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. 2016 Feb 11;164(4):681-94.
doi: 10.1016/j.cell.2015.12.034. Epub 2016 Feb 4.

Metabolic Heterogeneity in Human Lung Tumors

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Free PMC article

Metabolic Heterogeneity in Human Lung Tumors

Christopher T Hensley et al. Cell. .
Free PMC article

Abstract

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Figures

Figure 1
Figure 1. Image-guided assessment of glucose metabolism in human NSCLC
(A) Workflow for clinical study. (B) Pre-operative imaging of patient 1. FDG-PET/CT was obtained prior to enrollment, and all MRI was performed within a single session. Arrows indicate the grade 3 adenocarcinoma. (C) Plasma glucose enrichment during [U-13C]glucose infusion. The arrow indicates time of resection. (D) Histological features. Scale bar, 200μm. (E) Relative fractional enrichment of glycolytic and TCA cycle metabolites. Tumor values were normalized to values from adjacent lung, which were assigned a value of 1.0. Abbreviations: FDG-PET/CT, Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography; DWI, diffusion weighted imaging; DCE, dynamic contrast-enhanced; ADC, Apparent Diffusion Coefficient; H/E, hematoxylin/eosin; Ac-CoA, acetyl-CoA; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase.
Figure 2
Figure 2. Human NSCLC tumors have enhanced anaerobic and aerobic metabolism compared to adjacent lung
(A) Average relative enrichments (tumor/lung) from 9 patients. Average values were determined using one fragment from each tumor. Acetyl-CoA was not directly measured but estimated from other 13C enrichment data using tcaSIM. *, p<0.05 by Student’s paired t-test (B) Relative enrichments (tumor/lung) of individual fragments from all 9 patients. Note that for most tumors, several fragments were analyzed. No glutamate was detected in patient 2. (C) Density plots of standard deviations (SD) derived from all 13C enrichments derived from the same localized region of a tumor (SD between replicates) or all 13C enrichments derived from different tumors (SD between tumors). All fractional enrichments were first normalized against the tissue glucose enrichment.
Figure 3
Figure 3. Human NSCLC tumors have enhanced PDH-dependent TCA cycle flux relative to adjacent lung
(A) NMR spectra from the lung and tumor of patient 4. Insets show expansions of multiplets from lactate C2 (LAC2) and glutamate C4 (GLU4). GLN, glutamine; ALA, alanine; TAU, taurine; GLY, glycine; ASP, aspartate; SUC, succinate; S, singlet; D, doublet; Q, quartet (doublet of doublets). (B) Correlations between NMR-derived and GC/MS-derived labeling features in all fragments for which data were available using both techniques. Data are from 10 tumor fragments and 7 lung fragments from 5 patients. Cit, citrate; Glu, glutamate. (C) Correlation plots between selected mass isotopologues from 34 tumor and 9 lung fragments. (D) Tracer scheme illustrating the origin of M+3 species during [U-13C]glucose infusion, with average and SD of measurements from all tumor fragments relative to adjacent lung. (E) Average relative fractional enrichments (tumor/lung) of isotopologues related to the TCA cycle turnover labeling scheme in Figure S2F. (F) Fluxes for PDH and PC estimated using tcaSIM. CS, Citrate Synthase. *, p<0.05 by Student’s paired t-test (D, E, F).
Figure 4
Figure 4. Lactate metabolism in NSCLC in humans and mice
(A) Comparison of major labeled forms of glucose (M+6) and acetyl-CoA (M+2) in tumor and lung. Data are the average and S.D. of all fragments, excluding necrotic tumor fragments. * = p < 0.05 between tumor and lung acetyl-CoA enrichments (paired Student’s t-test). (B) Ratios of labeling in lactate and citrate to the glycolytic intermediates 3-PG and PEP, in lung and tumor. Data are the average and S.D. of all fragments, excluding necrotic tumor fragments. *, p < 0.05; ***, p<0.005 (paired Student’s t-test). (C) Tracer scheme illustrating a tumor lactate pool arising from both glycolysis and lactate import. (D) Plasma enrichment of glucose M+6 and lactate M+3 in patients 1 and 5. (E) Percent enrichments of informative mass isotopologues in tumor fragments from patients 1 and 5 (one fragment per patient). Solid green lines indicate glucose M+6 enrichment in the plasma and dashed orange lines indicate lactate M+3 enrichment in the plasma from each patient, as in panel D. (F) Tracer scheme illustrating [2-13C]lactate infusion in tumor-bearing mice. (G) Top, Plasma enrichment of lactate M+1 during [2-13C]lactate infusions in four mice each with A549 or HCC827 xenografts. Bottom, Fractional enrichments of tumor metabolites after infusion with [2-13C]lactate. Data are the average and S.D. of four tumors per cell line. *, p < 0.05 (A549); **, p<0.01 (A549); #, p < 0.05 (HCC827) (One-way ANOVA comparisons to 3-PG).
Figure 5
Figure 5. DCE MRI identifies areas of heterogeneous glucose metabolism in NSCLC
(A) DCE data acquired from the region of maximum diameter of each tumor. Tumors could easily be divided into groups with higher and lower DCE signal. (B) Initial area under the curve for the first 60 seconds (iAUC60) of the time courses in Figure 5A. This semi-quantitative marker of DCE also generates two distinct groups of tumors. Data are average and S.D. of all tumors in the study. (C) Relative fractional enrichments (tumor/lung) for isotopologues related to glycolysis (3-PG and lactate M+3), PDH and the first TCA cycle turn (Citrate, Glutamate and Malate M+2), subsequent turns of the TCA cycle (Malate and Citrate M+1) and PC activity (Malate and Citrate M+3). Data are average and S.D. of all tumor fragments. (D) Fluxes for PDH and PC modeled using tcaSIM. Data are average and S.D. of all tumor fragments. (E) Top, Sagittal pre-contrast image of an adenocarcinoma (patient 8). Areas assessed by DCE MRI and sampled for metabolic analysis are indicated. Bottom, DCE curves for superior and inferior aspects of the tumor. The curves plot the average of gamma variate functions fit to three distinct slices from each region of interest. (F) Relative fractional enrichments (tumor/lung) for isotopologues related to glycolysis (3-PG and lactate M+3), PDH and the first TCA cycle turn (Citrate, Glutamate and Malate M+2). Data are average and S.D. of three fragments each from the superior and inferior aspects of the tumor. *, p<0.05; **, p<0.01; ***,p<0.005, Student’s unpaired t test.
Figure 6
Figure 6. Gene expression signatures in tumors with high and low DCE signal
(A) Unsupervised clustering of RNA-Seq data from tumor fragments. Data included quantitation of all detected transcripts (left) or a subset of transcripts from 2,756 genes involved in metabolism. (B) Results of GSEA using transcripts from metabolic genes. The 20 most enriched gene sets are shown for the high and low DCE groups. (C) GSEA plots for three gene sets enriched in low DCE tumors. (D) Proposed model for utilization of alternative fuels in NSCLC tumors with regional differences in perfusion.

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