Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231

Abstract

Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50°C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism. IntroductionMammalian metabolomics has received increased attention in recent years mainly because of its potential in the prognosis and diagnosis of cancer and for assessing treatment efficacy by analysing cells, fluids or tissues for specific biomarkers in experimental, translational and clinical studies. Adherent mammalian cell-lines are used extensively in oncology research as preclinical models but to date there is insufficient information on the application of metabolomics for the analysis of cultured mammalian cell lines.1,2 To obtain reliable metabolomics data for adherent cells, an optimised workflow must be identified which will provide maximum coverage for all classes of metabolites with minimum leakage. Breast cancer (BC) is a highly heterogeneous cancer for which morphologically and clinically distinct subgroups of cell lines have been established. 3 Triple negative breast cancer (TNBC) is characterised by the absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor (HER-2). TNBC represents approximately 20% of all BC and is typically associated with poor prognosis. Moreover, due to its aggressive phenotype, TNBC only partially res...

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Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions

“…Metabolomics analysis of cultured cells has emerged as an important technology for studying cellular biochemistry that provides an instantaneous snapshot of ongoing cellular metabolism. The major bottlenecks associated with metabolomics cell samples preparation workflow are efficient sampling, quenching and metabolites extraction in order to preserve the internal metabolite signatures [87].…”

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures

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