The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.
Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.
Otto Warburg observed that cancer cells acquire the unusual ability to take up glucose and ferment it to produce lactate in the presence of adequate oxygen—a phenomenon termed as Warburg effect or aerobic glycolysis1,2. Mitochondrial respiration defects are speculated as the underlying basis for aerobic glycolysis in cancer cells3. Indeed, the Warburg effect is the basis for tumor imaging by fluorodeoxyglucose (FDG)-positron emission tomography (PET), which is widely used in clinical practice4,5. A high rate of aerobic glycolysis is considered a key feature of cancer and has been recently adopted as one of the well-known “hallmarks of cancer,” as described by D. Hanahan and B. Weinberg6. Somatic mutations in oncogenes and tumor suppressor genes—such as HRAS/KRAS/NRAS, EGFR, BRAF, MYC, TP53, isocitrate dehydrogenase (IDH), and fumarate hydratase (FH)—have been linked to specific metabolic changes in cancer cells, believed to be a result of the Warburg effect7.
Metabolomic analysis is a promising approach not only to understand metabolic regulation in cancer cells but also to identify early-stage cancer biomarkers and chemotherapy response prediction. Following treatment of sensitive or resistant cancer cells with anticancer compounds, tracking of their metabolic responses facilitates identification of metabolic biomarkers to predict efficacy of specific anticancer therapies in cancer patients8,9,10,11. In this article, cancer cell lines derived from a lung adenocarcinoma with an EGFR mutation treated with diamide—which causes oxidative stress—were used as models for metabolomic analysis. The advantage of this analytical method using capillary electrophoresis-mass spectrometry (CE-MS) is its comprehensive measurement of charged metabolites with the mass range m/z 50-100012,13. The purpose of this article is to provide novices a detailed stepwise visual protocol for preparation of aqueous metabolites from cultured cancer cells and subsequent metabolomic analysis, particularly by CE-MS.
1. Cell culture on day 1
NOTE: Each sample for metabolite extraction should be prepared from a single 100 mm tissue culture dish that is moderately but not fully confluent (containing approximately 2–5 million cells). Calculate the number of dishes needed for the assay and prepare them accordingly.
2. Preparation of reagents
3. Pre-washing centrifugal filter units
4. Cell culture on day 2
5. Extraction of metabolites from cultured cells
6. Ultrafiltration of cell extracts
7. Sample evaporation
8. Metabolomic analysis by CE-MS
Since metabolite concentrations in cancer cells (pmol/106 cells) are normalized to the number of viable cells, experimental conditions should be set up with care so as to minimize variation in the number of viable cells between conditions. For example, diamide treatment was at a relatively high concentration (250 μm) but for a short time to allow all the cells to grow as equally as possible, thereby equalizing the number of viable cells analyzed. Under these experimental conditions, HCC827 and PC-9 cells grew equally for 3 h (Figure 1). CE-MS analysis of diamide-treated cells compared with PBS-treated (control) cells revealed 175 and 150 differential metabolites in HCC827 and PC-9 cells, respectively. Among these, several intermediates in the pentose phosphate pathway (PPP) and in upper glycolysis were significantly higher in the diamide-treated conditions in both cell lines, whereas a few tricarboxylic acid (TCA) cycle intermediates were lower in the treated conditions (Figure 2 and Figure 3).
The PPP generates reducing equivalents in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is used for redox homeostasis maintenance and fatty acid biosynthesis15. Following diamide treatment, the level of gluconic acid—an oxidized glucose—increased 12-fold in HCC827 cells and 10-fold in PC-9 cells; similarly, following diamide treatment, the level of glucose 6-phosphate (G6P)—a phosphorylated glucose and the first hexokinase-catalyzed glycolysis product—also increased 6.3- and 3.5-fold in HCC827 and PC-9 cells, respectively (Figure 4). In addition, following diamide treatment, the levels of 6-phosphogluconate (6PG)—the first intermediate in PPP—dramatically increased 89-fold in HCC827 cells and 231-fold in PC-9 cells compared to the levels seen in the PBS controls (Figure 4). In contrast, levels of other glycolytic intermediates, such as fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (F1,6P), did not change in the diamide experimental condition (Figure 4). Total nicotinamide adenine dinucleotide phosphate (NADP+) levels were nearly equivalent between diamide treatment and PBS control conditions (Figure 4), suggesting that glucose was mainly catabolized via the PPP.
