS for the Center for Environmental and System Biochemistry (CESB)

Authorship: Richard M. Higashi (1,2,3), Andrew N. Lane (1,2,3), Ronald C. Bruntz (1,3), Marc O. Warmoes (1,3), Qiushi Sun (1,3) Teresa W-M. Fan (1,2,3)
1)Center for Environmental and System Biochemistry, University of Kentucky, (UKY) Lexington KY USA, (2) Dept of Toxicology and Cancer Biology, UKY, (3) Markey Cancer Center, UKY

Short Abstract

Elucidating metabolic reprogramming in cancers requires functional omics, especially metabolomics. Most metabolites have numerous roles, such that their chemical identity alone cannot establish their BIOchemical identity. Stable isotope resolved metabolomics (SIRM) can distinguish among pathways and functions. Using SIRM we have mapped glucose C utilization in resected tumor and non-tumor tissues from lung cancer patients infused with [U-13C]-glucose, plus C,N utilization in their tissue slices with [U-13C]-glucose or [U-13C,15N]-glutamine. This revealed many details, including up regulation of pyruvate carboxylase anaplerosis. The paired human tissue slice platform is being used with drug candidates to test impacts on metabolic reprogramming.

Long Abstract


Diseases such as cancers have nutrient utilization and metabolic reprogramming at their core, which requires functional omics, and functional network metabolomics is a bedrock approach. Metabolites are often referred to as "nuts and bolts" of metabolism; the analogy is appropriate, as most metabolites are used in numerous roles, so that their chemical identity alone cannot establish their BIOchemical identity. This is especially true for investigations of metabolic reprogramming in cancers. Thus, despite excellent analytical data signal-to-noise ratio (s/n) of modern analytical techniques, the INFORMATION s/n of each metabolite is less than unity due to their participation in multiple pathways. Stable isotope resolved metabolomics (SIRM) can distinguish pathways and functions among otherwise chemically identical metabolites. Using SIRM, we have demonstrated the capability over the last decade to elucidate key pathway/network metabolic reprogramming in cancers.


SIRM achieves functional proteomics effectively via in situ enzyme assays, unambiguously assigning up/down regulation of pathways in metabolic reprogramming, as well as C,N resource allocation to guide signaling and other omics. This is accomplished by incubating cell cultures with e.g. [U-13C]-glucose or [U-13C,15N]-glutamine (1), infusing human lung cancer patients with [U-13C]-glucose and resecting their tumor and non-tumor tissues (2,3), and incubation experiments of their resected tissue slices with [U-13C]-glucose or [U-13C,15N]-glutamine (3). Some experiments simultaneously incubate the 13C and 15N and/or 2H. Analyses of polar and non-polar metabolites are by multidimensional, multinuclear NMR (e.g. HSQC) combined with LC, ion chromatography (IC), and direct infusion nanospray ultra-high resolution FTMS; the latter requires fulltime resolving power >400,000 to analyze the simultaneous elemental isotopologues (4).

Example of Findings Using SIRM

For example, glucose vs glutamine sources of carbon in cancers has revealed key aspects of resource allocation, including the up regulation of anapleurosis via pyruvate carboxylase in relation to glucose vs glutamine utilization (1). In fact, we initially elucidated this pathway in lung cancers resected from 13C glucose infused human subjects (2), and further detailed the metabolic reprogramming in humans, tissue slices, and cell cultures (3). This demonstrates the unique ability of SIRM for untargeted discovery of specific metabolic pathways directly in humans for maximal relevance, comparing the identical experiments in animal and cell models for unprecedented congruence of results. Among the key findings from simultaneous 13C, 2H experimentation is the mapping of preferred pools of metabolites for e.g. nucleotide vs protein synthesis; these apparent “channeling” pathways are previously unknown, and significant enough to impede metabolic modeling.

The Future

Of course many technical challenges remain some which will be discussed, however the greatest challenges are a new horizon revealed by SIRM: that of complex metabolic pools corresponding to previously unknown dynamic compartmentation. This is a crucial area poorly addressed by all of the omics, even in combination. This is in part due to lack of extremely detailed, dynamic metabolic phenotype information, which SIRM can help to remedy.

References & Acknowledgements:


1) Le, A., Lane, A.N., Hamaker, M., Bose, S., Gouw, A., Barbi, J., Tsukamoto, T., Rojas, C.J., Slusher, B.S., Zhang, H., Zimmerman, L.J., Liebler, D.C., Slebos, R.J.C., Lorkiewicz, P.K., Higashi, R.M., Teresa W. M. Fan, T.W-M., Dang, C.V. (2012) Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B-cells. Cell Metabolism 15, 110-121. PMC3345194

2) Fan TW, Lane AN, Higashi RM, Farag MA, Gao H, Bousamra M, Miller DM. (2009) Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM). Mol Cancer 8:41, 2009; PMC2717907

3) Sellers, K., Fox, M., Bousamra II, M, Slone,S., Higashi, R., Miller, D., Wang, Y., Yan, J., Yuneva, M., Deshpande, R., Lane, A., and Fan, T. (2015) Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation. J Clin Invest. 125(2):687–698. doi:10.1172/JCI72873.

4) Lorkiewicz, P., Higashi, R.M., Lane, A.N., Fan, T. W-M. (2012) Method for nucleotide detection using direct-infusion FT-ICR MS for the purpose of Stable Isotopic Resolved Metabolomics (SIRM) analysis. Metabolomics. 8, 930-939. PMC3477816


This work was supported in part by National Science Founda-tion EPSCoR infrastructure grants EPS-0447479 and EPS-0132295; NIH National Center for Research Resources (NCRR) grants 5P20RR018733, 1R01CA118434-01A2, 1RO1CA101199-01, 3R01CA118434- 02S1, and 1R01ES022191-01, P01CA163223- 01A1, and 1U24DK097215-01A1 ; the University of Louisville CTSPGP/ARRA grant 20044; the Kentucky Lung Cancer Research Program grants OGMB090354B1 and OGMB101380; the Robert W. Rounsavall Jr. Family Foundation; the Kentucky Challenge for Excellence and Drive Cancer Out Campaign. We thank Katherine Sellers for major experimentation on one of the publications discussed, Jin Lian Tan, Alex Belshoff, Radhika Burra, and Tao Xu for technical assistance; Pawel Lorkiewicz for help with FT-ICR-MS analysis; and Melissa Hall and Bridgett Curry for clinical support.

Financial Disclosure

Board Memberno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: