Supplementary MaterialsSupplementary Information 41467_2017_1518_MOESM1_ESM. glycogen, lipids, and proteins in mouse tissues
May 8, 2019
Supplementary MaterialsSupplementary Information 41467_2017_1518_MOESM1_ESM. glycogen, lipids, and proteins in mouse tissues during 12 to 48?h of 13C6-glucose feeding. Applying this approach to patient-derived lung tumor xenografts (PDTX), we show that the liver supplies glucose-derived Gln via the blood to the PDTX Baricitinib cost to fuel Glu and glutathione synthesis while gluconeogenesis occurs in the PDTX. Comparison of PDTX with ex vivo tumor cultures and arsenic-transformed lung cells versus xenografts discloses differential glucose metabolism that could reflect distinct tumor microenvironment. We further found differences in glucose metabolism between the primary PDTX and distant lymph node metastases. Introduction Stable isotope tracer approaches, here termed Stable Isotope Resolved Metabolomics (SIRM) are becoming widely used for studying malignancy metabolism1, 2. These approaches not only improved understanding of known metabolic phenotypes in cancer cells such as enhanced glycolysis (the Warburg effect3) and glutaminolysis1, 4, but also revealed novel metabolic reprogramming crucial to Baricitinib cost tumor growth or survival to enable discovery Baricitinib cost of new therapeutic targets2, 5, 6. Most tracer studies have been performed in vitro using cell lines, which lack the tumor microenvironment and 3D architecture that may be crucial to recapitulate the transcriptional and metabolic programs in vivo. However, in vivo tracer studies remain challenging, due in part to the overt stress induced by animal handling with existing techniques, which also limits the duration of tracer delivery. There are several Baricitinib cost established means of administering 13C-glucose tracers in vivo, including bolus injections via the tail vein7 and continuous infusion via cannulation8, 9. These methods may suffer from the well-documented effects on metabolism due to anesthesia10, 11 and/or physical trauma12, 13, thereby confounding the metabolic signatures of tumors. Although these invasive methods can achieve high 13C-glucose levels in mouse plasma, tracer experiments are transient in nature (e.g., 30C150?min) to reduce stress responses induced by anesthesia or physical constraint. As such, 13C labeling is usually often limited to metabolites of the faster turnover pathways such as glycolysis and the Krebs cycle7, 8 but not the metabolome of more extended pathways such as de novo synthesis of lipids, proteins, and nucleotides. A better method of introducing stable isotope tracers is needed that avoids overt stress, while enabling long-term tracer administration for deeper pathway coverage in vivo. Here, we report a noninvasive method of administering 13C6-glucose to mouse models via liquid diet feeding to achieve deep metabolic network coverage. We have applied this method to non-small cell lung cancer patient-derived tumor xenografts and arsenite-transformed lung cell xenografts to assess the influence of the microenvironment on cancer cell metabolism. Results A liquid diet for in vivo 13C Rabbit polyclonal to AKR1C3 enrichment of metabolites Here we describe a new method to introduce 13C-glucose via an ad libitum liquid diet that is stress-free, highly reproducible, and achieves relatively high enrichment of complex carbohydrates, nucleotides, lipids and proteins in major organs of NOD/SCID/Gamma (NSG) mice in 24?h. We designed a liquid diet formula in which glucose is the primary source of carbohydrate (Fig.?1a). Mice were habituated to an unlabeled glucose liquid diet for 2 days to achieve stable daily food intake (Fig.?1b). On day 3, the dietary carbohydrate was replaced with 13C6-glucose. On the basis of the daily mouse-feeding pattern (Fig.?1c), we supplied the enriched liquid diet at 1600 hours, Baricitinib cost followed by necropsy at 1000 hours on the next day. Mouse organs were dissected, flash frozen in liquid nitrogen (LiqN2), and extracted for polar metabolites, lipids and proteins, then analyzed by NMR and mass spectrometry. Open in a separate windows Fig. 1 Procedure for tracing glucose metabolism in vivo. a Composition of liquid diet used for 13C6-glucose tracer feeding (presented as g/kg and %kcal for each ingredient). The constituted liquid.