This methodology uses the integration of different indicators to assess the degree of transformation of any cell model. This technique provides researchers with a set of time-consuming but simple-to-use tools for assessing cell transformation in vitro. This allows the method to be used in most laboratories.
Demonstrating the procedure will be Teresa Anglada, a post-doctoral researcher from my laboratory, and Joan Repulles, a microscopy specialist. When the breast primary epithelial cell culture reaches 90%confluency, transfer the supernatant from the culture flask to a 15 milliliter tube containing two milliliters of fetal bovine serum and wash the cells with PBS. Treat the cells with one milliliter of trypsin for five minutes at 37 degrees Celsius.
When the cells have detached, inactivate the trypsin with the reserved medium and collect the cells by centrifugation. Flick the tube to resuspend the pellet and add one to two milliliters of fresh medium for counting. After determining the cell concentration, add 300, 000 cells to a new tube and bring the final volume to five milliliters with additional fresh culture medium.
Seed cells into a new T25 flask and return the cells to the cell culture incubator. Then use the formula to calculate the number of accumulated population doublings. To assess the proliferation rate of the cells, plot the accumulated number of population doublings for a specific time interval.
An increased slope indicates an increased cell proliferation rate. To set up a 3D cell culture, rinse wells with a 1.9 square centimeter surface area with cold sterile PBS and coat the bottom of each well with 100 microliters of basement membrane matrix. Then place the plate in the cell culture incubator for about 20 minutes to allow the matrix to solidify.
In the meantime, harvest mammary cells as just demonstrated and dilute the cells to a 400, 000 cells per milliliter of medium concentration. Mix the cells with fresh medium supplemented with 8%basement membrane matrix at a one-to-one ratio and add 500 microliters of the cells to each well of solidified matrix. Incubate the cells at 37 degrees Celsius for a few minutes, then add 500 microliters of medium supplemented with 4%basement membrane matrix to each well.
Place the cells in the cell culture incubator for 14 days. The seeded cells will group and proliferate forming acini-like structures. For the immunofluorescent protein detection in the 3D cultured mammary cells, use a cutoff P200 pipette tip to remove the basement membrane matrix from each well and smear approximately 50 microliters of disaggregated matrix from each well onto individual glass slides.
When the samples have completely dried, fix the cells with a one-to-one methanol-acetone solution at minus 20 degrees Celsius for 30 minutes. After fixing, block samples with blocking solution for two hours at room temperature. At the end of the incubation, add 30 microliters of the primary antibody working solution of interest and cover the slides with a strip of laboratory wrapping film for an overnight incubation at four degrees Celsius in a humid chamber.
The next morning, wash the slides three times with PBS for one hour per wash and label the samples with the appropriate secondary antibody solution as just demonstrated. At the end of the incubation, wash the samples two times in PBS for two hours per wash before counter-staining with DAPI diluted in anti-fade mounting medium and covering each slide with a coverslip. Then analyze the fluorescent signal distribution for each acinus on a confocal microscope.
To perform an anchorage-independent assay, first cover the bottom of the appropriate number of wells of a six-well plate with 1.5 milliliters of 0.6%agar in medium solution and let the agar solidify at room temperature. Next, prepare a five times 10 to the fourth cells per milliliter solution as demonstrated and filter the cells through a 40 micron strainer into a sterile 50 milliliter tube. Holding the tube at a 45 degree angle, add an equal volume of 0.6%agar in medium solution down the wall of the tube.
And after thorough mixing, layer one milliliter of the cells in agar suspension to each well of the previously prepared six-well plate. Use an inverted microscope to confirm that the cells have been homogeneously dispersed throughout the agar. When the agar has completely solidified, carefully add one milliliter refresh medium to each well without disturbing the agar and incubate the cells for three weeks, changing the medium twice per week.
At the end of the colony formation period, replace the medium with one milliliter of one milligram per milliliter MTT per well for a 24-hour incubation in the cell culture incubator. The next day, remove the MTT from each well and acquire images of each well on the inverted microscope. When all of the images have been acquired, open the images in ImageJ and click image, adjust, threshold, and apply to obtain a binary mask through thresholding of the original image.
When well-delimited colonies have been obtained, select plugins and biovoxxel to launch the extended particle analyzer to identify the MTT-positive colonies. Over a specific interval of time, partially and fully transformed cells achieve a higher number of population doublings compared to non-transformed cells, indicating that the cell division rate is increased with the transformation process. Over the two-week 3D cell culture period, the cells aggregate and proliferate, increasing the acini size.
The proper polarization of each acinus can be accurately assessed using protein immunofluorescent detection in conjunction with three-dimensional signal location by confocal microscopy. While all of the acini formed by non-transformed breast cells are properly organized, a loss of polarization is observed in acini formed by partially and fully transformed cells. After three weeks of embedded agar culture, cells with an anchorage independent growth capacity give rise to colonies composed of multiple cells.
Only metabolically active cells are capable of cleaving the tetrazolium MTT ring, resulting in purple MTT formazan crystals after 24 hours. Measurement of the colony diameter by automatic image analysis allows the filtering of low proliferative colonies or individual cells. The number of colonies formed by non-transformed cells is negligible compared to those from partially and fully transformed cells.
Colony size can also be used to discriminate between cells with different degrees of transformation. These assays can be used to screen cell lines prior to any inoculation as only those cell lines that have tested positive in vitro should be inoculated into mice.
