The overall goal of this procedure is to utilize the three dimensional culture system as a means to distinguish the benign breast phenotype from the malignant breast phenotype. This is accomplished by first plating the control and experimental cells in triplicates on a layer of growth factor reduced matrigel in an eight well chamber slide. The second step is to give fresh complete media changes with 2%GFR Matrigel every fourth day, allowing the asinine like colonies to grow for 10 to 15 days.
Next, the phase contrast images of the aser like colonies grown in 3D are acquired at a five x resolution. The final step is to process the aser like cultures for immuno cyto chemical analysis with relevant molecular markers, followed by the acquisition of confocal images of in C two immuno stain aser like colonies at midsection. Ultimately, the phase contrast and confocal images of the cells grown in 3D culture are used to identify cellular phenotypic and molecular changes and thus differentiate the benign breast cells from the malignant cells.
The main advantage of this technique over existing methods like two dimensional cultures, invasion or migration assays, is that it can recapitulate the tissue architecture of the benign breast and its loss of organization during malignant transformation. Thus, this technique can be effectively used to differentiate the benign breast cells from the malignant breast cells. This method can help answer key questions in the breast cancer field, such as defect of signaling pathways on cell phenotype, and in identifying new oncogenic and or tumor suppressor pathways.
To begin the experiment thaw growth factor reduced or GFR matrigel at four degrees Celsius overnight. Next, warm up complete HME media at 37 degrees Celsius and place an eight well chamber slide on ice to cool. Add 40 microliters of GFR Matrigel to the center of each well of the pre cooled slide.
Spread the gel as evenly as possible using a P 200 pipette tip, avoiding bubbles and over spreading, which can lead to meniscus formation. Then solidify the matri gel by incubating the slide at 37 degrees Celsius and 5%CO2 while the matri gel is solidifying trypsin eyes, the HME cells, then collect them in a 15 milliliter tube and spin the cells at 150 times gravity for four minutes, count the cells and dilute them to 12, 500 cells per milliliter. In complete media, add GFR matrigel to the cell suspension at a final concentration of 2%and add 400 microliters of the solution to each well of the pre-coded chamber Slide.
Then place the slide in a CO2 buffered incubator. Replace the media with fresh complete media every four days for 15 days. For inhibitor studies, add small molecule inhibitors to the complete media at the time of plating, followed by fresh media changes supplemented with the inhibitors every three days to begin growth cells and triplicates for each treatment set.
Take phase contrast images of aser structures from each well at five x resolution using a phase contrast microscope attached to a slide shifter and a computer. Take the images by moving the slide from one frame to another, covering the entire well of the chambered slide wide. Count the total number of aser structures, the number of single round flat asinine, and the number of multi aser structures or haphazardly growing structures lacking organization.
And with emanating branching processes, calculate the percentage of single round flat aser structures versus malignant structures and plot the information. As a column graph. Calculate the significance by the two-tailed unpaired student T test.
Prepare reagents for immunofluorescence staining as described in the accompanying text protocol. Next, use a P 200 pipette to carefully aspirate the conditioned media. Then fix the ASIN or structures with freshly prepared 2%para formaldehyde at room temperature for 20 minutes after fixation, permease the cells with 0.5%tritton X 100 for 10 minutes at four degrees celsius.
Then wash the cultures three times with 100 millimeter glycine for 10 to 15 minutes for each wash. Next, block the cells in primary block buffer for one hour at room temperature. Then incubate the cultures in 20 micrograms per milliliter of goat anti mouse fab two fragment in primary block buffer, also called the second blocking solution for 30 minutes to block the amino react mouse IgG species in the matrigel.
After blocking, incubate the cells with primary antibody in second blocking solution overnight at four degrees Celsius. Wash the cells three times with immunofluorescence buffer or I buffer with gentle rocking for 10 to 15 minutes for each wash. Then incubate the cells with fluorescent conjugated secondary antibodies in if F buffer for 45 minutes.
Next, wash the cells three times for 10 minutes each with if F buffer and gentle rocking. Mount the slides with DPI containing anti fade reagent to visualize the nuclei. Allow the slides to dry overnight at room temperature.
Then acquire confocal images at the colony midsection of cells growing on top of the matrigel and a series of optical sections throughout the entire length of the asner structure by z stacking confocal images of DPI stained HME controlled and C CN six knockdown cells on day five show that knockdown cells lack organization. On day 15, the control cells become growth arrested with differentiated ASIN or structures and well-defined lumen diverging from the knockdown cells, which present an invasive branching and tubular phenotype. Immunofluorescent staining shows the loss of expression of laminin v alpha six integrin and ECA herrin indicating the disruption of basement membrane, loss of basal polarity and cell cell contact respectively.
In malignant cells, control cells display an intact layer around the asinine basal expression of alpha six integrin apical expression of GM one 30 and eca. Here at the cell cell junction, the malignant phenotype of CCN six knockdown HME cells was reversed with exogenous treatment of purified recombinant human CCN six protein two highly metastatic breast cell lines, some 1 49 and M-D-A-M-B 2 31. Were treated with P 38 map kinase inhibitors, SB 2 0 3 8 50, and SB 2 0 2 1 90, which reduced malignant behavior.
After watching this video, you should have a good understanding of how to grow breast cells in 3D culture and to effectively use sino like growth on matrigel as a readout to differentiate the benign from malignant breast cell phenotypes.
