The overall goal of this procedure is to transplant MA stromal cells and epithelial cells to study the effects of stromal epithelial interactions on tumor formation following transplantation. This is accomplished by first harvesting tail tissue and extracting donor collagen from C 57 black six mice donor fibroblasts and epithelial cells are next isolated and cultured from normal and or M-M-T-V-P-Y-V-M-T transgenic mice. On the C 57 black six background, the cells are then embedded into the collagen and plated onto tissue culture plates as circular plugs.
Finally, the collagen embedded cells are transplanted into the fat pads of the inguinal memory glands of the recipient mouse. There's several advantages to using the system over existing approaches such as generation of conditional knockout mice or transplantation into immunocompromised mice. One advantage is that the system is relatively easy to establish.
Another advantage is that the recipient mice have an intact immune system allowing researchers to analyze the role of specific molecules and stromal epithelial interactions during mammary tumor progression. Demonstrating these techniques will be Diana Lambert, a research assistant from my laboratory. After sacrificing five to seven mature normal female C 57 black six mice using approved iacuc methods, harvest the tails and soak them in 70%ethanol for 45 minutes to sterilize them.
Dry the tails with tissue paper. Wrap them in aluminum foil and store them at minus 20 degrees Celsius until needed. In a laminar flow hood, using scissors first, cut the tip of the tail off, then split the skin at the tail root and peel it away from the tail.
Remove 0.5 to one centimeter from both ends of the tail and divide the remaining tail into three or four pieces. Dissect the tendons from the tails and separate them into individual fibers. Using a scalpel or razor blade, place the fibers into a small webo boat.
Next, transfer the tendon fibers to a sterile container and wash with sterile distilled water. Then aspirate off most of the volume. Pour the remaining water containing the fibers into a whey boat, and then transfer them to a 50 milliliter conical tube containing 35 milliliters of glacial acetic acid.
Diluted one to 500 in sterile deionized water with penicillin, streptomycin and amphotericin. Place them on a rocker at four degrees Celsius for one week to extract the collagen. Spin down the debris in a tabletop centrifuge at 3000 Gs at four degrees Celsius for 15 minutes.
Transfer the supernatant to two polycarbonate tubes, adapted for the Beckman 50.2 TI rotor and spin it 35, 000 Gs and four degrees Celsius for one hour to concentrate the supernatant to one to two milligrams per milliliter. Transfer it to an ultra cell 50 K AmCon filtration unit and centrifuge at 3000 RPM for 15 minutes at four degrees Celsius. Repeat the spin until the volume is reduced to five to six milliliters.
Transfer the filtrate to a sterile conical tube and save at four degrees Celsius. Check the protein concentration using a standard Bradford assay. The purity of the extract can also be checked by resolving a sample on a 6%SDS poly acrylamide gel to isolate donor memory cells and fibroblasts sacrifice.
Age matched, 10 week old female normal or P-Y-V-M-T transgenic mice whose tumors are evident and palpable Using approved iacuc methods, make an upside down T incision between the nipples of the thoracic and inguinal memory gland and pull the skin flat back to expose the memory glands. Remove the memory tissues and mince into small pieces using a razor blade or surgical scissors. Prepare 100 milliliters of an enzymatic digestion cocktail.
Refer to the written protocol for detailed instructions. Incubate the tissue in the cocktail overnight on ice. And then for three hours at 37 degrees Celsius, add five milliliters of PBS containing 10%fetal bovine serum to each sample and centrifuge at 1500 RPM for five minutes at four degrees Celsius.
Repeat this wash two more times. Resus suspend the cell pellet and 10 milliliters of DM EM containing 10%FBS and antibiotics and plate in 10 centimeter dishes coated with collagen. Type one, incubate the cells at 37 degrees Celsius for three hours.
Separate the fibroblasts and epithelial cells by selective fibroblasts are more loosely adhered than epithelial cells. Check for fibroblast detachment under the microscope after two minutes in trypsin, cells should be floating in suspension or rounded up while epithelial foci should still be adhered to the surface. Reflate the suspended cells onto collagen coated 10 centimeter dishes and repeat the selective trypsin ization process until fibroblasts and epithelial cells are completely separated.