Figure 1. Unchanged cell numbers upon diamide treatment. Cell growth responses to 250 μm of diamide were measured using trypan blue staining. Cell numbers of (A) HCC827 and (B) PC-9 cells treated with PBS (blue) or diamide (red; 250 μm) for 1 or 3 h are shown. Data are shown as the mean ± SD (n = 6). Please click here to view a larger version of this figure.
Figure 2. Representative MS peaks of metabolites. Electropherograms annotated as (A) gluconic acid, (B) glucose 6-phosphate (G6P), (C) 6-phosphogluconate (6PG), and (D) nicotinamide adenine dinucleotide phosphate (NADP+) obtained by CE-MS analysis. Each line indicates the cell line (solid, HCC827; dotted, PC-9) and treatment (blue, PBS; red, diamide) used. Please click here to view a larger version of this figure.
Figure 3. Metabolome profiles of intracellular metabolites. Fold changes of metabolites in (A) HCC827 and (B) PC-9 cells treated with diamide are shown as log2(diamide/PBS). In total, 175 and 150 metabolites were annotated in HCC827 and PC-9 cells, respectively. Please click here to view a larger version of this figure.
Figure 4. Up-regulation of PPP upon diamide treatment. Intracellular concentrations (pmol/106 cells) of key metabolites involved in glycolysis and the pentose phosphate pathway (PPP) after treatment with diamide are shown. Metabolites were extracted from HCC827 and PC-9 cells treated with PBS (blue) or diamide (red, 250 μm) for 30 min. Representative metabolites such as gluconic acid, glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), 6-phosphogluconate (6PG), and nicotinamide adenine dinucleotide phosphate (NADP+) are shown. Please click here to view a larger version of this figure.
Here, we describe a widely accessible methodology to prepare metabolites from cultured cancer cells for CE-MS-based metabolomic analysis. One of the most critical points in this protocol is the proper preparation of cancer cells, because measured metabolite concentrations are normalized to the number of viable cells. For accurate estimation of cell number, it is necessary to prepare at least one additional culture dish per experimental group to count the number of viable cells in parallel with the extraction of metabolites for metabolomic analysis. In addition, the same number of cells should be seeded in each dish for the replicates and in the dish for counting; in the future, this would be aided by a quick and stressor-free (e.g., trypsin-free) cell counting protocol that allows the same dish to be used for both counting viable cells and extracting metabolites. Care should be taken during washes so that cells do not detach from the surface of the dishes. Severe cytotoxicity tests and other experiments that reduce cell adhesion may be unsuitable for this extraction protocol due to potential loss of cells during the washing procedure.
It is important to use a 5% mannitol solution as the wash buffer for extracting metabolites from cultured cells for CE-MS-based metabolomic analysis, because salt-based buffers, such as PBS, interfere with metabolomic analysis and adversely affect measurement.
Two or three dishes can be combined as a single sample by individually extracting metabolites from each dish and then pooling samples; however, combining multiple dishes often increases residual mannitol in the extracted metabolite solution. This may also interfere with metabolomic analysis by CE-MS. Hence, it is recommended to not use multiple dishes or wells as a single sample.
This metabolomic analysis method using CE-MS has been developed for comprehensive measurement of charged molecules with molecular weights between 50 and 1000 Da; thus, this protocol is optimized for extraction of aqueous, low molecular weight compounds. Therefore, this protocol is not suitable for extracting hydrophobic metabolites such as lipids or macromolecules such as proteins and nucleic acids. Since there is an increasing demand for comprehensive lipid analyses or lipidomics of cultured cell samples, the development of an easy and effective protocol for simultaneous extraction of both hydrophilic and hydrophobic metabolites is needed.
