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.
Highlights
Breast cancer (BC) is a highly heterogeneous cancer for which morphologically and clinically distinct subgroups of cell lines have been established.[3]
We have evaluated metabolite leakage in the adherent metastatic breast cancer cell line MDA-MB-231 using a range of washing and quenching solvents
Estimation of extracellular ATP levels in response to various quenching solvents results in less leakage of ATP using methanol supplemented with 70 mM HEPES, and this treatment appears to preserve the membrane integrity of MDA-MB-231 cells compared to the other quenching solvents evaluated
Summary
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. Due to its aggressive phenotype, TNBC only partially responds to chemotherapy. There is a lack of clinically established targeted therapies and a resultant increase in patient fatalities.[4,5,6] In order to sustain growth and proliferation of tumour cells, they constantly require supplements of macromolecular precursors and an exhibition of altered metabolism compared to 2038 | Analyst, 2017, 142, 2038–2049
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