Establishment of Stable Isotope-Resolved Metabolomics as Test System to Assess Membrane-Associated Drug Targets in Cancer

Abstract

Stable isotope-resolved metabolomics (SIRM) is a powerful technique to gain insights into cancer metabolism, characterize drug targets and discover novel biomarkers for potential diagnosis and prognosis. The scope of this work was to establish SIRM as a test system to assess cancer metabolic network activities in vitro and to enable the characterization of drug induced alterations in the absence and presence of the selective PI3Kβ inhibitor AZD8186. The work entailed (I) the establishment and optimization of a sample preparation workflow including assessment of metabolism quenching, sample storage and stability, cell washing and pre-analytical normalization, and (II) the functional characterization of a PTEN (phosphatase and tensin homolog) wildtype and a PTEN-null triple-negative breast cancer (TNBC) cell line to enable dose finding of AZD8186 as a prerequisite for studying the effect of PI3Kβ inhibition on metabolic pathway activities using SIRM. The SIRM platform was established with (III) [U13C] glucose as tracer including a combination of targeted isotopologue feature extraction with a non-targeted routine based on X13CMS to enable identification of isotope-enriched features that are significantly altered between control and treatment conditions (i.e., untargeted differential tracing). The platform was further applied using (IV) [U15N] and [U13C, 15N] glutamine to assess altered metabolic fluxes in lower parts of central metabolism (e.g. purine and pyrimidine nucleotide biosynthesis, amino acid metabolism, hexosamine pathway). Lastly, (V) employing untargeted differential tracing, differences in AZD8186-induced enrichment of labeled isotopes between the epithelial cancer cell line HCC70 and MDA-MB-468 were investigated, thereby demonstrating isotope-enriched features that were significantly altered in the presence of AZD8186 both jointly and uniquely for each cell line.