Check for cell purity by immunofluorescent. Staining for epithelial markers such as CK 14 and CK 18 and fibroblast markers such as alpha smooth muscle actin for one graft. Begin by mixing 100 microliters of collagen with 25 microliters of setting solution in an einor tube.
Please see the written portion of this protocol for the setting solution details. Pipette the sample up and down to mix thoroughly. Add collagen or setting solution at five to 10 microliter increments and mix thoroughly until the phenol red dye in the mixture changes to a light pink to orange color reflecting a neutral pH.
Keep the mixture on ice to prevent polymerization. Remove cultured fibroblasts and epithelial cells from plates by trypsin. Count each cell suspension using a hemo cytometer pellet the cells then resus.
Suspend them in complete medium to a concentration of 100, 000 cells per 100 microliters. Mix 250, 000 stromal cells and 100, 000 tumor cells in a separate tube micro fuge for one minute at 1500 RPM. Then remove the supra natin by pipetting.
Add 50 microliters of the adjusted collagen solution to the cells pipette up and down to disperse the cells evenly. Pipette the cell suspension into a sterile six centimeter tissue culture plate. To form a circular drop, incubate the graft at 37 degrees Celsius for 15 minutes.
To polymerize gently add five milliliters of complete medium by tilting the plate and slowly pipetting the medium into one side. Tilt the plate around until the medium covers the graft. Incubate the cells for 24 hours at 37 degrees Celsius.
Then transplant immediately to surgically implant the cells into mice. First fabricate a thin glass rod by heating a glass pasture pipette using a buns and burner using forceps. Stretch the end of the pipette out to two to three millimeters in thickness.
Let cool. Then sterilize the rod in 70%Ethanol. Sterilize surgical instruments and gut sutures by autoclaving.
Anesthetize the recipient mouse with two to 3%isof fluorine. Perform a toe pinch to check for the proper level of anesthesia. Lay the mouse on its back with its nose inside the iso fluorine dispensing cone and immobilize the limbs with tape or other removable adhesive.
Remove the hair with a chemical hair remover such as nare. Wipe the area clean with water, then disinfect with ethanol and Betadine. Next, hold up a part of the skin by the MA gland using blunt forceps with one hand with the other hand.
Use fine surgical scissors to make an upside down teen incision between the number nine and number 10 nipples or the number four, number five, nipples of the inguinal mammary glands. To expose the memory glands, make a pocket incision under the lymph node or memory artery with small surgical spring scissors. Using forceps, remove the collagen graft from the tissue culture dish and drain the excess liquid by gently dabbing it onto a sterile wipe.
Slide the collagen graft completely into the pocket using a thin glass rod. Use number two, gut absorbable sutures or wound stables to suture the skin flap. Let the mouse recover in the cage.
Monitor the mice twice weekly at a minimum palpating for tumors. Sacrifice the mice when the tumors reach one centimeter in diameter. Then harvest the tissues for analysis I extraction of collagen protein from five to seven.
Mouse tails yields approximately one to 1.5 milligrams per milliliter in a six milliliter final volume, or six to nine milligrams of protein by kumasi Blue staining bans corresponding to 90 kilodaltons and 130 kilodaltons are detected in the lanes loaded with samples extracted from mouse tails indicating the presence of collagen type one and pro collagen respectively. Ppy VMT carcinoma cells and fibroblasts can be distinguished by differences in cell morphology and expression of specific epithelial and mesenchymal markers. P-Y-V-M-T carcinoma cells are identified by cobblestone shape and co-expressed CK 18 luminal epithelial marker and CK 14 a basal epithelial marker mammary fibroblasts larger cells with a spindle shaped phenotype and express high levels of alpha SMA transplantation recipient.
Mice are sacrificed when tumor diameter reaches 1.0 centimeters in diameter or approximately 60 days while transplantation of P-Y-V-M-T cells alone results in palpable tumors after 30 to 40 days reaching a mean tumor mass of 0.3 a two grams at 60 days cot Transplantation of P-Y-V-M-T carcinoma cells with memory fibroblasts results in a mean tumor mass of 0.628 grams indicating an enhancement of tumor growth by fibroblasts Following this procedure. Other methods such as drug treatment can be performed in order to answer additional questions such as determining the role of targeted therapies on strong epithelial interactions during mammary tumor progression.
