This protocol describes the orthotopic transplantation of breast tumor fragments into the mammary fat pad and provides valuable approaches for investigating tumor biology, drug responses, and mechanism of drug resistance. This technique is minimally invasive and ensures proper placement of the tumor tissues within the mammary fat pad, accessibility for counting per measurement, and animal mobility maintenance. This technique allows the transplantation of dozens of animals from a single patient derive's inter graft, for the evaluation of multiple candidate cancer therapies simultaneously in a preclinical trial format.
This method is superior to subcutaneous tissue implantation for establishing patient derives inter-graph models, and can also be used when orthotopic implants are not possible. The most difficult stuffs are applying the appropriate amount of pressure to peel the fat pad from the body wall and making the tissue pocket. Visualization of the fat pad peeling and the cracked anatomical placement of the tissue fragment, will help you ensure a successful performance of the procedure.
To thaw cryopreserved mammary tumor tissue fragments for transplantation. First, thaw a cryo vial from liquid Nitrogen storage containing the tumor fragments of interest in a 37 degrees Celsius water bath, with occasional gentle flicking. When the fragments have thawed, mix the fragments with gentle inversion, and dry the outside of the vial.
Spray the vial with 70%ethanol, and place it in a vial-safety hood. Transfer the cryo vial contents to a 15 milliliter conical tube. Then add 10 milliliters of cold DMEM, and invert the tube to mix.
Allow the fragments to settle, and replace a supernatant two times with 10 milliliter aliquot, so, fresh cold DMEM. Then place the tube on ice. To collect a mammary tumor tissue sample, spray the tumor region of the tumor bearing mouse with 70%ethanol and use serrated forceps to pinch and lift the skin surrounding the tumor.
Use scissors to make a small incision, and separate the tumor from the skin, taking care to avoid hair contamination. Trim off any remaining mouse fat pad tissue from the outer surface of the tumor, and place the tumor in a 15 milliliter conical tube containing five milliliters of cold DMEM. In a bio-safety hood transfer the dissected tumor into a sterile 10 centimeter Petri dish, containing enough DMEM to prevent drying, and use a UV sterilized blade to cut the tumor into one cubed millimeter fragments under aseptic conditions.
Then transfer the tumor fragments to a 15 milliliter tube containing cold DMEM on ice. After confirming a lack of response to pedal reflex, apply ointment to the anesthetized recipient mouse's eyes, and shave the abdomen around the fourth memory gland. Then disinfect the exposed skin three times, using a circular motion and starting in the center of the surgery site while moving outward toward the outer edges, with povidone-iodine surgical scrub, followed by scrub removal with a 70%isopropyl alcohol pad.
For transplantation of the tumor tissue fragments, into the fourth inguinal mammary fat pad, cover the animal with a sterile fenestrated surgical drape, and use serrated forceps to pinch and lift the skin at the fourth nipple. With the blunt side of the tips facing down, use scissors to make an approximately one centimeter parasagittal incision from the number four nipple toward the head. Use a cotton tip applicator to separate the skin from the peritoneum on the medial side of the incision, and gently peel the number four memory fat pad from the skin.
Use a sterile 27 gauge needle to secure the skin close to the animal's body, and use serrated forceps to gently extend the fat pad away from the body. Locate the inguinal lymph node at the intersection of the major vessels of the gland, and carefully cauterize the vessels medial to the lymph node and the fat adjoining the fourth and fifth fat pads. Using micro dissecting scissors, cut each cauterized vessel one at a time, til the section of the fat pad that contains the lymph node is removed, and use blunt forceps to grasp the fat pad.
Using the other hand and a curved motion, insert the close tip of a pair of angled fine forceps into the middle of the fat pad above the remaining vessel close to the wall of the peritenoneum. Retract the tips and reinsert the tips into the original hole, opening the forceps to stretch the hole into a small pocket. Remove the forceps from the hole and use them to insert a piece of tumor fragment into the pocket.
Slowly open the tips to release the tumor fragment into the fat pad pocket and carefully withdraw the forceps. After confirming that the fragment has been deposited into the fat pad pocket, starting from the base of the incision, collect the on each side of the incision with serrated forceps and lift the skin slightly. Uncurl the edges of the incision with claw forceps and pinch the edges together to form a continuous surface at the top.
Holding the two sides with the serrated forceps, use a wound clip applicator to place a clip in the center of the incision, and place the animal in a clean cage on a warming surface with monitoring until full recumbency. Typically, patient derived xenograft or genetically engineered mouse model tumors can be palpated from about two to eight weeks post transplantation. H&E staining can be performed to analyze the tumor pathology while immunohistochemistry can be used to monitor markers for a specific cell lineages of interest, or cell viability and functional status.
It is important to make sure that the tumor fragment is placed securely in the pocket, so, that it doesn't fall out and grow within the subcutaneous space. Once the tumors have grown, drug treatment can be administered to assess tumor responses. The tumors can also be removed to determine the metastatic potential of the cancer cells.
This basic technique has been used since the 1950s. Our modification provides a more efficient and less invasive manner in which to conduct larger scale developmental biology in cancer studies.
