Cancer Metabolism is a developing Shared Resource within the SBP Cancer Center. The scientific focus of the core is to investigate the role of metabolism in cancer on both the cellular and organismal level, combining in vitro and in vivo analysis. To accomplish this, we integrate a number of instruments and methodologies to build an overall model of cellular metabolism. These approaches range from the relatively simple (such as rapidly measuring basic metabolic substrates from media or plasma) to rather complex (tracing the metabolism of stable isotope-labeled substrates in vitro or in vivo). All of the instruments and approaches are available on a fee-for-service basis to Cancer Center scientists, but also (as capacity permits) to outside users. Samples for most services can be shipped, but Seahorse users need to be local so they can bring live cells for analysis.
The primary technology and tools in the La Jolla core are:
- GC/MS-based analysis of metabolites such as amino acids, keto acids and fatty acids. Measuring both the abundances of these metabolites and the rate of stable isotope labeling enables determination of the relative activity of a number of metabolic pathways involved in tumor progression, including glycolysis, the pentose phosphate pathway, the TCA cycle, de novo fatty acid biosynthesis and amino acid biosynthesis. In addition, the metabolism or exchange of other isotope labeled metabolites can be tracked as warranted.
- Measurement of cellular respiration and glycolysis using a Seahorse XFp. This instrument determines the rate of glycolysis and respiration of adherent cells in a 6-well format. Glycolysis is observed via the acidification of the tissue culture media. Respiration is observed by measuring total oxygen consumption. With proper experimental design, the rates of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial oxygen consumption may be determined.
- Rapid and accurate measurement of major metabolites (glucose, glutamine, lactate, and glutamate) using the YSI 2950 analyzer. This instrument is able to rapidly (~1 min per metabolite) determine the concentration of these important metabolism substrates from a small volume of sample (~200 µL).
In addition, the core has access to HPLC, LC/MS and NMR instrumentation and experts in SBP, La Jolla, LC/MS/MS at the Lake Nona site.
- The YSI 2950 metabolite analyzer is able to measure glucose, glutamine, lactate, glutamate, ammonia and potassium in media or other liquid samples in 2-3 minutes for all 4 metabolites. It is able to handle 96-well plates or a rack of 24 Eppendorf tubes. The minimum volume needed is 100 μl for plate samples and 400 μl for tubes. A full 96-well plate maybe analyzed in 3-4 hours of hands-free automation.
- The XFp Analyzer can measure respiration in most cell types, including primary cells, adherent cells, and suspension cells. With a new 6 well format, the XFp Miniplate is ideal for pairwise comparisons (in triplicate) and the analysis of precious samples, such as patient-derived and other rare samples. We mainly focus on the Cell Mito Stress Test to measure the key parameters of mitochondrial function: basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity. Other protocols using alternate substrates or permeabilized cells are available.
- The Shimadzu QP2010 gas chromatograph-mass spectrometer is used for broader metabolic flux analysis including determination of stable isotope ( 13C, 15N) labeling rates of various intra- and extracellular metabolites, as well as determining the abundance of amino, keto and fatty acids, short-chain fatty acids, cholesterol and sugars. Analysis of cells, tissues, tumors, and serum or plasma can be performed.
- A Shimadzu Prominence HPLC is available for the measurement of metabolites not generally accessible by GC/MS. In particular, assays have been developed to detect ATP, ADP, AMP and NAD species. The HPLC is also ideal for bulk amino acid analysis via automated HCl hydrolysis. Detectors include a dual UV/Vis , fluorescence and refractive index. Both the column and autosampler are temperature controlled.
Additional SBP Analytical Resources Available for Metabolomics
- Lake Nona Metabolomics Core - Utilizes the Duke Steadman modules for analysis of Acylcarnitines, Organic acids, and Amino acids. The core is working to expand its repertoire to include focused analysis, and very broad Orbitrap-based profiling.
- Informatics core in Lake Nona with particular expertise in analysis of metabolic data, and integration of these data with other data types such as genomics.
- Lake Nona (in vivo) Metabolic Phenotyping Core – CLAMS metabolic cage system (food, water, excretion, O2/CO2), exercise metabolism, NMR body composition analysis, and extensive glucose/insulin studies.
- NMR-based analysis - Can be accessed in La Jolla through collaboration with J. Smith Lab and the NMR Facility.
- Lipidomics - Shotgun lipidomics, in collaboration with X. Han lab at Lake Nona.
- Seahorses (both XF24 and 96) – Common equipment at Lake Nona.
- Other LC-MS Instruments in La Jolla – API 3000 LC/MS/MS, and several other LC/MS systems in the Proteomics core facility.
- Scripps LC-MS/MS facility - (Center for Metabolomics and Mass Spectrometry) for broad profiling of metabolites.
For a complete list of services, please call (858)646-1000 ext. 3941 or email us.
|Cancer Metabolism Services||Internal
|Polar metabolite quantification (per sample)||each||$40||$50||$54||$105.20|
|Polar metabolite 13C analysis (per sample)||each||$40||$50||$54||$105.20|
|Fatty acid analysis (per sample)||each||$40||$50||$54||$105.20|
|Cholesterol/sterol quantification (per sample)||each||$40||$50||$54||$105.20|
|Seahorse XFp (per plate)||each||$200||$250||$270||$526|
|YSI metabolite analysis (per plate)||each||$60||$75||$81||$157.80|
David Scott, Ph.D.
David Scott focuses on the development and application of methods for metabolomics, enhanced by the use of stable isotope substrates. Stable isotopes (mainly 13C) provide an added layer of information to metabolomic studies – not only is static pool information obtained, but it is also possible to infer the origins of metabolites and to track flux through metabolic pathways. Methods used include the analysis of small polar metabolites (mainly amino acids and carboxylic acids) and fatty acids by gas chromatography-mass spectrometry. Other techniques are being developed to analyze and quantify an expanded range of (labeled) metabolites. We have applied these methods in a study of the central carbon metabolism in Escherichia coli, and, in several papers, on melanoma metabolism. Investigations of melanoma cells showed the importance of glutamine metabolism under hypoxia, identified a novel route for the utilization of the carbon backbone of glutamine for fatty acid synthesis, and defined pathways for proline synthesis. Other recent work uses alternately labeled sugars to simultaneously track different inputs to glycans in normal cells and cell lines from patients with glycosylation defects.
Please call (858)646-3100 ext. 3941 or use the button below to send us an email.