Materials and cell lines
Human breast cancer cells, MCF7 and MDA-MB-231 were from the ATCC (American Type Cell Collection, Manassas, VA, USA). Fresh frozen human breast tissues were collected from University Hospital of Wales under the approval of the local ethical committee, obtained immediately after surgery and stored at -80°C until used.
Antibodies to human FAP-α (sc-100528 and ab5066), FAK (sc-1680) and pFAK (sc-11765-R were from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA or Abcam, Cambridge, UK). ROCK inhibitor was from Santa-Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA), ERK inhibitor, Wortmannin, and Wiskostatin were from Calbiochem (Nottingham, UK). Matrigel (reconstituted basement membrane) was purchased from Collaborative Research Products (Bedford, MA, USA). Transwell plates equipped with a porous insert (pore size 8 μm) were from Becton Dickinson Labware (Oxford, UK). RT-PCR reagents and plasmid extraction kits were from Sigma (St. Louis, MO, USA).
Construction of expression vector of human FAP-α and transfection of breast cancer cells
Touch-down PCR was used to generate the cDNA of FAP-α from human prostate tissues with primers 5’-TTAGTCTGACAAAGAGAAACACTG and 5’-ATGAAGACTTGGGTAAAAATCG. The cDNA of FAP-α was subsequently cloned into a pEF6/V5-His vector (Invitrogen, Paisley, Scotland, UK). The new plasmid, named pEF6/V5- FAP was amplified in E. coli and verified by PCR reaction by using a pair of different primers 5’-AGAGCTTTAGCAATCTGTGC and 5’-TCCCTTGCTAATTCAAGTGT.
Breast cancer cells MCF7 and MDA-MB-231 were cultured in DMEM media. The cells were transfected with plasmid pEF6/V5- FAP-α by electroporation. Following selection of transfected cells with blasticidin (used at 5 μg/ml) and verification by PCR, the stably transfected cells were established: FAP-α over-expression cells MCF7exp and MDA-MB-231 exp, plasmid only control cells MCR7pef and MDA-MB-231pef and the wild type cells MCF7wt and MDA-MB-231wt. The transfected cells thus created were always kept in a maintenance medium which contained 0.5 μg/ml blasticidin. Pooled populations of genetically manipulated cells from multiple clones were used in the subsequent studies.
In vitro cell function including cell growth, adhesion, invasion, and migration assay
Cell growth assay: cells were plated into 96-well plated at 2,000 cells/well. Cells were fixed in 10% formaldehyde on the day of plating, and the day3 and day 5 subsequently. 0.5% crystal violet (w/v) was used to stain cells. Following washing, the stained crystal violet was dissolved with 10% (v/v) acetic acid and the absorbance was determined at a wavelength of 540 nm using an ELx800 spectrophotometer (Bio-Tek, ELx800). Absorbance represents the cell number.
Adhesion assay: a 96-well plate was pre-coated with 5 μg of Matrigel and allowed to dry overnight. Following rehydration with serum-free media, 20,000 cells were seeded into each well. After 40 min of incubation, non-adherent cells were washed off using BSS buffer. The remaining cells were fixed with 4% formalin and stained with 0.5% crystal violet. The number of adherent cells was then counted under microscopy.
Invasion assay: transwell inserts (upper chamber) with 8 μm pore size were coated with 50 μg of Matrigel (Collaborative Research Products, Bedford, Massachusetts, USA) and air-dried. Following rehydration with serum-free media, cells were seeded at a density of 30,000 per insert. After 3 day’s incubation, cells that had migrated through the matrix and adhered to the other side of the insert were fixed in 4% formalin, stained with 0.5% (weight/volume) crystal violet, and counted under a microscope.
Migration/wounding assay: cells were seeded at a density of 250,000 per well into a 24-well plate and allowed to reach confluence by overnight culture. The monolayer of cells was then scraped with a fine gauge needle to create a wound of approximately 200 μm. The movement of cells to close the wound was recorded for 4 hours. The movement of cells were analyzed by tracking cell boundary, for each frame in a series, using the Optimas 6.0 motion analysis (Meyer Instruments, Houston, Texas).
Electric Cell-substrate Impedance Sensing (ECIS) based cell adhesion and motility assay
Electric Cell-substrate Impedance Sensing (ECIS, Applied Biophysics Inc, Troy, NY, USA) instrument ECIS Zθ (Theta) was also used to record both cell adhesion and migration abilities which were shown here as the changes of resistance. 96W1E arrays were incubated with complete medium for 1 hour. 50,000 cells of breast cancer cells were seeded into each well. The electric changes were continuously monitored for up to 24 hr while an electric wounding was performed after 6 hours. Multiple conditions of frequency 1000 Hz, 2000Hz, 4,000 Hz, and 8,000 Hz were used to screen the nature of resistance changes.
