In Vitro Tracking of Intracellular Metabolism-Derived Cancer Volatiles via Isotope Labeling

[Image: see text] Cancer detection relying on the release of volatile biomarkers has been extensively studied, but the individual biochemical processes of the cells from which biogenic volatiles originate have not been thoroughly elucidated to date. Inadequate determination of the metabolic origin of the volatile biomarkers has limited the progress of the scientific and practical applications of volatile biomarkers. To overcome the current limitations, we developed a metabolism tracking approach combining stable isotope labeling and flux analysis of volatiles to trace the intracellular metabolism-derived volatiles and to reveal their relation to cancer metabolic pathways. Specifically, after the (13)C labeling of lung cancer cell, the isotopic ratio of whole cellular carbon was measured by nanoscale secondary ion mass spectrometry-based imaging. The kinetic modeling with the time-dependent isotopic ratio determined the period during which cancer cells reach the metabolic steady state, at which time all of the potential volatiles derived from intracellular metabolism were fully enriched isotopically. By measuring the isotopic enrichment of volatiles at the end-stage of isotopic flux, we found that 2-pentadecanone appeared to be derived from the metabolic cascade starting from glucose to fatty acid synthesis. Furthermore, this biosynthetic pathway was determined to be distinct in cancer, as it was upregulated in colon, breast, and pancreatic cancer cells but not in normal cells. The investigation of the metabolic footprint of 2-pentadecanone demonstrates that our novel approach could be applied to trace the metabolic origin of biogenic volatile organic compounds. This analytical strategy represents a potential cutting-edge tool in elucidating the biochemical authenticity of cancer volatiles and further expanding our understanding of the metabolic network of airborne metabolites in vitro.