Cell culture, cell lines, and chemicals
We used human cell line RMF-621, which corresponds to an hTert-immortalized fibroblasts derived from a reduction mammoplasty; obtained via a generous gift from Dr. Charlotte Kuperwasser (Tufts University, MA). Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Invitrogen, Carlsbad, CA), supplemented with antibiotics and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and maintained in a humidified atmosphere of 37°C, 5% CO2 (15). MCF-7 and MDA-MB 231, both human breast cancer cell lines, were purchased in ATCC (Manassas, VA), cultured in DMEM/F12 supplemented with 10% FBS and maintained in the same condition as above. MCT1 inhibitor AR-C155858 was acquired from Chem Express (Princeton, NJ) and diclofenac was purchased on Sigma (St. Louis, MO).
Plating of cells in matrices of different stiffness.
In all the experiments in which RMF 621 cells were cultured in matrices with different stiffness, cells (2 X 105) were pre-cultured for 3 days in media enriched with 10°/o FCS plated on 0.2 kPa low rigidity matrix (Advanced Biomatrix, San Diego, CA) previously coated with 50 µg/cm2 of type I collagen, to simulate the normal (soft) condition of breast tissue. After this, cells were released with trypsin and transferred to plates with increased stiffness (0.2–32 kPa) for an additional 72 h period before being lysed for western blot. In experiments in which media conditioned from these cultures were collected, enriched culture media was replaced by serum-free media during the last 24 h of culture before collection.
Preparation of protein-coated gel substrate.
To measure the effect of ECM rigidity on glucose and lactate transport, we used compliant elastic polyacrylamide gels as substrate material to which ECM molecules can be covalently-linked . For these studies, preactivated coverslips were used to prepare matrices which are cross-linked with 2 mM sulfo-SAMPAH and activated with 7,500 J of UV light for 8 min. After rinsing with PBS, matrices were incubated overnight at 4ºC with 50 µg/cm2 of type I Collagen (Advanced Biomatrix, San Diego, CA) and then washed with PBS. Once collagen is coupled, matrices on coverslips were rinsed with sterile PBS and cells were seeded on top.
To evaluate glucose transport in cells seeded on coverslips coated with extracellular matrices with different stiffness, we used the FRET nanosensor (FLIIPglu60060Δ6) incorporated in adenoviral particles (Vector Biolabs, Philadelphia, PA) which exploit resonance energy transferred between a coupled pair of cyan and yellow fluorescent proteins (eCFP, eYFP). This system detects conformational changes induced by a glucose-binding domain derived from chemotactic receptors of bacteria, namely the glucose/galactose-binding protein of Escherichia coli (MglB) .
To evaluate lactate transport in living cells, we used the FRET nanosensor Laconic  incorporated in adenoviral particles (Vector Biolabs, Philadelphia, PA). Laconic is a fusion protein composed of a ligand-binding moiety (LldR) specific to lactate and the fluorescent proteins mTFP and Venus. Imaging was carried out using a Nikon Ti microscope with a 40 X objective equipped with a monochromator (Cairn Research, Kent, UK), which allows discrete excitation at 430 ± 10 nm. Two windows of emitted light were simultaneously collected at 490–520 nm (mTFP) and over 535 nm (Venus) by means of an optical splitter (Cairn Research). Images were digitized by a CCD camera (ORCA3, Hamamatsu, Japan) and data expressed as the ratio between mTFP and Venus fluorescence. Experiments were conducted 48–72 h after infection at room temperature (24–26°C) in KRH buffer (in mM: 140 NaCl, 4.7 KCl, 20 Hepes, 1.25 MgSO4, 1.25 CaCl2, pH 7.4) supplemented with lactate.
Cell motility assays
Low migrating mammary epithelial MCF-7 and highly invasive MDA MB-231 cell lines were used as a model of differential migration using a 6.5-mm Transwell chamber with a pore size of 8 mm (Corning, Corning, NY). In these experiments, 5 x 104 cells were allowed to migrate (MCF-7 for 24 hours and MDA MB-231 for 16 hours) to the stimulus of conditioned medium by RMF-621 cells previously cultured as described above, on increased stiffness conditions (0.2, 2.0, 16 and 32 kPa) diluted at 50% in culture media enriched with 1% FCS. To analyze the effect of stromal-derived lactate on epithelial migration and the role of MCT1 and MCT4 monocarboxylate transporters, a group of both cells were stimulated to migrate under the stimulus of stromal conditioned media in the presence of 1 µM AR-C155858 (inhibitor of MCT1 and MCT2) or 1 mM diclofenac a structurally-unrelated MCT1 & MCT4 blocker. After migration period, Transwell membrane was fixed in methanol and migratory cells were stained on the lower side of the membrane with 0.2% crystal violet . Migration values correspond to the average of three independent experiments by counting 16 fields from four pictures (X20) per chamber (two chambers per experimental condition).
