The overall goal of this procedure is to characterize the distinctive biology at the interface between malignant epithelial cells at the invasive tumor front and cancer associated stromal cells. This is accomplished by first establishing primary fibroblast cultures from colonic implants. The second step is to construct organotypic co-culture models using primary colonic fibroblasts and cultured colorectal cancer cells.
Next organotypic models are fixed in formin sectioned onto membrane mounted slides and stained with crestal violet. The final step is laser microdissection in order to separate invading tumor cells from non invading tumor cells and cancer associated stromal cells. Ultimately, molecular profiling techniques may be used to characterize the biology of invading and non invading tumor cells and stromal cells in the surrounding tumor microenvironment.
We first had the idea for this method when we realized how clearly distinguishable invading cells are from non invading cells in three-dimensional organotypic co-culture models of colorectal cancer. To begin, obtain samples of normal human colon mucosa tissue directly from the surgical operating theater in the laboratory. Place a specimen at the center of a 10 centimeter tissue culture dish, then wash the culture three times with phosphate buffered saline or PBS supplemented with penicillin, streptomycin, and fungi zone.
Do not aspirate after the final wash with sterile forceps and scalpel, cut the tissue into small pieces. Then place each piece at the center of a cross drawn with the scalpel blade. In a 12 well plate rock the scalpel gently over the tissue to allow it to adhere culture.
The specimens in 750 microliters of primary fibroblast growth media, which should submerge the tissue without enabling it to float. Then place the specimens into a 37 degrees Celsius, five to 10%CO2 incubator over the next five days. Fibroblasts will slowly start to grow out from the edge of the tissue at approximately seven days.
This will be more visible after approximately three to four weeks. And if there are sufficient fibroblasts growing out, add 750 microliters of trypsin to each well after cells have detached pool and transfer them to a T 25 tissue culture flask for the fibroblast impregnated organotypic gel. First, prepare a fibroblast cell suspension containing 500, 000 cells per gel in supplemented dmm.
Make up the organotypic gels on ice in the ratios found in the text protocol for nine gels at one milliliter per gel. Prepare 10 milliliters and mix gently to avoid bubbles. Then add one milliliter to each well of a 24 Well plate and incubate for one hour at 37 degrees Celsius in a humidified atmosphere.
To allow gels to set after one hour, add one milliliter of supplemented DM EM on top of the gels and return to the incubator overnight. The following day. Aspirate medium from the top of the gels and plate one milliliter of medium containing 500, 000 colorectal cancer epithelial cells for the gel coated nylon sheets.
Prepare sterile autoclaves nylon sheets measuring 1.5 centimeters by 1.5 centimeters in advance using sterile forceps. Place the required number of nylon sheets in a 10 centimeter culture dish. Prepare the gel mixture on ice as described in the text protocol and add 0.1 molar sodium hydroxide to the gel mix until it turns pink indicating the solution has been neutralized.
Then add 250 microliters of this solution to each nylon sheet. Then incubate at 37 degrees Celsius in a humidified atmosphere of 5%CO2 for 30 minutes to allow the gel to set. Next, add 10 milliliters of a 1%glutaraldehyde solution in PBS to each 10 centimeter culture dish.
Once the gel has set before incubating at four degrees Celsius for one hour, then wash the nylon sheets three times in PBS and once in supplemented dme. Incubate the sheets overnight in the same buffer at four degrees Celsius to raise the organotypic gels onto steel grids for invasion sterilized scaffolding grids that have been prepared by folding stainless steel sheets into a tripod formation. Then use forceps, sterilized and ethanol to place a steel grid into each well of a six well plate.
Place a gel coated nylon sheet with the collagen side up onto each grid. Carefully transfer the organotypic gels from the 24 well plate to the raised nylon sheet using a spatula, sterilized and ethanol. Fill the well with supplemented D mem until the nylon sheet is in contact with the medium but not submerged.
Make sure the medium does not touch the organotypic gel. Incubate for 14 days at 37 degrees Celsius in a humidified atmosphere of 5%CO2. Replacing the medium every two days.
Remove the whole organotypic including nylon sheet from the well and place it on clinging film. Bisect the organotypic and nylon sheet using a clean disposable scalpel and fix both halves in formaldehyde for 24 hours. At room temperature, replace formaldehyde with 70%ethanol after 24 hours and leave overnight prior to embedding in paraffin, sectioning and staining to perform laser microdissection.
First section, the organotypic to 10 micron thickness onto membrane mounted slides. After treating the sections as described in the text protocol, prepare to conduct laser microdisect using the chosen platform form. Place the glass slide with the stain section to be micros dissected.
Face down on the microscope stage mount a 0.5 milliliter micro centrifuge tube into the collection cassette and add 50 microliters of cell lysis buffer to the cap into which micro dissected tissue will collect under direct vision. Use the joystick to identify the tissue of interest and using the software interface, annotate the stained organic section, highlighting cells to be microdisect at the tumor invasion front. Instruct the laser to fire, which should both cut out the highlighted section and propel the microdisect tissue into the cap of the micro centrifuge tube.
Proceed to process samples by an appropriate method to extract the analyte of interest. Laser microdissection images of SW 480 colorectal cancer cells invading the organotypic stroma are shown here. Crestal violet is used to highlight the colorectal cancer epithelial cells.
The invasive tumor islands are then defined before laser dissection occurs and the cut piece is transported to a collection device. Non invading cells and stromal cells are identified and isolated using the same methodology. The three dimensional co cultures were analyzed by micro RNA profiling for comparison of differentially expressed microRNAs between cells at the invasive tumor front and those in the stratified tumor zones.
Data was sorted by full change and data values plus or minus twofold. Change from a representative experiment are presented showing differential micro RNA gene expression between the invasive margin cells and non-invasive margin cells. After watching this video, you should have a good idea of how to construct anatomically appropriate organotypic models and how to use laser microdissection to isolate individual populations of cells for further study.Whoa.
