A systematic evaluation of quenching and extraction procedures for
quantitative metabolome profiling of Hela carcinoma cell under 2D and 3D
cell culture conditions
Abstract
Metabolic reprogramming has been coined as a hallmark of cancer,
accompanied by which the alterations in metabolite levels have profound
effects on gene expression, cellular differentiation and the tumor
environment. Yet a systematic evaluation of quenching and extraction
procedures for quantitative metabolome profiling of tumor cells is
currently lacking. To achieve this, this study is aimed at establishing
an unbiased and leakage-free metabolome preparation protocol for Hela
carcinoma cell. We evaluated 12 combinations of quenching and extraction
methods from three quenchers (liquid nitrogen, -40°C 50% methanol,
0.5°C normal saline) and four extractants (80% methanol, methanol:
chloroform: water (1:1:1, v/v/v), 50% acetonitrile, 75°C 70% ethanol)
for global metabolite profiling of adherent Hela carcinoma cells. Based
on the isotope dilution mass spectrometry (IDMS) method, gas/liquid
chromatography in tandem with mass spectrometry was used to
quantitatively determine 43 metabolites including sugar phosphates,
organic acids, amino acids, adenosine nucleotides and coenzymes involved
in central carbon metabolism. Among 12 combinations, cells that washed
twice with phosphate buffered saline (PBS), quenched with liquid
nitrogen, and then extracted with 50% acetonitrile was found to be the
most optimal method to acquire intracellular metabolites with minimal
loss during sample preparation. Furthermore, a case study was carried
out to evaluate the effect of doxorubicin (DOX) on both adherent cells
and 3D tumor spheroids using quantitative metabolite profiling. Based on
this, quantitative time-resolved metabolite data can serve to the
generation of hypotheses on metabolic reprogramming to reveal its
important role in tumor development and treatment.