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Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102 Page 5 of 14

Glucose uptake and lactate production
Cells were cultured and transfected in 3-dimensional Matrigel with physiological modified medium for
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72 or 96 h and medium was changed daily. Glucose and lactate concentrations in the culture medium were
determined by fluorometric based Glucose Assay Kit and Lactate Assay Kit (BioVision, Inc., Milpitas, CA)
according to the vendor’s instructions using Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski,
VT). The amount consumed or produced by cells was determined by comparing the concentration in the
medium incubated without cells and then normalized to cell number. These assays were performed three
times with two experimental replicates for each cell type.

Western blot and antibodies
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Cells were collected form 3-dimensional Matrigel culture (as described previously) and lysed with RIPA
buffer (TEKnova, Hollister, CA) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo
Scientific, Rockford, IL). The lysates were clarified by centrifugation at 4 °C for 10 min at 12,000 ×g. The
concentration of protein in the supernatants was measured using BCA assay (Thermo Scientific Pierce,
Rockford, IL). Total proteins (20 μg/lane) were separated on a 10% SDS-PAGE and were transferred to
nitrocellulose membrane. The membranes were blocked with 5% BSA in 1× TBST (β-actin) or with 5%
non-fat milk (MCT1, MCT4 and Cleaved PARP) and subsequently incubated with primary and secondary
antibodies according to the vendor’s instructions. Blots were visualized by using Amersham ECL prime
(GE Healthcare Life Sciences, Buckinghamshire, UK) and imaged using G:box imaging system (SynGene,
Cambridge, UK). Primary antibodies to Cleaved PARP (1:1000) was given by Dr. Claffey from Cell Signaling
Technology (Danvers, MA); β-actin (1:2500) and MCT1 (1:1000) were purchased from Abcam (Cambridge,
MA). MCT4 (1:1000) was purchased from Millipore (Billerica, MA). All images were imported to Image J for
pixel grayscale intensity analysis. Western blot analysis was performed three times using two independent
protein samples for each cell group.

Nuclear magnetic resonance spectroscopy and data analysis
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Cells were collected from 3-dimensional Matrigel culture (as described previously) and lysed with 1:1:1
methanol/chloroform/water in sequence with 5 s of gyration between additions. In order to optimize
chemical extraction, non-trypsin-based methods were employed as follows . A 10 µL sample was then
[31]
removed for cell counting. Resulting lysed solution was then allowed to separate overnight at 4 °C to
form resultant aqueous and organic layers. Aqueous layer containing metabolites of concern were then
manually extracted and resulting solution was subjected to addition of Chelex (approximate 3 mg/mL)
for removal of divalent ions, which was then removed through vacuum filtration using Buchner filter.
Solution was then lyophilized overnight to form solid components. 800 µL of D2O with 10 mmol/L EDTA
and 1 mmol/L 4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added and solution was centrifuged
at 3000 rpm for 5 min prior to placement in standard 5 mm nuclear magnetic resonance (NMR) tube for
1H NMR.

1H NMR experiments were performed on 500 MHz Agilent spectrometer with HCN cold probe and a
VNMRS console. Default pre-saturation pulse sequences were utilized with the following parameters: pulse-
width = 7.2 µs, sweep-width = 8389.26 Hz, 2460 complex points, D1 = 1.5 s, SATPWR = 6 dB on H2O signal,
and 64 transients. Resulting 1H NMR underwent Fourier Transformation and data was further analyzed
in MestReNova NMR software, version 9.0.0. Processing template for all spectra had the following time
domain corrections: drift correction to tail points of 5% and apodization through an exponential of 1.5 Hz.
The frequency domain had global phase correction and a baseline correction using Bernstein polynomial
of n = 3. Reference signal was set as the singlet produced off of DSS at 0 ppm. Lactate and Pyruvate were
identified on the spectra through prior titration analyses and the absolute area under the curve was used for
their methyl groups with respect to methyl groups found off of DSS with the usage of Line-Shape analysis.
These areas were used to calculate concentrations by a ratio of the absolute area to the number of protons