Several in vitro assays have been developed to understand and elucidate the mechanisms by which malignant cells acquire an invasive phenotype under in vivo conditions (Shaw 2005). Invasion assays were performed in this study using a modified version of the Boyden chamber assay (Albini, Iwamoto et al. 1987) to compare the invasive potential of wtMCF-7 and Tam-R cells basally and following CD44-siRNA knockdown.
The invasion assay was carried out using special invasion chambers (6.5mm diameter inserts Corning Transwell® inserts possessing 8µm porous membranes) placed in a 12- well plate. 50µL of the synthetic basement membrane Matrigel™ (stock solution of 11mg/ml diluted 1 in 3 with ice cold wRPMI) was used to coat the upper surface of the porous membrane of each of the inserts. The Matrigel™ was allowed to set for a minimum of 2 hours at 37°C and appropriate media, with or without treatments, were added to the well housing the insert and the plate then incubated at 37°C. The cell line of interest was harvested and resuspended in appropriate medium, with or without treatments, and 200µL of this cell suspension was added to the insert at a cell density of 50,000 per well (Figure 2.3). Cells were then incubated for 48 hours. For CD44-siRNA experiments, the transfection protocol described in Section 2.6 was used prior to seeding siRNA-transfected cells into the Matrigel™ coated chambers .
Following this, the inserts were removed from the wells, the supernatant removed and Matrigel™ gently wiped off from the upper surface of the membrane using a cotton bud. The insert was then placed into a solution of formaldehyde (3.7% in PBS, 10 minutes) to fix the cells and subsequently washed with PBS (1 x 2 minutes) and allowed to air dry. The insert membrane was then carefully excised using a scalpel blade and prepared for
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staining by mounting it onto a glass slide, applying a fixative containing the nuclear stain DAPI (Vectashield®) and overlaid with a glass cover slip. The membrane mounted on the slide is then allowed to set overnight at 4°C.
A Leica DM-IRE2 fluorescence microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, UK) connected to a Hamamatsu C4742-96 digital camera (Hamamatsu Photonics UK Ltd, Hertfordshire, UK) and a PowerMAC G5 computer (Apple Computer Inc, California, USA) was used to assess the membranes and count the number of cells that had invaded the membrane. Quantification was performed by viewing 5 separate fields per membrane (20x magnification) and counting the number of cells in each field. Data were then plotted at mean cells per field ± SD for a minimum of 3 separate experiments, each performed in triplicate.
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Figure 2.3 Schematic representation of a Transwell®insert used for Boyden chamber
migration and Matrigel™invasion assays
A schematic diagram of a Transwell®insert used during the migration and invasion assays in this study as described in Section 2.7.2 and Section 2.8 is depicted above. The microporous membrane was coated with either Fibronectin (migration assay) or Matrigel™ (invasion assay) prior to adding the cell suspension and medium with or without treatments. Adapted from catalogue schematic in www.corning.com.
Transwell®insert Microporous membrane Outer well Cell layers Upper compartment Lower compartment
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2.9 CELL GROWTH ASSAY
Several assays have been designed to replicate cell growth in vitro in order to understand the mechanisms underlying this process. The growth assay used in this study is the cell counting assay standardised in our laboratory previously.
Cell growth measurements were performed by cell counting experiments using a Coulter Counter. Tam-R cells were harvested using trypsinisation, as described previously, and seeded into a 24-well plate at a density of 30,000 cells per well containing 1ml volume. Each treatment was set up in triplicate in three separate wells. Appropriate treatments were added after 24 hours, allowing the cells to adhere to the plate during this period. For siRNA experiments at least 72 hours was allowed for adequate siRNA transfection. Following treatment, the plate was incubated at 37°C in a humidified 5% CO2 atmosphere for 5 days. Medium was then aspirated from a well and 1ml of Trypsin/EDTA was added to each well using a Finnpipette. Cells were then incubated for a further 3 to 5 minutes until cells were in suspension. Using a 5ml syringe and an orange 25G needle the suspension was passed through the syringe twice to obtain a single cell suspension and finally aspirated back into the syringe. 1ml of Isoton® II was added to the well and aspirated into the syringe. This was repeated three times making up a total syringe volume of 4mls. The contents of the syringe were then transferred into a counting pot containing a further 6mls of Isoton® II making a final volume of 10mls. The number of cells in each well was then determined using a Coulter counter. The count was repeated twice and an average count calculated for each well. This average value was multiplied by 20 to give the average cell number per well. The mean cell number ± SD of the counts from the 3 wells was subsequently calculated for each experiment performed in triplicate.
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2.10 IMMUNOFLUORESCENCE STAINING
Immnuoflourescence is a technique based on the pioneering work by Coons and Kaplan which involves the use of fluorescently-labelled antibodies to tag proteins of interest in live or fixed cells. This allows for subsequent antigen detection using fluorescence microscopy (Coons 1941). The main benefit of this procedure is that it allows for dual staining, where two or more separate antigens can be labelled with different fluorophores. Image analysis of stained samples can be used to reveal whether these proteins are co- localised within the same cell or region of the cell.
