Abstract
An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non- cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g., 13C6-glucose or 13C5, 15N2-glutamine. These slices are viable for at least 24 h, and make it possible to eliminate systemic influence on the target tissue metabolism while maintaining the original 3D cellular architecture. It is therefore an excellent pre-clinical platform for assessing the effect of therapeutic agents on target tissue metabolism and their therapeutic efficacy on individual patients (Xie et al., 2014; Sellers et al., 2015).
Keywords: Tissue slices, SIRM, Metabolic pathway tracing, Preclinical testing, Cancer metabolism
Materials and Reagents
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Representative data
Figure 1 Example ex vivo tissue slice experiment. A. Example thin slices of non-cancerous lung tissue (NC, left) adjacent to a lung adenocarcinoma (CA, right); B. T25-flasks on a rocker inside a CO2 incubator; C. Representative 1D 1H{13C} HSQC NMR spectra (recorded at 14.1 T, 15 °C) of extracts of CA versus NC lung slices from an non small cell lung cancer (NSCLC) patient incubated for 24 h in the presence of 10 mM 13C6-glucose. The tissue slices were pulverized and extracted as described (Sellers et al., 2015; Fan, 2012) which produces three phases- an upper aqueous phase containing polar metabolites, a lower organic phase containing non-polar metabolites (mainly lipids) and an interfacial phase that contains protein. Here the upper phase was lyophilized and redissolved in a phosphate buffer containing 50% D2O and 25 nmol DSS-d6 that serves both as a chemical shift reference and a concentration standard (Fan and Lane, 2013). The HSQC spectrum detects protons attached directly to 13C, and thus gives a readout of the metabolites that have incorporated 13C from the source molecule (glucose in this instance). The spectra of cancer and non-cancerous tissues are recorded under identical conditions, and the absolute intensities are normalized to the tissue protein weight. Peak areas were determined using peak fitting functions in MNOVA (Mestrelab Research, Santiago de Compostela, Spain) Enhanced production of various 13C labeled metabolites in the CA tissue slice is evident, including 13C-lactate (Lac), which is consistent with the Warburg effect or accelerated glycolysis in tumor tissues (Warburg, 1956).
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Acknowledgments
This work was supported in part by the following grants: NIH P01 CA163223-01A1, NIH 5R01ES022191-04, NIH 3R01ES022191-04S1, NIH 1U24DK097215-01A1, and the Kentucky Challenge for Excellence. This protocol has been developed based on work described in Xie et al. (2014); Sellers et al. (2015) and Bousamra et al. (2012). The authors declare no conflicts of interest.
References
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