Establishment of stable cell lines and cell cultures
A3 cells (full name A337/311RP) and RsK4 cells were developed as described previously [30, 31]. The A3dnRhoA and A3dnMLC cell lines were prepared to stably express either a GFP-fused dominant negative RhoA (mutations F25N, T19N; [32]) from pEGFP-dnRhoA or a non-phosphorylable (dominant negative) GFP-fused MLC (mutations T18A, S19A; [33]) by selection in G418 at 400 μg/ml and subsequent FACS sorting of GFP-positive cells (FACSVantage SE, BD Biosciences). Cells A3GFP were prepared in the same fashion using only empty pEGFP vector. Rat sarcoma cells were cultivated in full DMEM medium: DMEM (GIBCO) with 4500 mg/l L-glucose, L-glutamine, and pyruvate, supplemented with 10% fetal bovine serum (Sigma), 2% antibiotic-antimycotic (GIBCO) and 1% MEM non-essential amino acids (GIBCO) kept at 37°C in a humidified atmosphere with 5% CO2. The cell lines PR9692, PR9692-E9 and PR9692-E9-MOCK were established as described previously [20]. The cell lines PR9692-dnRhoA, PR9692-dnMLCMLC, PR9692-MOCK, and PR9692-E9-caRhoA were prepared in the same fashion as PR9692-E9-MOCK using the appropriate SFCV-LE vectors (SFCV-GFP-dnRhoA, SFCV-dnMLC-GFP, SFCV-LE, SFCV-GFP-caRhoA, respectively), the PR9692 or PR9692-E9 cell lines, and the KUNDRA packaging cell line. After transfection, individual clones of G418 resistant cells were tested for the expression of appropriate constructs by immunoblotting. All chicken cells were maintained in Dulbecco‘s modified Eagle’s medium (DMEM, Sigma) supplemented with L-glutamine, penicillin, streptomycin, 4% fetal calf serum (PAA) and 2% chicken serum (Sigma) at 41°C in a humidified atmosphere with 5% CO2. Cells were treated with inhibitors 20 μM GM6001 (Sigma), 10 μM Y-27632 (Sigma), and 50 μM Blebbistatin (Sigma). The doubling time of cell lines was determined as a mean value of three doubling times counted in consecutive passages of cells in exponential phase of growth.
DNA constructs
SFCV-GFP-dnRhoA was constructed by introducing GFP-dnRhoA (EcoRI – NheI) from pEGFP-dnRhoA [32] between the XbaI and EcoRI sites of the pSFCV-LE vector. SFCV-GFP-caRhoA was constructed based on pEGFP-caRhoA (bearing RhoA with mutations F25N, G14V) [32] similarly to SFCV-GFP-dnRhoA. SFCV-dnMLC-GFP was constructed by introducing dnMLC-GFP (HindIII – NotI, Pfu polymerase blunted) from pEGFP-dnMLC [33] between the HindIII and EcoRV sites of the pSFCV-LE vector.
Immunoblotting
For protein analysis, cells were plated onto 100-mm dishes and grown until subconfluent. Before lysis, the cells were transferred into 15-ml tubes and centrifuged. The pellets were dissolved in 500 μl of LysBuf (20 mM Tris pH 6.8, 1% Triton X-100, 0.3% SDS, 5 mM EDTA, 10% glycerol, protease inhibitor cocktail Complete, EDTA-free from Roche) and the lysate was aspirated and transferred to a 1.5 ml tube. The lysates were cleared by centrifugation at 15,000 rpm for 20 min. Protein concentration was assayed by the bicinchonic acid method (Pierce), the lysates were diluted to equal concentration with LysBuf, mixed with 5× SB (300 mM Tris pH 6.8, 5% SDS, 360 mM 2-mercaptoethanol, 50% glycerol, 0.05% bromphenol blue) and incubated at 99°C for 10 min. Proteins (40 μg/lane) were separated on a 10% polyacrylamide gel by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Hybond-ECL). Membranes were probed with primary antibodies specific for GFP (sc-9996, Santa Cruz Biotechnology, 1:1,000), NPTII (#06-747, Millipore, 1:2,000) and MT-MMP1 (sc-12366, Santa Cruz Biotechnology, 1:1,000). Membranes were then incubated with the appropriate secondary antibodies (Jackson ImmunoResearch Laboratories) and subjected to enhanced chemiluminescence detection. For sequential detections, membranes were stripped with Re-Blot Plus Mild Antibody Stripping Solution (Millipore). Equal protein loading and transfer was verified by Ponceau-S staining of each membrane and by performing detection of GAPDH using antibody GTX30666 (GeneTex, 1:2,000) on the same membrane.
