Inferring subcellular compartmentalized flux in cancer cells: A new approach integrating isotope tracing with thermodynamic analysis

The inability to inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Numerous isozymes catalyze the same metabolic transformation in different compartment, having different flux, potentially in opposite directions – facilitating the shuttling of redox and energy co-factors across organelle membranes. The most direct approach for quantifying intracellular metabolic flux is isotope tracing coupled with computational Metabolic Flux Analysis (MFA). However, utilizing this approach with metabolic measurements performed on a whole-cell level typically limits its applicability to inferring whole-cell level metabolic flux – i.e. average flux through all subcellular organelles. Here, we developed a computational method for inferring cytosolic and mitochondrial specific metabolic fluxes based on whole-cell level measurements of metabolite isotopic labeling and concentrations. This is made possible by integrated modeling of compartment-specific isotope tracing as well as reaction and membrane transporter thermodynamics – where inferred Gibbs free energy of reactions in each compartment is associated with rates of isotope exchange (forward-to-backward flux ratio). While joint isotope tracing and reaction thermodynamics modelling is computationally hard, we provide an efficient iterative algorithm for inferring compartment-specific fluxes, concentrations, and reaction Gibbs free energy, as well as confidence intervals. We applied our method to several proliferating cancer cell lines, deriving a first comprehensive view of the interplay between mitochondrial versus cytosolic fluxes in central metabolism under physiological conditions. We expect this approach to be a highly useful tool for probing cytosolic and mitochondria metabolic dysfunction in cancer and other human diseases.