Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8⁺ T Cells During Tumorigenesis

The C57BL/6J mice (referred to B6 mice) and naive P14 transgenic mice^9,27 used were 6-10 weeks of age weighing 18-22 g. Both male and female were included without randomization or blinding. All animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Qingdao Agricultural University.

1. Preparation of medium and reagents

1. Prepare B16F10-GP melanoma cell culture medium, named D10 (complete DMEM medium) by adding DMEM, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine with an additional 100 U/mL puromycin. Maintain a sterile D10 and store for up to 2 weeks at 2-4 °C.
2. Prepare R2 medium (for termination of red blood cell lysis) by adding RPMI-1640 with 2% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.
3. Prepare fluorescence-activated cell sorting (FACS) buffer by adding 1x phosphate-buffered saline (PBS) with 2% FBS and 0.01% of sodium azide. The addition of sodium azide can prolong the storage time of FACS buffer, which can be stored for months at 2-4 ° C.
4. Prepare red blood cell lysis (RBL) buffer by adding 155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM ethylenediamine tetraacetic acid (EDTA) into double distilled water and adjust its pH to 7.3. Store RBL buffer at room temperature (RT) and it remains stable for up to 3 months.
5. Prepare the anesthetic, 2,2,2-tribromoethanol, as described below.
   1. Weigh out appropriate-amount of 2,2,2-tribromoethanol crystal in a 50 mL sterile conical tube wrapped with aluminum foil. Add appropriate amount of 2-methyl-2-butanol into the crystal to prepare the stock solution with the concentration of 500 mg/mL.
   2. Swirl it to mix and warm the tube in a 37 °C water bath until the crystal is dissolved. Make sure all crystals are dissolved.
   3. Mix the solution thoroughly. Filter the stock solution with a 0.22 µm filter into a sterile container. Store the stock solution frozen, shielded from light, at -20 °C or dilute with PBS into a working solution at the concentration of 12.5 mg/mL and stored at 4 °C.
      ​NOTE: It is best to use the prescribed concentration as higher concentrations of the liquid are irritating.

2. Preparation of B16F10-GP cell suspension

1. Thaw and culture a vial of 1 x 10⁶ B16F10-GP cells with 5 mL of D10 in a cell culture incubator at 37 °C and 5% CO₂. Subculture cells when they reached 80% to 90% density as described below.
   1. Discard the original culture medium by aspiration with a pipetting gun and wash the cells 2x with PBS. When cleaning adherent cells with PBS in a cell culture dish, move the PBS to the side wall or drop it into the dish.
   2. After aspiration of PBS, add 0.5-1 mL of 0.25% trypsin EDTA solution to the cell culture dish or flask. Gently shake to cover the entire cell surface. Place the cell culture dish or flask in an incubator at 37 °C for about 1 min or at RT until the cells are rounded and separated. Observe exfoliation of cells with the help of an inverted microscope.
   3. Add equal volume of freshly formulated D10 to terminate trypsin digestion. Purge the lower surface of the cell culture flask with a pipetting gun to ensure that all cells were separated.
   4. Transfer the B16F10-GP cell suspension to a 15 mL centrifuge tube and centrifuge at 163 x g for 4 min at RT.
   5. Aspirate the supernatant and discard. Resuspend the cells in 1-2 mL of D10 for precipitation. Transfer B16F10-GP cell suspension to a new cell culture dish or flask containing 8-10 mL of D10 and then incubate in a cell incubator at 37 °C at 5% CO₂.
2. On the day of tumor implantation, collect B16F10-GP cells with a density of approximately 90% as described in steps 2.1.1 to 2.1.4. After centrifugation, aspirate the supernatant and discard. Resuspend the cell precipitate in 1 mL of PBS.
3. Count viable cells using a 0.4% trypan blue hemocytometer. Add PBS to dilute the cell to 5 x 10⁵ cells per 100 µL. Place the cells on ice until use.

3. Ectopic inoculation of B16F10-GP cells in the bilateral inguinal region of mice

1. Aspirate 100 µL of prepared B16F10-GP cell suspension using a 1 mL syringe. Flick the tube wall to move the bubbles to the top and push the piston to move the top bubble out.
2. Hold the mouse in a supine position, exposing its abdomen. Press the right hind limb of the mouse in the supine position with a finger so that the skin in the right inguinal region is fully exposed (Same with the left inguinal region).
3. Remove the fur on the abdomen with depilatory cream, then clean the bilateral abdomen area with cotton containing 75% ethanol.
4. Before inserting the needle, make sure that the skin in the inguinal region is tightened. Insert the needle at a 45° angle in the subcutaneous space of the inguinal region of the upper thigh. Make sure the needle is beveled upward and the depth of the needle to 0.5-1 cm.
5. Apply gentle suction before injection, if there is no blood, inject the cells slowly injected into the subcutaneous tissue. At the same time, observe a small bolus (formation of fluid pocket) in the subcutaneous region.
6. After injection, remove the needle, put it in the sharps box, and return the mouse to its cage.

