Rapid probing of glucose influx into cancer cell metabolism: using adjuvant and a pH-dependent collection of central metabolites to improve in-cell D-DNP NMR

Abstract

Changes to metabolism are a hallmark of many diseases. Disease metabolism under physiological conditions can be probed in real time with in-cell NMR assays. Here, we pursued a systematic approach towards improved in-cell NMR assays. Unambiguous identifications of metabolites and of intracellular pH are afforded by a comprehensive, downloadable collection of spectral data for central carbon metabolites in the physiological pH range (4.0–8.0). Chemical shifts of glycolytic intermediates provide unique pH dependent patterns akin to a barcode. Using hyperpolarized ¹³C₁ enriched glucose as the probe molecule of central metabolism in cancer, we find that early glycolytic intermediates are detectable in PC-3 prostate cancer cell lines, concurrently yielding intracellular pH. Using non-enriched and non-enhanced pyruvate as an adjuvant, reactions of the pentose phosphate pathway become additionally detectable, without significant changes to the barriers in upper glycolysis and to intracellular pH. The scope of tracers for in-cell observations can thus be improved by the presence of adjuvants, showing that a recently proposed effect of pyruvate in the tumor environment is paralleled by a rerouting of cancer cell metabolism towards producing building blocks for proliferation. Overall, the combined use of reference data for compound identification, site specific labelling for reducing overlap, and use of adjuvant afford increasingly detailed insight into disease metabolism.