The overall goal of this procedure is to create a long-term culture of primary breast cancer tissue, and quantify the three dimensional growth pattern. This is accomplished by first extracting type one collagen from rat tails. Next, the cancer biopsy sample is prepared and embedded into the collagen matrix.
Following 20 days in culture, the breast cancer tissue is immuno fluorescently, stained and scanned using optical projection tomography, and the data is reconstructed and quantified. Ultimately, results can be obtained that show the pattern of invasion of an individual's tumor, which may be further quantified through three-dimensional analysis. The main advantage of ex Vivi cultures is that it uses real cancer materials in a physiological context.
Unlike cell line cultures, Visual demonstration of this technique is important as it is difficult to obtain primary cancer tissue. The correct technique will optimize the chances of the successful experimental outcome. Demonstrating this technique will be myself and Joanne Farrell from MRC Technology To begin extracting type one collagen.
Place frozen rat tails in 70%ethanol until thawed. Using a scalpel, deglove the overlying skin to expose the tendons, strip the tendons away from the tail and place them in 70%ethanol to sterilize. Weigh the collected tendons and transfer them to acetic acid.
Stir for 48 hours at four degrees Celsius. Next, centrifuge the tendon acetic acid mixture at 10, 000 times. Gravity for 30 minutes and discard the pellet.
Add an equal volume of 10%sodium chloride to the S supernatant and allow the mixture to stand overnight at four degrees Celsius. Pipette off and discard the top layer. Then collect the collagen rich insoluble bottom layer and centrifuge for 30 minutes at 10, 000 times.
Gravity then resuspend the collagen rich material in 0.25 molar acetic acid at four degrees Celsius and pipette into a dialysis bag. Dialyze against one to 1000. Acetic acid at four degrees Celsius for three days.
Changing the dialysis buffer twice daily. Collagen can be further sterilized with another centrifugation step and Resus suspension in acetic acid for this assay. Begin with multiple core biopsies that have been harvested from consenting patients at the time of curative surgical resection for invasive breast cancer by eye and using a scalpel.
Divide the cause into one millimeter cubed fragments, trim and discard any macroscopically distinct fat. Mounting the sample on a scalpel blade allows for better visualization within the hood and makes dissection both accurate and safe. Immerse the fragments in MEGM complete medium.
The tissue can be stored in this state for up to one hour to create a 0.3 milligrams per milliliter. Collagen gel mix on ice, one milligram per milliliter of rat tail collagen, one to 1000 filtered acetic acid 10 times D-M-E-M-F 12 and 0.22 molar sodium hydroxide pipette 1.2 milliliters of the collagen mixture into individual wells of a 24 well plate and place into an incubator at 37 degrees Celsius for 10 minutes. To initiate jelling, remove the plate from the incubator.
Place the tumor pieces onto the jelling collagen and use a 10 microliter pipette tip to gently push them into the center of the gel. Return the plate to the incubator for one hour. Supplement A-M-E-G-M complete medium with beta estradiol to a final concentration of 3.2 times 10 to the minus 10 molar.
Once the gel assays have incubated for one hour, carefully introduce 1.2 milliliters of the supplemented medium into each well. The final beta estradiol concentration will equilibrate to 1.6 times 10 to the minus 10 molar to recapitulate physiological estrogen levels found in breast tissue. Run a 10 microliter pipette tip around the inner edge of each well releasing each gel circumferentially, thus allowing each gel to float freely within the media.
Return the 24 well plate to the incubator exchange supplemented medium twice weekly until the termination of the experiment. Invasive growth may be observed in real time using a brightfield microscope here. A time delay capture extended depth of focus video demonstrates invasion of epithelium from the tumor edge into the surrounding collagen Over 60 hours from day seven through nine.
Terminate the experiment by removing the medium and add in a formula. Fixative for 48 hours to immunostain the 3D assay samples for optical projection tomography. First transfer formal in fixed specimens from wells to a vial.
Then wash them for 30 minutes. In PBS perform a stepwise dehydration of the gels using methanol diluted in PBS containing 0.05%Tween 20, incubate the tissue in freshly prepared methanol DMSO 30%hydrogen peroxide at a ratio of two to one to three at room temperature for 24 hours to quench the autofluorescence. Next, wash the samples twice for 30 minutes.
In methanol freeze thaw the samples from minus 80 degrees Celsius to room temperature five times for at least one hour each time to ensure that the antigens in the deeper parts of the tissue are rendered accessible. Then rehydrate the tissue back to PBS with 0.05%T between 20 using methanol as a diluent, incubate the tissue in blocking solution for 24 hours at four degrees Celsius to identify epithelial content, incubate the samples in rabbit anti cytokeratin primary antibodies diluted in 1.5 milliliters of blocking solution with 5%DMSO for 48 hours at four degrees Celsius. Wash the samples in PBS with 0.05%T between 24 times for 30 minutes.Each.
Incubate the tissue in fluorescent secondary antibodies, diluted in 1.5 milliliters of blocking solution containing 5%DMSO for 48 hours at four degrees Celsius. Wash the samples again in PBS with 0.05%between 24 times for 30 minutes each. To prepare samples for optical projection tomography mount the stain gel in 1%low melting point aase and dehydrated in 100%methanol for 24 hours.
Next, clear the specimen in BABB solution for 48 hours. Scan the samples using the OPT 3001 M scanner. Both target and autofluorescence are captured using the SI three and GFP one filter sets respectively.
The specimen is supported in a transparent cylinder of aro gel and rotated through angular steps of 0.9 degrees. A digital image of the static specimen is taken at each of the 400 rotated positions. Use the n recon software to reconstruct the set of angular projections acquired in OPT by creating a series of cross-section slices through the invasion assay.
Analyze the reconstructed data using velocity software to quantify epithelial volume by cytokeratin. Staining, set a minimal voxel intensity threshold to reduce interference from background fluorescence in the target channel.Here. This threshold is defined empirically at 9, 000 arbitrary units.
Finally, use a list of available parameters in the measurement application and the data from the Autofluorescent channel that demarcates the epithelium continuous an original core to quantify the volume of epithelium in a tumor outgrowth. A typical assay fixed after 20 days in collagen culture and subsequently stained for cytokeratin is shown here. Velocity software is able to recognize both the epithelial bulk, continuous with the original core and detached groups of invading cells and quantify each volume separately.
The original biopsies obtained were from a variety of tumors Following ex Vivi cultures. One could also use other techniques such as immunohistochemistry or immunofluorescence to determine rates of proliferation or apoptosis or anything similar. The development of this technique has allowed for researchers in the field of translational breast cancer research to explore methods of identifying drug resistance in individuals with breast cancer.