Influence of inhibitors of signalling pathway on adhesion and migration of breast cancer cells by ECIS assay
In order to explore the potential crosstalk of FAP-α and other adhesion and migration associated signalling pathway. We introduced inhibitors of FAK, ROCK, PLC-γ, and PI3K pathway in ECIS based cell adhesion and motility assay. 50,000 cells of breast cancer cells were suspended in 200 ul media with inhibitors of FAK, ROCK, PLC-γ, and PI3K respectively and the final concentration was 100 nm. The electric changes were continuously monitored for up to 24 hr under multiple condition of frequency while an electric wounding was performed after 6 hours.
Flow cytometric analysis of in breast cancer cells
In this study, we utilised the Vybrant® Apoptosis Assay Kit (Invitrogen, Inc., Paisley, UK) to perform the apoptosis assay. Cells including those suspended in the culture medium were harvested and washed in cold BSS buffer. After centrifugation, the cell pellet was resuspended in 1X annexin-binding buffer. Determine the cell density and dilute in 1X annexin-binding buffer to about 1 × 106 cells/ml. 5 μl of FITC annexin V and 1 μl of the PI working solution (100 μg/ml) were added to each 100 μl of cell suspension and incubated at room temperature for 5 min. After the incubation, 400 μl of 1X annexin-binding buffer was added, mixed gently and stored on ice. Cells were analyzed using the Partec CyFlow® SL flow cytometer and FlowMax software package (Partec GmbH, Munster, Germany), measuring the fluorescence emission at 530 nm and >575 nm.
Immunofluorescence staining in breast cancer cells
20,000 cells were seeded in each well of a 16-well chamber and cultured overnight. Then cells were fixed in 100% ethanol for 30 minutes. After blocked in a 10% horse serum solution, cells were incubated with primary antibodies overnight and were incubated for 30 min in the secondary FITC- and TRITC conjugated antibodies. Following extensive washings, the slides were mounted using Fluorsave™ mounting media (Calbiochem, Nottingham, UK) and allowed to harden overnight in the refrigerator before being examined. Slides were examined using an Olympus fluorescence microscope and photographed using a Hamamatsu digital camera. The images were documented using the Cellysis software (Olympus, Bristol, England, UK).
Western blotting and Immunoprecipitation
To detect the expression level of FAP-α in breast cancer cell lines, confluent cells were pelleted and then lysed using a lysis buffer containing 2.4 mg/ml Tris, 4.4 mg/ml NaCl, 5 mg/ml sodium deoxycholate, 20 μg/ml sodium azide, 1.5% Triton, 100 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin, for 45 min at 4°C. After lysis and centrifugation at 13,000 rpm for 15 min, protein concentrations for each sample were measured using an improved Lowary assay (DC Protein Assay kit, Bio-Rad). The samples were adjusted to equal concentrations with sample buffer and then boiled at 100°C for 5 min, before separated on a 10% polyacrylamide gel. Following electrophoresis, these separated proteins were transferred onto nitrocellulose sheets and blocked in 10% skimmed milk (w/v in TBS) for overnight. The membranes were then probed with the anti-FAP-α, anti-FAK antibodies, and anti-GAPDH antibody as internal control, followed by a peroxidase-conjugated secondary antibody. Protein bands were visualised using an ECL system (Amersham, UK), and photographed using an UVITech imager (UVITech, Inc). The proteins obtained from breast cancer cells were immunoprecipitated with 10 μl of anti-FAP-α and anti-FAK antibodies for 2 h at 4°C followed by the addition of 20 μl of protein A/G-agarose beads overnight at 4°C. The resultant pellet was subjected to SDS-PAGE and Western blotting by antibodies against the FAP-α and phosphatised FAK.
Human breast tissues
A total of 133 breast samples were obtained from breast cancer patients (106 breast cancer tissues and 27 associated background or related normal tissue). The anonymised breast tissue samples were obtained within the guidelines of the appropriate ethics committee (Bro Taf Health Authority 01/4303 and 01/4046). Informed patient consent was not applicable in this instance (as stated in the Human Tissue Act 2004, UK). The pathologist verified normal background and cancer specimens, and it was confirmed that the background samples were free from tumour deposit. These tissues after mastectomy were immediately frozen in liquid nitrogen.
Real-time quantitative Polymerase Chain Reaction (Q-PCR)
The assay was based on the Amplifluor system. It was used to detect and quantify transcript copy number of FAP-α in tumour and background samples. Primers were designed by Beacon Designer software, which included complementary sequence to universal Z probe (Intergen, Inc.). Each reaction contains 1 pmol reverse primer (which has the Z sequence), 10 pmol of FAM-tagged universal Z probe (Intergen, Inc.) and cDNA (equivalent to 50 ng RNA). Sample cDNA was amplified and quantified over a large number of shorter cycles using an iCyclerIQ thermal cycler and detection software (BioRad laboratories, Hammelhempstead, UK) under the following conditions: an initial 5 minute 94°C period followed by 60 cycles of 94°C for 10 seconds, 55°C for 15 seconds and 72°C for 20 seconds. Detection of GAPDH copy number within these samples was later used to allow further standardisation and normalisation of the samples.
All of the results are expressed as the means ± S.E. Cell growth, wounding or migration, adhesion, and invasion formation was analyzed using a Student's t test on log normalized data or Mann Witney for patient tissues (where required).