Lactate was evaluated in conditioned media by RMF-621 cells (1 x 105 cells in a 6 well plate) cultured on matrices of increasing stiffness of 0.2, 2, 16 and 36 kPa. To do so, we previously cultured RMF-621 cells on 0.2 kPa low rigidity matrix as described and then, cells were transferred to a set of dishes coated with matrices of increasing stiffness of 0.2, 2, 16 and 36 kPa. Lactate production was evaluated after 72 h of culture on these matrices of different stiffness levels. In the last four hours of culture, culture media was changed to phenol red-free media in the absence of serum, and lactate abundance in these media was evaluated by gas chromatography coupled with mass spectrometry, GC-MS equipped with a capillary column HP5M. Briefly, an internal standard was added to each sample and the organic acid was oximated and extracted twice with Ethyl acetate as previously described . The organic fraction obtained was dried under nitrogen and derivatized with BSTFA and TMS (1%). Afterwards, one microliter of derivatized sample was injected in the GC-MS. Each metabolite was identified based on its own mass spectra by matching with a spectral library of known metabolites from NIST, NIH. Quantification of lactic and pyruvic acids was carried out through prior elaboration of a calibration curve (quadratic equation) with increasing amounts of each metabolite normalized to Tropic Acid (Internal Standard).
Matrix stiffness measurements
To measure the elastic modulus of matrices used as cell support, an indentation method was implemented. A glass sphere that is pushed against the matrix at constant speed was selected to ensure a defined geometry for indentation. Compression force was determined through a load cell, Futek LBS200 S-Beam, operating in the range of 2N, provided with a signal conditioning module and the respective interface for the conversion and transmission of data to the computer, through the USB computer port. The sample (1 mm thick and 1 cm diameter) was positioned horizontally under the spherical indenter (5 mm diameter). To press the sample, the indenter is displaced by means of a micro-control system, with 1 µm resolution, at speed of 10 µm/s.
The resulting force, F, as a function of the penetration distance, δ, is modeled by the Hertz’s relation,
With , where E is the Young’s modulus and ν is the Poisson’s coefficient of the sample, which is assumed to be close to ν = 0.5. R is the indenter’s radius of curvature at sample contact. E is assessed through a standard fitting procedure of the F vs δ curve that is well represented by the Herzt’s formula. The values obtained for substrata prepared at two distinct reticulation periods were:
Sample 1: E = 4.5 ± 0.5 kPa
Sample 2: E = 31.0 ± 0.5 kPa
All force measurements were obtained in samples immersed in PBS buffer under the same conditions to those of the cell cultures.
Western blot and antibodies.
The expression of MCT4 and Glut 1 protein in RMF-621 cells was evaluated by Western-blot. Briefly, cells were lysed in lysis buffer (30 mM Tris-HCl pH 7.5, 5.0 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycolate, 0.1% SDS and 10% glycerol) supplemented with complete protease inhibitors (Roche, Mannheim, Germany). Pellets were incubated for 1 h in lysis buffer at 4°C, and then centrifuged at 14000 g for 15 m at 4°C, keeping the supernatants. The protein concentration of cell lysates was determined using Pierce BCA Protein Assay kit (Thermo, Rockford, IL). Protein extracts were denatured in sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis loading buffer 4 X (240 mM Tris–HCl, pH 6.8, 8% SDS, 40% glycerol, and 20% 2-mercaptoethanol), incubating the samples for 1 h at 37°C. Equal amounts of protein from different treatments were resolved by SDS–polyacrylamide gel electrophoresis in 10% acrylamide gels and electrotransferred to polyvinylidene difluoride membranes using a buffer containing 24 mM Tris, 194 mM glycine, and 20% methanol. Proteins were further analyzed using the Supersignal West Dura Extended Duration Substrate (Thermo, Rockford, IL). Immunoreactions were achieved by incubation of the membranes, previously blocked with a solution containing 5% bovine serum albumin in Tris-buffered saline and 0.05% Tween 20 (Sigma, St. Louis, MO), with anti MCT4 (D-1) mouse monoclonal antibody (sc-376140) from Santa Cruz (Santa Cruz, CA), anti MCT1 rabbit polyclonal antibody (M4470-01B) from US Biological (Salem MA), anti Glut1 mouse monoclonal antibody (MAB14181) from R & D (Minneapolis MN) and mouse anti-alpha tubulin (T5168) from Sigma (St. Louis MO). Densitometric analysis of western blot bands was performed using C-Digit Blot Scanner and Image Studio Digits software v.5.2 from LI-COR Biosciences (Lincoln, NE).
Human Breast Cancer Samples. Breast cancer formalin-fixed samples and paraffin-embedded samples were obtained from the repository of the Arturo Lopez Perez Foundation (FALP, Santiago, Chile). Informed consent was obtained from all individual participants included in the study with the supervision and approval of the Ethics Committees at INTA, University of Chile and FALP. Samples were obtained before the beginning of chemotherapy.
Unless otherwise specified, experiments were repeated between three and five times. All data are presented as means ± standard error of the mean (SEM). Statistical analyses were carried out using GraphPad Prism software version 8.0.2 (GraphPad Software Inc.). Differences between groups were evaluated with the Kruskal-Wallis or Friedman analysis followed by Dunn’s multiple comparisons test. A p-value lower than or equal to 0.05 was considered statistically significant and indicated with one, two or three asterisks: * = P ≤ .05, (**) = P ≤ .01 and (***) P ≤ .001.