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Materials and Methods
Chemicals
MMP inhibitor III is a reversible inhibitor of MMPs with higher specificity for MMP-1 and MMP-2. Before MMPs inhibition assays, different concentration of the MMP inhibitor where added to cells, at different time-points and cell viability was studied. Since no toxic effects were observed, further cells were treated, for 48 hours, with 10nM and 100nM of MMP III inhibitor or only with DMSO (which was used as drug vehicle).
Recombinant human P-cadherin/Fc chimera (hrPcad, R&DSystems, Inc,Minneapolis,USA) is the extracellular domain of human P-cadherin fused to the carboxi-terminal 6x histidine-tagged Fc region of human IgG, via a polypeptide linker. The hrPcad was added to the cells at a final concentration of 20g/ml.
Wound-healing Assay
For the wound-healing assay, cells were seeded in a 6-well plate. When these became confluent, wounds were carefully made across the cell monolayer, so that the surrounding cells were not disturbed. The medium was replaced by fresh complete medium and cells were incubated at the referred conditions. The cells were monitored along time, in order to observe their migratory behaviour. Quantitative measurements were made at several time-points of culture: 0, 1, 2, 3, 4, 5 and 6 hours, by determining the distances between the wound-edges. Photographs were taken under the phase contrast microscope, using a 400X magnification.
To exclude that difference in cell migration was due to cell proliferation, BrdU incorporation proliferation assay was done. Briefly, cells were cultured in coverslips, incubated during 1h with BrdU and fixed with 4% formaldehyde (during 20 minutes). After washing with PBS, cells were treated with HCl 2M for 20 min at RT. Following three more washes with PBS-0,5% Tween-0,05%BSA, cells were incubated with 1h with anti-BrDU primary antibody (Dako Cytomation, Carpinteria, CA) and after incubated with anti-mouse FITC secondary antibody. After a wash with PBS, each sample was mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA) containing 4,6-diamidine-2-phenylindolendihydrochrolide (DAPI) and number and percentage of proliferating cells wascalculated.
Time-lapse Microscopy for Wound-healing Assay and Motility Assay
Migration and motility of culture cells were monitored and analysed using time-lapse microscopy. Cells were seeded in a 60mm glass bottom dish and were introduced in a micro-chamber module, connected to a temperature and CO2controllers (Temp control 37-2 digital and CTI-controller 3700 digital, Pecon, Leica, Bensheim, Germany). A camera and time-lapse controller (Leica FW 4000) were attached to an inverted microscope (Leica DMIRE 2), and sequential and single images, at 400X magnification, were acquired at intervals of 5 minutes, during 6 hours.After the complete acquisition of time-lapse images, movies were edited with the series of captured and sequential images (see supplementary data). For the motility assay, the position of individual cells was determined manually, based on the centre of the nuclei. Then, the distance moved by the cells was determined, as well as the cell speed (m/hour). For the wound-healing assays, measurements were done between the wound edges at several time points.
Immunofluorescence and Confocal Microscopic Analysis
In order to observe cell phenotype and the aggregation capacity of the cells, these were stained cells by immunofluorescence. Briefly, cells were cultured on glass coverslips, and fixed with 4% formaldehyde (during 20 minutes). After fixation, cells were treated with 50mM NH4Cl for 10 minutes, washed with PBS, and permeablilized with 0.1% Triton X-100 in PBS for 5 minutes, at room temperature. Non-specific binding was blocked by cell treatment with PBS containing 5% BSA, for 30 minutes, at room temperature. Cells were then stained with rhodamine-phalloidin (Invitrogen) in order to visualize F-actin cytoskeleton or with a rabbit polyclonal antibody for P-cadherin, during 1 hour and conjugated with an Alexa 488 as secondary antibody. To study cell aggregates, cells were stained with fluorescein isothyocianate (FITC)-phalloidin (Sigma, St Louis, MO) for 15 minutes and with a mouse monoclonal antibody to stain p120ctn expression for 1h, which was then stained with goat Texas Red-conjugated anti-mouse secondary IgG (Dako Cytomation), for 1 hour, at room temperature. After a wash with PBS, each sample was mounted with vectashield with DAPI. The p120ctn and F-actin dual cell staining was observed with a Leica DM 2000 microscope or with an Olympus Fluoview 1000 Confocal microscope (Olympus, Germany). For confocal microscopy, the 40X lens was used and successive 0.5 m optical sections were taken. Images were then processed and analysed with FV10-ASW 1.4 viewer software (Olympus). For P-cadherin and F-actin single staining, a Zeiss microscope (Imager Z1)with apotome was used, and images were taken using the Axiovision software.
