Cell culture and reagents
Human BC cell lines, MCF-7 (ATCC® HTB-22), BT-474 (ATCC® HTB-20), SK-BR-3 (ATCC® HTB-30), MDA-MB-468 (ATCC® HTB-132), MDA-MB-231 (ATCC® HTB-26), HCC1937 (ATCC® CRL-2336) derived from different breast cancer subtypes were purchased from ATCC (Manassas, VA, USA), and SUM149 (Asterand SUM-149PT) from Asterand (Hertfordshire, UK) in 2013.The cell lines were authenticated by provider with morphology, karyotyping, and PCR based approaches. The subtypes of each cell lines were further authenticated with determining ER, PgR and HER2 expression in a recent paper (27). Cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Nakalai, Kyoto-shi, Kyoto-fu, Japan) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, GE Healthcare, Logan, UT, USA), 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (Gibco, Gaithersburg, MD, USA) in a humidified atmosphere with 5% CO2 at 37°C. Cells in logarithmic growth phase were seeded at an appropriate density, and used for all experiments. 2-deoxy-D-glucose (2-DG) (Sigma-Aldrich, St. Louis, MO, USA), 3-Nitropropionic Acid (3-NA) (Cayman Chemical, Ann Arbor, MI, USA), CB-839 (Cayman Chemical), Phloretin (Tokyo Chemical Industry, Nihonbashi, Tokyo, Japan) were the inhibitors used in this study.
Transwell Invasion assay
The invasive potential of seven BC cell lines were examined as previously described (28). Briefly, matrigel was added to culture insert of transwell chambers containing a 6.5-mm filter with a pore size of 8 μm (Corning, NY, USA), and 1x10^5 cells were suspended with serum-free RPMI were seeded onto it. RPMI with 10% FBS was added to the lower well, and the invasion assay was performed for 24h. Invasive cells that reached the undersurface of the transwell membrane were fixed and stained with Diff Quick (Sysmex, Kobe, Japan). The transwell membrane was photographed under bright field using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) using a Nikon Plan Apo 4x/0.2 lens. Numbers of invaded cells in each field were counted with the particle counting application of ImageJ software (Version 1.52q) (28). Percentage of invaded cells was calculated by dividing the number of invaded cells with the number of seeded cells on the transwell membrane.
For inhibitor studies, cells were pre-treated with 2-DG, 3-NA, or CB-839, and incubated for 16 h. Cells were then trypsinized, suspended in serum-free RPMI with appropriate inhibitor, and used for the invasion assay. Inhibitor was also added to the lower well, and the invasion assay was performed for 24 h. In addition, surviving fraction of cells treated with each inhibitor for 16 h was examined by counting viable cells using trypan blue staining.
INV preparation and re-invasion assay
To prepare the invaded cells of SUM149, transwell invasion assays were performed as described previously (29). Briefly, cells were trypsinized and viable cell numbers were counted using trypan blue staining to exclude dead cells and separated into two groups; one set was for the whole cultured cells (WCC) group, the other set was for preparing the invaded cells (INV). For WCC, cells were suspended in RPMI with 10% FBS, and plated on the culture dish at the appropriate density. For preparation of INV, cells were suspended into serum-free RPMI, and 5 × 10^5 cells were seeded into upper well of each transwell chambers (the 24 mm transwell insert diameter with a pore size of 8 µm, Corning) coated with Matrigel. RPMI supplemented with 10% FBS was added to the lower well as a chemoattractant. After 24 h, the non-invasive cells remaining on the Matrigel-coated side were wiped off with a cotton swab, and the cells that moved through to the undersurface of the transwell membrane were collected by incubating the cells with Accutase (Innovative Cell Technologies, San Diego, CA, USA) for 30 min at room temperature. INV collected from several transwells were pooled, suspended in RPMI with 10% FBS and plated on the culture dish at the appropriate density. For the re-invasion assay, WCC and INV were trypsinized on day 1, 4, 7, 12, or 19 after cultivation, and the transwell invasion assay was repeated with these cells as described previously (30, supplemental figure 1).
Sample preparation for the metabolome analysis
INV and WCC (1 x 10^5 cells/sample for the invaded cells, and 1 x 10^6 cells/sample for the whole cultured cells, respectively) were used for the extraction of intracellular metabolites (29). Cells were collected by centrifugation at 1,000 × g for 5 min at room temperature and washed twice with 5% mannitol solution. Cells were then treated with methanol to inactivate enzymes. Cell extract was treated with milliQ containing internal standards (Human Metabolome Technologies, Inc., Tsuruoka, Yamagata, Japan). The extract was centrifuged at 2,300 × g and 4 ºC for 5 min, and the aqueous layer was filtered through a Millipore 5-kDa cutoff filter (Merck Millipore, Billerica, MS, USA) at 9,100 × g and 4 ºC for 120 min. The concentrated filtrate was then re-suspended in 50 µL of milliQ for the CE-MS analysis.
Metabolome analysis by CE-TOFMS
CE-TOFMS was performed using an Agilent CE Capillary Electrophoresis System with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany), as described in previous papers (31- 33). The systems were controlled by the Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). The metabolites were analyzed by using a fused silica capillary with the electrophoresis buffer (Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 37.5mmHg (50mbar) for 10 seconds in cation analysis and 25 seconds in anion analysis. The scanning of spectrometer was performed from m/z 50 to 1,000.
