Prospects for clinical cancer metabolomics using stable isotope tracers

Abstract

Metabolomics provides a readout of the state of metabolism in cells or tissue and their responses to external perturbations. For this reason, the approach has great potential in clinical diagnostics. For more than two decades, we have been using stable isotope tracer approaches to probe cellular metabolism in greater detail. The ability to enrich common compounds with rare isotopes such as carbon ((13)C) and nitrogen ((15)N) is the only practical means by which metabolic pathways can be traced, which entails following the fate of individual atoms from the source molecule to products via metabolic transformation. Changes in regulation of pathways are therefore captured by this approach, which leads to deeper understanding of the fundamental biochemistry of cells. Using lessons learned from pathways tracing in cells and organs, we have been applying this methodology to human cancer patients in a clinical setting. Here we review the methodologies and approaches to stable isotope tracing in cells, animal models and in humans subjects.

Figure 1. Simple metabolic scheme

Carbon flow from [U-¹³C]-glucose through glycolysis, Krebs cycle, PPP, pyruvate carboxylation, malate/Asp shuttle, and synthesis of GSH and pyrimidine nucleotides. Solid oval represents the plasma membrane and the dashed oval represents the mitochondrial space. Double-headed arrows: exchange or reversible processes. Glc = glucose, G6P = glucose-6-phosphate, Rib = nucleotide ribose, DHAP, GAP = dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, PEP = phosphoenolpyruvate, Pyr=pyruvate, Lac = lactate, Cit=citrate, 2=OG = 2-oxoglutarate, Mal=malate, OAA = oxalacetate, U = uracil base. Critical enzymes are shown in cyan. HK= hexokinase (entry to glycolysis), G6PDH = glucose-6-phosphate dehydrogenase (entry to the oxidative branch of the pentose phosphate pathway), TK, TA transketolase and transaldolase (non –oxidative pentose phosphate pathway), PK = pyruvate kinase, PDH = pyruvate dehydrogenase, PC = pyruvate carboxylase, AAT = aspartate amino transferase, MDH= malate dehydrogenase, ME = malic enzyme. Glutaminolysis is the pathway from Gln to pyruvate via ME, leading to unlabeled malate, Asp, Ala and lactate.

Figure 2. Time course of glucose consumption and lactate secretion from A549 cells

Human lung adenocarcinoma cells A549 were grown at 37 °C in RPMI 1640 medium containing [U-¹³C]-glucose in place of natural ¹²C glucose in 5% CO₂ and 10% dialyzed FCS. Medium was sampled at different times, extracted with cold trichloracetic acid (Fan et al. 2008; Lane et al. 2008) and lyophilized. The polar metabolites remaining were redissolved in D₂O and analyzed by NMR at 600 MHz. A. 1D spectrum showing the presence of ¹³C lactate. B. Time course of ¹²C (○) and ¹³C (●) lactate production and consumption of ¹³C glucose (■), Gln (□), Val (◆) and Thr (✧). Average of 4 parallel experiments. C. The fraction of lactate that is U-¹³C, and the fraction of glucose consumed converted to lactate was calculated as previously described (Lane et al. 2008). Average of 4 parallel experiments.

Figure 2. Time course of glucose consumption and lactate secretion from A549 cells

Human lung adenocarcinoma cells A549 were grown at 37 °C in RPMI 1640 medium containing [U-¹³C]-glucose in place of natural ¹²C glucose in 5% CO₂ and 10% dialyzed FCS. Medium was sampled at different times, extracted with cold trichloracetic acid (Fan et al. 2008; Lane et al. 2008) and lyophilized. The polar metabolites remaining were redissolved in D₂O and analyzed by NMR at 600 MHz. A. 1D spectrum showing the presence of ¹³C lactate. B. Time course of ¹²C (○) and ¹³C (●) lactate production and consumption of ¹³C glucose (■), Gln (□), Val (◆) and Thr (✧). Average of 4 parallel experiments. C. The fraction of lactate that is U-¹³C, and the fraction of glucose consumed converted to lactate was calculated as previously described (Lane et al. 2008). Average of 4 parallel experiments.

Figure 3. 2-D ¹H TOCSY identified unlabeled and ¹³C-labeled metabolites in extracts

TCA extract of A549 cells grown in the presence of 5 mM [U-¹³C]-glucose for 24 h, as described for Figure 2. Boxes show the ¹³C satellite peaks of selected metabolites (Lac, Ala, Glu and Asp).

Figure 4. NMR spectra of plasma and tissue of NSCLC cancer patient

A patient was infused with 10 g [U-¹³C]-glucose 3 h prior to resection of the lung. The tumor and non-tumorous lung tissue was flash frozen and then extracted with cold TCA as described (Gradwell et al. 1998; Lane et al. 2008). A. 1D tissue: normal and cancer B. TOCSY spectrum of cancer

Figure 4. NMR spectra of plasma and tissue of NSCLC cancer patient

A patient was infused with 10 g [U-¹³C]-glucose 3 h prior to resection of the lung. The tumor and non-tumorous lung tissue was flash frozen and then extracted with cold TCA as described (Gradwell et al. 1998; Lane et al. 2008). A. 1D tissue: normal and cancer B. TOCSY spectrum of cancer

Figure 5. FT-ICRMS spectra of lung tumor lipids

Part A shows FT-ICR-MS spectra of phospholipid extracts, from the lungs of a single human subject. Note that overall, the spectra appear similar to each other, which is expected since most of the phospholipids serve functional roles as membrane structure. Part B shows the expansion of the region indicated in Part A, illustrating the three general categories of results for each of the hundreds to thousands of peaks obtained from each 10 minute sample run. Thus, this is a high-information throughput (HIT) technique. As illustrated, the most abundant lipids tend to be similar in profile, while many of the less abundant lipids decrease or increase in lung cancer relative to the non-cancerous lung tissue from the same patient. In this particular example, there are >1,000% differences in the indicated lipids, which are only two out of dozens of differences at this magnitude that are present.