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

Overview

The video demonstrates the breast cancer orthotopic inoculation technique in a mouse model. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
Micro Dissection Scissors	Roboz	RS-5983	For cancer cell innoculation and mastectomy
5-0 silk sutures	Look	774B F
Roswell Park Memorial Insitute 1640	Gibco	11875093	For cell culture
Fetal Bovine Serum	Gibco	10437028	For cell culture
Trypsin-EDTA (0.25%)	Gibco	25200056	For cell culture
Dry sterilant (Germinator 500)	Braintree Scientific	GER 5287-120V	For cancer cell innoculation and mastectomy
Needle Holder	Roboz	RS-7830	For cancer cell innoculation and mastectomy
Adson Forceps	Roboz	RS-5233	For cancer cell innoculation and mastectomy
Clipper	Wahl	9908-717	For cancer cell innoculation and mastectomy
Matrigel	Corning	354234	For cancer cell innoculation