This protocol can be used to understand the molecular basis for the role of fibroblast as regulators of tumor growth. The protocol described can provide more insight into functional role of cancer-associated fibroblasts in tumor growth and progression. This technique can help to develop new therapies for melanoma.
While our study is focused on melanoma, this technique can be extended to other types of cancer as well. Begin by placing the bottle containing DMEM supplemented with 10%FBS in a water bath at 37 degrees Celsius. Once the medium is warm, bring the bottle into the biosafety cabinet and aliquot 10 milliliters of the medium into 15 milliliter conical tubes.
Remove the required number of vials of frozen cells from the liquid nitrogen freezer and quickly thaw them in a water bath while gently swirling under sterile conditions. Pipet the cells into the tubes containing DMEM and centrifuge the cells at 180 x g for five minutes. Gently remove the supernatant with sterile suction or a pipette and resuspend the cell pellet in DMEM containing 10%FBS.
Then transfer the cells into a labeled tissue culture plate. Using sterile 10 milliliter pipettes, pipette medium with serum into the plate wells and incubate the plate and a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide. Remove the medium from the incubated tissue culture plate containing cells and gently add 10 milliliters of PBS without disrupting the cells.
Swirl the plate. Then gently aspirate the PBS. Next, pipet five milliliters of trypsin-EDTA in PBS solution onto the cells and incubate for five minutes at 37 degrees Celsius.
Dislodge the cells from the plate with gentle taps and pipet them into a conical tube containing DMEM with 10%FBS. Centrifuge the tubes at 180 x g for five minutes and remove the supernatant by aspiration or pipetting. Wash the cell pellet twice with PBS and remove the PBS by centrifugation after each wash.
Resuspend the washed cell pellet in one milliliter of PBS. Next, clean the hemocytometer slide with alcohol and dry it. Then add 10 microliters of trypan blue to 10 microliters of cell mixture into a fresh tube.
Gently fill the hemocytometer glass chambers underneath the cover slip by pipetting the cell mixture. Focus on the grid lines of a 10x objective under a microscope and count the blue cells and live unstained cells in one set of 16 squares. Then count all four sets of 16 squares.
Average the count of unstained cells and multiply it by 10, 000. Then multiply by the appropriate dilution factor to obtain the final concentration in cells per milliliter. After confirming that there are sufficient melanoma cells and fibroblasts for all of the injections planned, assuming at least 20%loss of sample during the injections, transfer the required number of cells to a new conical tube and pellet them by centrifugation.
Then discard the supernatant, resuspend the cell pellet of the required concentration in an appropriate amount of USP-grade PBS and place the cells on ice until further use. After anesthetizing the mouse, pinch its toe to ensure it's in the appropriate surgical plane of anesthesia. Next, wipe the skin of the mice with alcohol swabs.
Fill a sterile syringe with the desired volume of cells or cell mixture without air bubbles and cap it with a 27 gauge needle. Insert the needle into the intradermal region of the skin on the animal's flank and gently inject the cells inside. Monitor the mice until they recover from an anesthesia and provide them with extra insulating materials if required.
Approximately every three days after tumor formation, measure the tumor length and width using calibers and calculate its volume. After euthanizing the mouse, excise the entire tumor using a sterile scalpel and surgical scissors. Trim the non-tumor tissue with surgical scissors.
Then weigh the tumor on an analytical balance. The plot of tumor volume over time shows the comparison of melanoma growth in the presence and the absence of co-injected fibroblasts. The tumor volumes were monitored on 12, 14, 16, 19, and 21 days post-injection.
The images of the excise tumors upon the euthanasia show that the melanoma and fibroblast tumors are larger than the melanoma tumors alone. The presence of fibroblasts result in significantly larger tumors as evidenced by the final weight of tumors at the end of the experiment on day 21. Cancer-associated fibroblasts promote tumor growth by creating a tumor promoting microenvironment.
This protocol can provide more insight into the functional role of CAF in tumor growth and progression.