4. Adoptive transfer of P14 T cells into tumor-bearing mice

1. Perform adoptive transfer in tumor-bearing mice 6-8 days after tumor implantation, when the tumors are palpable (approximately 3-5 mm in diameter). Intraperitoneally inject 4 mg cyclophosphamide the day before transfer^28,29,30.
   NOTE: The aim of CTX injection is to create space in the lymphatic compartment for adoptively-transferred T cells by transiently eliminating proliferating lymphocytes.
2. Isolate lymphocytes from the spleen and LNs of naïve P14 transgenic mice as described below^31,32 (6-10 weeks old, the same sex as tumor-bearing mice to avoid rejection issues).
   NOTE: Perform the following procedures in a biosafety cabinet to ensure strictly sterile conditions.
   1. Prepare two 6 cm dishes and add 3 mL of R2 medium to one of the dishes and 3 mL of RBL buffer to the other dish. Place a 70 µm cell filter in the Petri dish containing RBL buffer.
   2. Euthanize P14 mice in airtight containers containing isoflurane followed by cervical dislocation. Adjust the number of P14 mice according to the number of recipient mice.
   3. Harvest the spleen, inguinal, and axillary lymph nodes from the euthanized mice and transfer them to 6 cm dishes containing 4 mL of R2 medium and placed on ice.
   4. Place the spleen in a strainer soaked with 3 mL of RBL buffer, grind the spleen with the putter inside of the 1 mL syringe. Incubate the cells at RT for 1-2 min, then terminate the reaction with 3 mL of cold R2 medium.
   5. Grind the LNs with the putter inside of the 1 mL syringe until only connective tissue remains in the strainer with a 70 µm cell filter. Rinse the filter with cold R2 medium and transfer the cell suspension to a new 15 mL centrifuge tube. Centrifuge the samples at 500 x g for 6 min at 4 °C.
   6. Discard the supernatant and resuspend the cells with 3 mL of PBS. Use a 70 µm cell filter to remove flocculent material from the cell suspension.
   7. Centrifuge the cell suspension at 500 x g for 6 min at 4 °C. Resuspend the cells with 3 mL of PBS and place the tube on ice. Take a small sample, mix with trypan blue, and count cells using a blood cell counting plate.
3. Determine the percentage of P14 (live/dead^–CD45.1⁺CD8⁺Vα2⁺) cells by flow cytometry. Ensure the transferred donor cells exhibit distinct congenic marker with recipient mice (tumor-bearing B6 mice is CD45.2⁺). Perform staining prior to transfer to verify the correct phenotype of the transferred cells.
   1. Add 5 x 10⁴– 1 x 10⁵ cells to a 1.5 mL centrifuge tube containing 1 mL of FACS buffer. Centrifuge the cell suspension at 500 x g at 4 °C for 3 min.
   2. Discard the supernatant, flick the bottom of the tube to disperse the cells, and place the tube on ice.
   3. Prepare the following conjugated antibody mixture^9,32 diluted in 100 µL of FACS buffer: anti-CD8, 1:200; anti-TCR Vα2, 1:100; anti-CD45.1, 1:200; Live/dead stain, 1:400. (Table of Materials).
   4. Centrifuge the antibodies mixture at 15,000 x g for 3 min to aggregate the particles. Place the mixture on ice and shield it from light by wrapping it in foil. Only take the supernatant to avoid the aspiration of particles.
   5. Resuspend the cells in 100 µL of the antibody mixture and thoroughly mix by flicking the tube wall. After wrapping in tin foil, incubate the tube for 30 min on ice.
   6. Wash the cells 2x with FACS buffer. Centrifuge the tube at 500 x g at 4 °C for 3 min. Resuspend the cells with 200 µL of FACS buffer and transfer the cell suspension to a flow tube.
   7. Run the flow tube with stained cells on a flow cytometer to determine the percentage of live/dead^–CD45.1⁺CD8⁺Vα2⁺ cells.
      NOTE: Generally, over 90% of CD8⁺ T cells from P14 transgenic mice harbored Vα2⁺TCRs, which are specific to H-2D^b GP_33-41 epitope of LCMV (lymphocytic choriomeningitis virus).
4. Calculate the absolute number of live/dead^–CD45.1⁺CD8⁺Vα2⁺ cells by multiplying its percentage and the live cell number obtained in steps 4.2 and 4.3.
5. Aspirate 200 µL of P14 cell (5 x 10⁵ cells) suspension with a 100 U insulin syringe and remove bubbles. The initial number of transferred naive P14 cells (5 x 10³ – 5 x 10⁵) did not affect the phenotype of transferred cells⁹.
6. Place the mice in a cage and warm with an infrared lamp for 5-10 min to expand the tail vein. Hold in place with an appropriately sized mouse fixator, straighten the tail, and wipe it with a cotton ball containing 75% ethanol to make the veins visible.
7. Insert the needle parallel to the tail vein and gently pull back the plunger. If there is blood flowing into the syringe, slowly push the cell suspension into the vein.
   NOTE: If there is resistance or swelling of the tail during injection, the injection position needs to be adjusted. The injection site should start at the distal end.
8. After the injection is completed, pull out the needle quickly and press the injection site gently with a cotton ball. Return the mouse to a new clean cage and closely observe it for several minutes for any adverse effects.