Tam-R cells were cultured in 35mm dishes on glass cover slips with W+5% until cells reached log phase. The medium was then aspirated and the coverslips transferred to fresh 35mm dishes. Fixation was carried out with 3.7% formaldehyde in PBS for 15 minutes followed by permeabilisation with 0.2% Triton X-100 in PBS for 7 minutes. Coverslips were then treated with 10% normal goat serum for 30 minutes as a blocking agent followed by wash with PBS (x2) and treatment with antibodies diluted in PBS containing 1% BSA as detailed in Table 2.8. Coverslips were then washed after use of Alexa Flour® conjugates and mounted onto microscope slides using a hard set mounting medium (Vectashield®, Vector Laboratories Inc, Peterborough, UK) containing the nuclear stain DAPI (4',6-diamidino-2-phenylindole). Cells were viewed at 63x magnification using a Leica DM-IRE2 inverted fluorescent microscope and images taken for further analysis.
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Table 2.8 List of antibodies used in immunofluorescence staining
Antibody Source Dilution Incubation conditions
Supplier
Primary Antibodies
CD44 Mouse 1:1500 2 hours RT Stratech Scientific Ltd, Suffolk, UK
T-HER2 Rabbit 1:100 2 hours RT Dako Ltd, Cambridgeshire, UK T-HER3 Rabbit 1:100 2 hours RT Santa Cruz Inc, CA, USA
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2.11 IMMUNOPRECIPITATION
Immunoprecipitation involves precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. NT and CR44-siRNA transfected Tam-R cells were treated with HA for 5 minutes and cell lysates were obtained as described in Section 2.4 for SDS-PAGE. Lysates were centrifuged at 12,000rpm for 15 minutes, the supernatant pipetted into a fresh eppendorf and protein concentration of the supernatant determined using a Bio-Rad Dc Protein Assay Kit (Bio- Rad Laboratories Ltd, UK) using a modification of the original Lowry method (Section 2.4.2). The protein lysate was diluted such that 500µL of the solution contained 500µg protein and this was then divided into two parts in two different eppendorfs. One eppendorf was prepared for the specific immunoprecipitating anibody and the other for a matching isotype control antibody. Into each eppendorf, 5µL of the appropriate antiobody was added (from the antibody stock tube), lids sealed with Parafilm and tubes were left in a rotator overnight at 4°C to allow antigen-antibody complexes to form.
The following day, 20µL dye-conjugated agarose beads (EZViewTM Red, Sigma) were placed in a series of eppendorf tubes (corresponding to the number of immunoprecipitation samples) and washed by adding 750µL lysis buffer to each tube, vortexed and centrifuged at 8200rpm for 30 seconds at 4°C. Tubes were then returned to ice, supernatant discarded and wash repeated two more times. Cell lysates containing the antibody-antigen complexes were then removed from the rotator, briefly centrifuged at 8200rpm and contents transferred to the eppendorfs containing the equilibrated EZViewTM Red beads. Eppendorf lids were sealed with Parafilm and placed again in rotator for 2 hours at 4°C for the antigen-antibody complexes to attach to the agarose beads. Following this, tubes were returned to an ice filled container, briefly centrifuged at
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8200rpm and the supernatant discarded. Pellets were washed with 750µL lysis buffer (containing protease inhibitors), vortexed gently and left on ice for 5 minutes. Samples were then centrifuged at 8200rpm for 5 minutes at 4°C and supernatant discarded. This wash was repeated three times and on the final wash, attempt was made to remove as much supernatant as possible without discarding any beads. The next step involvesd eluting the antigen-antibody complexes from the beads and this was carried out by adding 20µL lysis buffer and 20µL of 2x loading buffer (containing DTT) to each tube and boiling samples at 100°C for 5 minutes. Samples were then briefly vortexed and centrifuged again at 8200rpm for 5 minutes. At this point, samples were considered ready for SDS-PAGE analysis and Western blotting as described in Section 2.4.4. The Ponceau S stain step was avoided as only some IgG bands were usually noted and hence, was not considered essential. Blots were immunoprobed with the antibody of interest and detected using chemiluminescent reagents as described in Section 2.4.5.
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Table 2.9 List of antibodies used in immunoprecipitation
Antibody Source Dilution Incubation conditions
Supplier
CD44 Mouse 1:1500 2 hours RT Stratech Scientific Ltd, Suffolk, UK
T-EGFR Rabbit 1:100 2 hours RT Cell Signaling Technology Inc, USA
T-HER2 Rabbit 1:100 2 hours RT Dako Ltd, Cambridgeshire, UK T-HER3 Rabbit 1:100 2 hours RT Santa Cruz Inc, CA, USA
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