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.
According to the number, amount, and reproducibility of extracted
metabolites, we found that 50% acetonitrile was an excellent extractant
for Hela cells while 1:1:1 methanol: chloroform: water behaved the worst
extraction efficiency. The results showed that the total amount of the
intracellular metabolites in cell extracts obtained using different
sample preparation procedures with the IDMS method ranged from 21.51 to
295.33 nmol/million cells. Consistent with previous study, metabolite
leakage was found to be maximal with cold methanol as the quencher.
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.
In addition, the same conclusion was drawn as these 12 combinations were
applied to obtain quantitative metabolome data from three-dimensional
(3D) tumor spheroids. 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. Extracellular
metabolite concentrations showed that 3D cells performed an enhanced
glucose consumption and lactate excretion rate than two-dimensional (2D)
cells before and after the addition of DOX. While, the addition of DOX
did not interfere with extracellular glutamine concentration in both 2D
and 3D cells. Pathway enrichment analysis using targeted metabolomics
data showed that DOX exposure would significantly affect amino acid
metabolism-related pathways, which might be related to the mitigation of
redox stress. Strikingly, our
data suggested that compared to 2D cells the increased intracellular
glutamine level in 3D cells benefited replenishing the tricarboxylic
acid (TCA) cycle when the glycolysis was limited after dosing with DOX.
Taken together, this study provides a well-established quenching and
extraction protocol for quantitative metabolome profiling of Hela
carcinoma cell under 2D and 3D cell culture conditions. 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.
Keywords : Hela; metabolomics; sample preparation; 3D tumor
spheroids; quenching; extraction; isotope dilution mass spectrometry