The first step of metabolite extraction—aspirating medium and washing cells with mannitol—should be conducted as quickly as possible to minimize changes to the metabolic profile of the cells. Treatment of cells with methanol after washing with mannitol is assumed to denature proteins and thereby prevent enzymes from catalyzing further metabolic reactions. However, even after methanol treatment, non-enzymatic chemical reactions—such as redox reactions, some decarboxylation processes, and thiol linkages—may take place. As such, any concentrations of metabolites involved in these reactions measured by this protocol should be interpreted with caution. In contrast to the genome or transcriptome, the metabolome consists of molecules with a wide variety of chemical properties; hence, no single protocol can extract all metabolites without any loss or disturbance. For more accurate measurements of such highly reactive metabolites, a protocol specifically designed to extract certain groups of metabolites, which requires fractionations and derivatizations, should be consulted. The protocol presented here, however, describes a simple and quick extraction of aqueous metabolites from cultured cell samples for metabolomic analysis by CE-MS. In this paper we could not describe how to set up CE-MS in detail because the focus of the present manuscript is different, however, describing detailed steps to set up CE-MS may require a separate dedicated article.
The authors have nothing to disclose.
We thank all the members of the Shonai Regional Industry Promotion Center for their help. This work was supported in part by research funds from Yamagata Prefecture and Tsuruoka City, by the National Cancer Center Research and Development Fund [grant number 28-A-9], and by the Japan Society for the Promotion of Science (JSPS) KAKENHI [grant number 17K07189] to HM.
Automated cell counter | Thermo Scientific | AMQAX1000 | Countess II automated cell counter |
Automatic integration software | Agilent Technologies | MassHunter G3335-60041 | version B.02.00 |
CE system | Agilent Technologies | Agilent 7100 CE system | |
CE/MS adapter kit | Agilent Technologies | G1603A | |
CE-ESI-MS Sprayer kit | Agilent Technologies | G1607A | |
Cell counting chamber slide | Thermo Scientific | C10282 | Countess cell counting chamber slides |
Centrifugal filter device, 5 kDa | Human Metabolome Technologies | ULTRAFREE MC PLHCC, UFC3LCCNB-HMT | |
Conical sterile polypropylene tube, 15 ml | Thermo Scientific | N339651 | |
Conical sterile polypropylene tube, 50 ml | Thermo Scientific | N339653 | |
Costar stripette, 10 ml | Corning | 4488 | |
Costar stripette, 5 ml | Corning | 4487 | |
D(-)-Mannitol | Wako | 133-00845 | 500 g |
Dulbecco's phosphate buffered saline (DPBS) | Sigma-Aldrich | D8537-500ML | |
Electrophoresis buffer | Human Metabolome Technologies | H3301-1001 | for cation analysis |
Electrophoresis buffer | Human Metabolome Technologies | H3302-1021 | for anion analysis |
Fetal bovine serum | Biowest | S1780 | |
Filter tip, 1000 μl | Watson | 124P-1000S | |
Filter tip, 20 μl | Watson | 124P-20S | |
Filter tip, 200 μl | Watson | 1252-703CS | |
Fused silica capillary | Polymicro Technologies | TSP050375 | 50 μm i.d. × 80 cm total length |
HCC827 | American Type Culture Collection | CRL-2868 | |
Internal standard solution | Human Metabolome Technologies | H3304-1002 | |
Isocratic pump | Agilent Technologies | Agilent 1100 Series Isocratic Pump | |
Methanol | Wako | 138-14521 | 1 L, LC/MS grade |
Microtube, 1.5 ml | Watson | 131-415C | |
Operating Software | Agilent Technologies | ChemStation G2201AA | version B.03.01 for CE |
PC-9 | RIKEN Bio Resource Center | RCB4455 | |
RPMI-1640 | Sigma-Aldrich | R8758-500ML | |
Sterile tissue culture dish, 100 mm | Corning | 430167 | |
Time-of-flight mass spectrometer | Agilent Technologies | Agilent G1969A Time-of-Flight LC/MS | |
Trypan blue solution, 0.4% | Thermo Scientific | T10282 | |
Trypsin-EDTA solution | Sigma-Aldrich | T4049-100ML | |
Ultrapure water | Merck | Milli-Q water | 18.2 MΩ・cm pure water |
Volumetric flask, 50 ml | Iwaki | 5640FK50E | TE-32 |