Orthotopic Injection: Implanting Tissue Specific Cancer Cells into an Adult Mouse

1. Preparation of Cells (for 10 Mice)

NOTE: The cells should be inoculated within 1 h after being detached from the dish to avoid decreased cell viability. Specifically, the cell suspension should be mixed into the gelatinous protein mixture within 15 min after detaching the cells from the dish to maintain their viability.

1. Culture 4T1-luc2 cells, a mouse mammary adenocarcinoma cell line expressing luciferase, in RPMI 1640 media with 10% FBS in a humidified incubator at 37 °C in 5% CO₂.
2. Wash the adherent 4T1-luc2 cells in a 10-cm dish with phosphate-buffered saline (PBS) using a 10 mL serological pipette. Add 1 mL of 0.25% trypsin using a P1000 pipette and, then, incubate the sample at 37 °C for 5 min. Then, add 4 mL of growth media (RPMI-1640 with 10% FBS) using a 5 mL serological pipette and transfer the cell suspension to a 15-mL conical tube in a tissue culture hood. Centrifuge the cell suspension at 180 x g for 5 min.
3. Aspirate the supernatant and resuspend the cells in 2 mL of PBS; then, count the cells using the hemocytometer.
4. Suspend 2 x 10⁶ 4T1-luc2 cells in 40 μL of cold PBS (pH 7.4, 4 °C) in the tissue culture hood.
5. Mix the 40-μL cell suspension with 360 μL of the gelatinous protein mixture in a 1.5 mL microcentrifuge tube on ice in the tissue culture hood.
   NOTE: The final concentration is 1 x 10⁵/20 μL (1:9 PBS:gelatinous protein mixture). For the orthotopic model (no mastectomy), 1 x 10⁴/20 μL of the final concentration was used, to avoid reaching euthanasia criteria (tumor size > 2 cm) within two weeks.

2. Cancer Cell Inoculation

1. Put the mice in the anesthesia induction chamber with 2-4% isoflurane and 0.2 L/min oxygen flow until the mice breathe calmly (2–3 min).
2. Grasp the mouse and inject 0.05 mg/kg buprenorphine into its shoulder subcutaneously.
3. Confirm adequate anesthesia by the lack of reaction to a toe pinch. Insert the mouse’s nose into the hole of the mouse mask, which allows inhalational anesthesia with 2-4% isoflurane and 2 L/min oxygen flow attached to a charcoal canister unit.
4. Restrain the mouse’s limbs using lab tape and sterilize its skin using chlorhexidine, iodine, and 75% ethanol, using cotton swabs.
5. Make a 5 mm skin incision in the middle of the anterior chest wall utilizing sterile microdissection scissors, lift the right-side skin next to the incision and detach the skin from the chest wall using the scissors, and then, invert the skin to expose the right #2 mammary fat pad.
6. Carefully inject 20 μL of cancer cell suspension using a 1 mL insulin syringe with a 28.5 G needle into the fat pad under direct vision through the wound.
   NOTE: The needle goes through the wound, not the skin. Keep holding the needle in the fat pad for 5 s prior to pulling it out, which allows time for the gelatinous protein mixture to solidify.
7. Close the skin incisions by stitching, using sterile 5-0 non-absorbable sutures.
8. After surgery, return the animals to a clean cage and monitor them until they have recovered and are moving freely (after ~1–2 min). If an animal does not appear to be in good health within 24 h of surgery, administer buprenorphine (0.2 mg/kg).
9. Remove the sutures under anesthesia (see step 2.1) 7 d after the surgery.