This method can help answer the key questions in tumor immunology, including whether there is a replenishment of tumor-specific immune cells from peripheral tissues, the transitions between newly infiltrated and already existing immune cells within tumor microenvironment, and their responses to immunotherapy. This is convenient and easy-to-follow technique to distinguish the periphery-derived cells from tumor-infiltrated immune cells and track the dynamic, phenotypic, and functional changes of these cells in longitudinal assays. Begin by withdrawing 100 microliters of the prepared B16F10-OVA cell suspension into a 1 mL tuberculin syringe.
Tap the barrel to move any bubbles to the top and gently push the plunger to remove air bubbles. Restrain the mouse to expose its abdomen. Press the left hind leg with the little finger to tighten the skin of the left inguinal region.
Remove the mouse's hair from its left lower abdomen with an electric shaver. Use cotton soaked in 75%ethanol to clean the posterior quadrant of the left abdomen. Hold the syringe at a shallow angle between 0 to 15 degrees and, with the bevel of the needle facing upwards, insert it at the side of the left upper thigh.
Advance the needle 0.5 to 1 centimeter through the subcutaneous tissue into the inguinal region. Pull back on the plunger before injecting. If there is negative pressure, depress the plunger entirely and observe the formation of a small fluid pocket, or a bolus, in the subcutis.
Remove the needle after the injection is carried out. Release and place the mouse back into the cage. On day six to eight after B16F10-OVA implantation, measure the tumor size with a vernier scale.
Select the mice with an approximately three-millimeter diameter or a mung bean-sized tumor, and divide them equally and randomly into two groups. Withdraw 200 microliters of OT-1 cell suspension into a 100-unit 29-gauge insulin syringe and remove bubbles. Place the mouse separately in a cage, with an infrared lamp over the cage for 5 to 10 minutes to dilate the tail vein.
Immobilize the mouse with a restraining device of appropriate size. Hold the tail to straighten it and spray 75%ethanol to make the vein visible. Hold the syringe parallel to the vein and insert it into the vein at an angle of 0 to 15 degrees.
Pull back the plunger slightly, and if blood enters the barrel, slowly and steadily inject the suspension at a rate of no more than one milliliter per minute. After the injection is completed, remove the syringe and press the injection area gently for three to five seconds to stop the bleeding. Return the mouse to the cage and closely observe it for a few minutes for adverse reactions.
If it has normal mobility and nasal discharge, place it back in the company of the other mice. 8 to 10 days after the adoptive transfer, select donor mice bearing comparable tumor mass of approximately five-millimeter diameter for transplantation surgery. Place a 100-millimeter-by-20-milliliter dish in a biosafety cabinet and add 10 milliliters of sterile, ice-cold PBS.
After euthanizing the mouse, immerse it in 75%ethanol for three to five minutes. Place the mouse in the supine position on a dissection board covered with clean absorbent paper in the biosafety cabinet. Restrain the mouse limbs with dissection needles.
Cut the skin along the midline from above the urethral orifice to the xiphoid with scissors. Stretch the skin towards the left side of the mouse body with tweezers and restrain the skin with dissection needles. Excise the tumor, keeping its capsule as intact as possible.
Carefully and gently remove the connective tissue near the tumor with surgical scissors, then place the tumor tissue in the dish containing sterile, ice-cold PBS for subsequent transplantation. Use veterinary ointment on eyes to prevent them from drying. Shave the left flank of the mouse with an electric shaver, then apply a depilatory cream to remove the remaining hair.
Place the mouse inside the biosafety cabinet in a prone position on a dissection board covered with clean absorbent paper, with the mouse's vertical axis parallel to and its head to the right side of the experimenter. Rub the skin of the shaved area with cotton soaked in povidone-iodine. Lift the skin at the midpoint between the mouse hip joints with surgical tweezers.
Use the scissors to make a five-millimeter-long vertical excision and extend the cut rostrally along the dorsal midline to around 10 to 15 millimeters. Perform a sharp dissection by inserting the closed tips of the scissors into the incision and then opening to separate the peritoneum of the left flank from the skin and soft tissue. Perform sharp dissection several times to generate a skin pocket on the left flank.
Deposit the encapsulated, intact, donor-derived tumor mass into the pocket. Close the incision by an interrupted suture, using two or three stitches for each incision. Disinfect the skin around the cut with cotton soaked in povidone-iodine.
Place the mouse in the lateral position in a clean and warm cage. Monitor it continuously until it has regained sufficient consciousness to maintain sternal recumbency. Administer buprenorphine subcutaneously at a dose of 0.1 milligram per kilogram of body weight every eight hours, for a total of three doses.
Return the transplant recipient to the company of other animals only after it has fully recovered. Using flow cytometry, two populations of CD44-negative and CD8-positive tumor antigen-specific T cells were easily identified in the tumor microenvironment, including CD45.1-positive donor-derived and CD45.1-negative and CD45.2-positive recipient-derived tumor-infiltrating CD8-positive T cells. At day two post-transplantation, there were approximately 83%of donor-derived antigen-specific CD8-positive T cells within the transplanted tumor, more predominant than their recipient-derived counterparts.
The proportion of recipient-derived OT-1 cells was elevated in the late stage of tumorigenesis, exceeding tumor-inherent OT-1 cells derived from the donor. The most important thing to remember is that to perform a gentle dissection during subcutaneous tumor transplantation to avoid injuries on surrounding tissues, especially in granular lymph node.