5. Resection of the primary tumor

NOTE: Ensure all surgical instruments are autoclaved before use. Sterilize the operating area inside the biosafety cabinet with 75% ethanol, followed by UV irradiation for at least 30 min. Wear clean gowns, hats, masks, and sterile gloves during surgery.

1. When the tumor is palpable (approximately 5 mm in diameter), resect the primary tumor.
2. Anesthetize the mice with intraperitoneal injection of Ketamine (75 mg/kg). Pinch foot pad to evaluate the degree of anesthesia, if there is no pain reflex, it indicates the right timing for surgery. If the hind limbs were withdrawn, provide another dose of 10-30 µL.
   NOTE: Alternatively, intraperitoneal medetomidine (1 mg/kg body weight; Domitor) is recommended to anesthetize mice.
3. Apply the drying preventive ointment on mouse eyes. Remove the fur on the abdomen with depilatory cream to fully expose the surgical field.
4. Place the mouse in a biosafety cabinet and place it on an anatomical board covered with clean absorbent paper in a supine position so that the longitudinal axis of the mouse is parallel to the experimenter.
   NOTE: To maintain a strict sterile environment, the following procedures must be performed in a biosafety cabinet.
5. Disinfect the abdomen of the mice with a cotton ball soaked in povidone-iodine. Incise the skin near the tumor-bearing site with a sterile scalpel or opthalmic scissors. Insert closed scissors tip into the incision to clearly expose the tumor. When incising the skin, make sure not to damage the inguinal lymph nodes.
6. During tumor removal, keep the capsule as intact as possible. Carefully and gently remove the connective tissue adjacent to the tumor with sterile scissors. Remove the tumor in situ completely, otherwise it may recur.

6. Intranodal injection of B16F10-GP cells in the inguinal lymph node

NOTE: After bilateral tumor clearance, B16F10-GP cells were injected into unilateral inguinal lymph node and PBS was injected into the other side.

1. Aspirate 20 µL (5 x 10⁴ cells) of B16F10-GP cell suspension with a 100 U insulin syringe, remove bubbles and then inject into one inguinal lymph node. Inject an equal volume of PBS into the inguinal lymph node on the other side.
   1. During the injection, insert the needle from the distal end of the lymph node, and then slowly insert the needle to the center of the lymph node. At this time, if the fluid is accurately injected into the lymph node, the lymph node can be seen to swell significantly.
      NOTE: When the needle is inserted from the distal end of the lymph node, make sure not to puncture the node.
2. Suture the incision with 2-3 stitches using a 3-0 suture. Disinfect the skin surrounding the wound with cotton impregnated with povidone iodine. Take care to bypass lymph nodes during suturing.
3. Place the mouse in a clean cage and keep warm using infrared light in the lateral decubitus position. Monitor continuously until consciousness is recovered. Ensure that the mice that has undergone surgery is not returned to the company of other animals until fully recovered.
4. Give buprenorphine every 4-6 h for 3 consecutive days after the operation to relieve postoperative pain (at a dose of 0.1-0.5 mg/kg, SQ or IP). Monitor the feeding, drinking, movement, and activity areas of the mice. Typically, mice recover from surgical trauma within 3 days.
   NOTE: If mice are unable to resume normal feeding and activities and show any signs of infection, consult a veterinarian for intervention or euthanize it.
5. Sacrifice mice at different time points: day 8 and day 18 after intranodal injection of tumor cells. Recover activated donor cells using flow cytometry analysis.