Metabolome data analysis
Raw data measured by CE-TOFMS was analyzed using the MasterHands software (Keio University, Tsuruoka, Japan), and peak information including m/z, migration time for CE-TOFMS measurement (MT), and peak area were obtained (34). Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded, and the remaining peaks were annotated with the putative metabolites from the HMT metabolite database (Human Metabolome Technologies) based on their MTs and m/z values determined by TOFMS. The tolerance range for the peak annotation was configured at ±0.5 min for MT, and ±10 ppm for m/z. In addition, peak areas were normalized against those of the internal standards, and the resultant relative area values were normalized by the sample amount. Principal component analysis (PCA) were performed by the software, SampleStat (Human Metabolome Technologies).
Immunoblotting
Immunoblotting was performed as described previously (35). Briefly, cells were lysed in 2´ Laemmli sample buffer, followed by electrophoresis using the Novex® NuPAGE® SDS-PAGE Gel system (ThermoFisher Scientific, Waltham, MA, USA). Primary antibodies against Pyruvate Dehydrogenase E1-alpha subunit (9H9AF5) (PDH), phosphorylated-Pyruvate Dehydrogenase E1-alpha subunit Ser300 (pPDH S300), Ser232 (pPDH S232), or Ser239 (pPDH S239) (Abcam, Cambridge, UK) were used along with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Biosciences; Buckinghamshire, UK). Bands were detected by enhanced chemiluminescence and visualized with a Lumino image analyzer, LAS 4000 (Fujifilm, Tokyo, Japan) using the ImageQuant LAS 4000 Control Software.
Flow cytometry
In order to examine the glucose uptake into living cells, 2-NBDG (2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, Cayman Chemical), a fluorescent derivative of glucose, was used. For WCC, cells were separately cultured as 3 groups; control, 2-NBDG treating, or 2-NBDG + Phloretin (inhibitor for the glucose transporter) treating groups. On the day of flow cytometry experiment, cells were washed with PBS and medium were changed to fresh RPMI supplemented with 0.5% FBS with or without 100 μM Phloretin. After 4 h incubation, 60 μM 2-NBDG was added to 2-NBDG group, and 2-NBDG + Phloretin group, and were incubated another 1 h. Cells were then washed with PBS, incubated with Accutase for 15 min (Innovative Cell Technologies Inc.), collected and used for flow cytometry analysis in accordance with the manufacturer’s instructions (CytoFLEX S with Analysis Software; Beckman Coulter). For INV, invasion assay was performed one day before the flow cytometry experiment. After 24h INV cells underneath the transwell were directly treated with 2-NBDG or 2-NBDG + Phloretin. WCC were also subjected to same treatment groups. Cells were collected with Accutase for 15 min and used for the flow cytometry analysis.
Immunofluorescence labeling and image acquisition
Immunofluorescence labeling, and image acquisition was performed as described previously with some modifications (28). Briefly, cells were cultured on glass slide chamber with phenol red free RPMI at appropriate density. On the day of immunofluorescence experiment, cells were washed with PBS, and medium was changed to fresh phenol red free RPMI with or without 3-NA. After 5 h incubation at 37℃/5% CO2, 5, 5', 6, 6'-tetrachloro-1, 1', tetraethylbenzimidazolocarbocyanine iodide(JC-1, mitochondrial membrane potential detection indicator, ThermoFisher Scientific)and NucBlue® Live ReadyProbes (Nuclear staining solution, ThermoFisher Scientific) were added to each well, and incubated for 30 min. Cells were then washed with PBS and fresh phenol red free RPMI was added, and images were acquired using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) using a 10X PlanFluor NA 0.30 Ph1 lens with BZ filters for TRITC, GFP-B, and DAPI. Representative images were uniformly processed in Creative Cloud Photoshop CC using the brightness and contrast tools.
Lactate measurement
Cells were separately cultured as 3 groups; control, 0.3 mM 2-DG treatment, or 1 mM 2-DG treatment groups. On the day before the experiment, cells were washed with PBS and medium was changed to fresh RPMI with appropriate concentration of 2-DG. After 16 h incubation, conditioned medium was collected and used for the assay according to the manufacture’s protocol (Lactate Assay Kit-WST, Dojindo).
Spheroid invasion assay
Spheroid invasion assay was performed in ultra-low attachment multiple 96 well plates (Sigma-Aldrich) (30). Cells (5 x 10^3) were plated in each well and incubated in 37℃ CO2 incubator. After 48 h, spheroids were stained with JC-1 (1:50 concentration) for 30 min, embedded into phenol red-free collagen solution (custom version 3D Ready Atelocollagen, KOKEN CO., LTD., Bunkyo-ku, Tokyo, Japan), followed by 1 h incubation at 37℃/5% CO2 to allow solidification of collagen solution. Image of spheroid was captured at 1 h and 24 h after embedding into collagen gel and fluorescent signal was visualized and photographed with a BZ-9000 fluorescence microscope using a 4X PlanApo λ NA 0.20 lends with BZ filters for TRITC, GFP-B (Keyence, Osaka, Japan). Representative images were uniformly processed in Adobe Photoshop using the brightness and contrast tools.
Statistical analysis
All results are expressed as the mean +/- SD. Statistical analyses were performed using unpaired Student’s t-test. P value of <0.05 was considered significant.