Gelatin and -Casein Zymography
The conditioned medium collected from the several cell cultures, which were grown 6-well plates coated with collagen type I, was analysed for proteinases activity using gelatin and β-casein zymography.Gelatin gels were loaded with 12 μg of protein per sample and β-casein gels with 100μg of protein per sample. Samples were mixed with sample buffer [0.03% bromophenol blue, 0.25M Tris-HCl pH 6.8, 10% SDS (w/v) and 4% sucrose (w/v)] and electrophoresed, under non-reducing conditions, on 10% polyacrilamide gels containing 0.1% (w/v) gelatin or β-casein from bovine milk (Sigma). After electrophoresis, gels were washed twice, for 30 minutes, in 2% (v/v) Triton X-100 (Sigma) at room temperature, in order to remove SDS. Then they were incubated in a Substrate Reaction Buffer for 20h in case of the gelatin gels 50 mM Tris-HCl, 5 mM CaCl2, pH 7.5 or 72h for β-casein gels 0.2M NaCl, 5 mM CaCl2, 1% (v/v) Triton X-100 in 50mM Tris-HCl, pH 7.4,and finally stained with Coomassie Blue Staining Solution [0.1% (w/v) Coomassie Blue R250 in 10% (v/v) acetic acid and 40% (v/v) methanol], for 25 minutes. The gels destaining was performed in a solution with 20% methanol and 10% acetic acid, until bands start to become visible. Enzymatic activity was visualized as a clear band against the blue background of stained casein gels, and MMPs were identified by their molecular weight. Quantification of band density was carried out using the Quantity One software (version 4.0, BioRad, Hercules, CA).
Immunoprecipitation and Western Blot
Protein lysates were prepared from cultured cells, using catenin lysis buffer [1% (v/v) Triton X-100 and 1% (v/v) NP-40 (Sigma) in deionised PBS] supplemented with 1:7 proteases inhibitors cocktail (Roche Diagnostics GmbH, Germany). Cells were washed twice with PBS and were allowed to lyse in 500 μl of catenin lysis buffer for 10 minutes, at 4°C. Cell lysates were submitted to vortex 3 times and centrifuged at 14000 rpm and 4°C, during 10 minutes. Supernatants were collected and protein concentration was determined using the Bradford assay (BioRad protein quantification system).
For sP-cad immunodepletion, 300µl of conditioned medium, from collagen-cultured MCF-7/AZ.Pcad cells, was incubated with 1µg of P-cadherin antibody (NCC-CAD-299), in a rotor shaking, at 4°C and overnight. After this, 40µl of IgG-coated beads (Amersham Biosciences, Buckinghamshire, UK)were added to each sample, and incubated for 1 hour at 4°C. A second run of immunoprecipitation was performed under the same conditions, during 2 hours.
Proteins were dissolved in sample buffer [Laemmli with 5% (v/v) 2-β-mercaptoethanol and 5% (v/v) bromophenol blue] and boiled for 5 minutes at 95°C. Samples were separated by an 8% SDS-PAGE, and proteins were transferred into nitrocellulose membranes (Amersham Hybond ECL) at 130 V for 1 hour. For immunostaining, membranes were blocked with 5% (w/v) non-fat dry milk in PBS containing 0.5% (v/v) Tween-20. These were subsequently incubated with primary antibodies, during approximately 1-2 hours, followed by four 5 minutes washes in PBS/Tween-20 (PBS-T) and incubation with horseradish peroxidase-conjugated secondary antibodies, during 45 minutes. Proteins were detected using ECL reagent (Amersham), as a substrate, and blots were exposed to an autoradiographic film. Quantification of western blots was performed using Quantity One software (BioRad), and the ones selected to show are representative experiments.
Matrigel Invasion Assay
Matrigel invasion assay was performed using 8 µm pore size BD BioCoat™ Matrigel Invasion Chambers (BD Biosiences, NJ, USA). In the upper compartment of the chamber, 3.5 x 104 cells were added, whereas in the lower compartment, only fresh medium supplemented with 10% FBS was present. After 24h (for BT-20 cells) or 48 hours (for MCF-7/AZ cells) of incubation at 37°C, the upper surface of the filter was cleared from non-migratory cells with a cotton swab and washed with PBS. The remaining (invasive) cells, which were attached to the lower surface of the filter, were fixed with cold methanol and stained with DAPI. Invasive cells were scored by counting the cells in the filter with a fluorescence microscope (Leica DM 2000), at 200X of magnification. The highly invasive CHO cell line was routinely included as a positive control for the invasion assays. Invasion index expresses the ratio between invasive cells from the different conditions relative to non-invading MCF-7/AZ control cells.