In-gel gelatin zymography
In total, 2 × 105 cells were plated per well in a 24-well plate. After 16 h, cells were washed with PBS and incubated in 300 μl of serum-free medium for 72 h. Aliquots (25 μl) of the conditioned medium were loaded for zymography on a 10% SDS-PAGE gel containing 1 mg/ml gelatin. Briefly, gel proteins were washed for 1 h in 50 mmol/l Tris–HCl (pH 7.5), 0.1 mol/l NaCl, and 2.5% Triton X-100 and then incubated at 37°C in 50 mmol/l Tris–HCl (pH 7.5), 10 mmol/l CaCl2, and 0.02% sodium azide for 17 h. The gels were stained with Coomassie blue and destained in 7% acetic acid/5% methanol.
In vitro cell invasion assays in 3D collagen
The 3D collagen invasion assay was analyzed as described previously [34]. Briefly, cell suspension (2 × 105 cells/ml) was added on top of a collagen gel in a multiwell plate, and after 48 hours the level of invasion was measured as the average invasion depth of the cells in the selected field of view using a Nikon-Eclipse TE2000-S (20×/0.40 HMC objective) and NIS-Elements software. For each experiment, invasion was analyzed in 3 wells and in 6 fields of view per individual well. In order to compare individual experiments, the average invasion depth was normalized to that of untreated cells. Three independent experiments were analyzed for each condition. Significance of differences was analyzed with ANOVA followed by Tukey’s honest significant difference test. The analysis was performed in version 2.15.3R (R Core Team, 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/).
Cell morphology assays in 3D collagen
To analyze cell morphology in 3D collagen, cells were trypsinized, washed in complete medium, counted, and then 105 cells were mixed with 500 μl of 3 mg/ml Collagen R in complete medium. This suspension of cells in collagen (500 μl) was loaded into a well of a 12-well plate, the gel was allowed to polymerize at 37°C for 30 min, and was then overlaid with complete medium. After 24 h the morphology of cells in 3D collagen was analyzed using the Nikon Eclipse TE2000-S microscope (20×/0,40 HMC objective). Cell morphology was classified on the basis of the elongation index. The elongation index was calculated as the length divided by the width. Cells whose elongation index was greater than 3 were considered elongated. Intermediate cells had an elongation index of 2–3; for rounded cells, the index was 1–2. Dividing cells were excluded from the analysis. Three independent experiments (at least 300 cells per experiment) were analyzed for each condition. As the data have the form of counts in categories, the Pearson's Chi-squared test was used to reveal statistically significant differences.
Dermis-based matrix
For immunofluorescence staining the following procedure was used: two days before use the dermis-based matrix (XeDerma®; BIO SKIN a.s.) was cut into small pieces (approx. 1x1 cm), placed into 12 wells plates with HBSS buffer, and just prior to use washed twice with DMEM medium and cells were seeded on the epidermal side of the dermis. After incubation the dermis was washed with PBS and fixed. For labeling the dermis was pre incubated for two days in HBSS buffer. The conjugation of FITC (1 μg/ml; Molecular Probes) was performed in 0.1 M sodium bicarbonate buffer, pH.9.0 for 30 min and then the unconjugated dye was washed off three times with PBS and two times with DMEM.
Scanning electron microscopy
Cells in full DMEM medium were grown on dermis-based matrix for 24 hours, and for the next 24 hours in serum free DMEM. The dermis-based matrix with cells was washed two times in PBS, fixed in 2.5% glutaraldehyde, and washed again three times. Dehydration in increasing concentrations of ethanol (10 min. each for 30%, 50%, 70%, 80%, 90%, 95 and 100%) was followed by critical point drying using CPD 030 (BAL TEC), coated with 3 nm gold on Sputter Coater SCD 050 (BAL TEC) and visualized by SEM on a JEOL 6380 LV. Rat fascia freshly removed from an adult rat was immobilized in a frame, positioned inside 6-well plates, and cells were seeded on top. After 2 days the whole frame was fixed and processed the same ways as dermis-based matrix.
Immunofluorescence microscopy
Cells on epidermal side of dermis-based matrix were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X 100, washed thrice with PBS, and stained for 15 min with Alexa 594 phalloidin (Molecular Probes) and then washed thrice with PBS and mounted in Mowiol containing 4',6-diamidino-2-phenylindole (DAPI, Sigma). Images were acquired by a Leica TCS SP2 microscope system using a Leica 20×/0.7 oil objective.
Animals
Experiments were done with the Prague inbred chicken line CC.R1 [35]. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of the Academy of Sciences of the Czech Republic. Chicks were kept under standard laboratory conditions with free access to food and water.
Monitoring of tumor weight and metastases
Chickens were inoculated by injection into the outer area of the pectoral muscle at an age of 3 weeks with 5×105tumor cells that had been freshly harvested from the tissue culture and resuspended in 0.2 ml of cultivation medium. The weight of the primary tumor and spontaneous metastatic activity of each tumor cell line were determined by examining chickens autopsied from 21 to 35 and from 28 to 45 days after inoculation with cells derived from the PR9692 cells and PR9692-E9 cells, respectively. The time of autopsy reflected the health status of the animals. Metastases were observed by gross inspection and using a dissection microscope. The experiments were performed several times with a total number of at least 33 animals in each group, except for the control groups where the numbers were slightly reduced (26) to spare animals.