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Stable isotope tracing to assess tumor metabolism in vivo

Abstract

Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [13C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [13C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method’s versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.

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Fig. 1: Patient infusion workflow.
Fig. 2: Mouse infusion workflow.
Fig. 3: Mouse infusion apparatus.
Fig. 4: Confirmation of [13C] peaks in HRMS.
Fig. 5: Data from [U-13C]glucose infusions in patients and mice.
Fig. 6: Examining metabolic features among tumor and tissue types.

Data availability

Previously unpublished data are included in the Supplementary Information. The remaining supporting data can be found in refs. 4,5,21.

Code availability

The software can be found at https://github.com/wencgu/nac.

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Acknowledgements

R.J.D. is a Howard Hughes Medical Institute (HHMI) Investigator, the Robert L. Moody, Sr. Faculty Scholar at UT Southwestern and Joel B. Steinberg, M.D. Distinguished Chair in Pediatrics. S.J.M. is an HHMI Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the Kathryn and Gene Bishop Distinguished Chair in Pediatric Research, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. The research was supported by the Cancer Prevention and Research Institute of Texas (RP170114 and RP180778), the National Institutes of Health (R35 CA220449; U01 CA228608) and the Robert A. Welch Foundation (I-1733). A.T. was supported by the Leopoldina Fellowship (LPDS 2016-16) from the German National Academy of Sciences and the Fritz Thyssen Foundation. B.F. is supported by the National Institutes of Health (K99/R00 CA237724-01A1). Figures were generated using BioRender.com.

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Authors and Affiliations

Authors

Contributions

B.F., A.T., S.J.M., T.P.M and R.J.D. wrote and edited the manuscript. T.P.M established HRMS methodology.

Corresponding authors

Correspondence to Brandon Faubert or Ralph J. DeBerardinis.

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Competing interests

R.J.D. is an adviser for Agios Pharmaceuticals and Vida Ventures, and a founder and adviser for Atavistik Bio. S.J.M. is an adviser for Frequency Therapeutics and Protein Fluidics.

Additional information

Peer review information Nature Protocols thanks Monica Montopoli and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key data used in this protocol

Faubert, B. et al. Cell 171, 358–371 (2017): https://doi.org/10.1016/j.cell.2017.09.019

Tasdogan, A. et al. Nature 577, 115–120 (2020): https://doi.org/10.1038/s41586-019-1847-2

Hensley, CT. et al. Cell 164, 681–694 (2016): https://doi.org/10.1016/j.cell.2015.12.034

Supplementary information

Reporting Summary

Supplementary Data 1

Supplementary data tables for patient and mouse 13C glucose infusions

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Faubert, B., Tasdogan, A., Morrison, S.J. et al. Stable isotope tracing to assess tumor metabolism in vivo. Nat Protoc 16, 5123–5145 (2021). https://doi.org/10.1038/s41596-021-00605-2

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