Chemical-induced Two-stage Skin Carcinogenesis Model: An Experimental In Vivo Mouse Model of Skin Cancer

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Experimental Animals, Reagents, and Equipment

1. Use age and sex-matched mice. Start the study at 7-9 weeks of age because the skin in most mice is in telogen (the resting phase) around that age.
2. Observe the behavior of the animals during the study and if they fight, which often happens with males, house them separately. Fights may cause cuts in the skin, which promote tumor formation. Female mice are preferred due to their less aggressive behavior. The typical experimental group size varies between 8-20 animals per group.
   NOTE: Power calculations based on the biological variance seen in earlier studies help to choose a sufficiently large group size. The strains used as examples in this article include Balb/c and C57BL/6. However, many other mouse strains, such as SENCAR and FVB, have been used with the DMBA-TPA-model and Wistar and Sprague-Dawley rats. A license from the national or local committee of animal work is needed before the initiation of the study. In addition to general welfare considerations, the model-specific endpoints are typically squamous cell carcinoma (SCC) and infection of the skin. Scratches in the skin due to itching after applying the tumorigenic chemicals in acetone are typical, but otherwise, the animals should show no signs of discomfort. Weighing regularly (e.g., 2x a month) helps evaluate the animals' welfare.
3. Use DMBA and TPA, both diluted in acetone. The working concentration of DMBA is 250 g/L. A dose for one animal is 50 µg of DMBA in 200 µL of acetone. The stock concentration of TPA is 125 g/L and the treatment concentration 25 g/L. A TPA dose for one animal is 5 µg in 200 µL of acetone.
   CAUTION: DMBA is harmful if swallowed and may cause cancer. Acetone evaporates rapidly and is flammable. It may cause dizziness and irritate the eyes. Use a breathing mask and/or work under a vacuum flow. Change gloves after handling any of these chemicals. Individually ventilated cages prevent the spread of the chemicals during the housing of the mice. After application, gather the pipette tips used for handling the DMBA and dispose them as dangerous waste.
   NOTE: The DMBA must be protected from light. The diluted TPA is stored at -20 °C, preferably protected from light.
4. Procure the following equipment: a scale, an ordinary shaver for the fur, pipettes, and tips of an appropriate size, an ordinary ruler, a digital camera, a notepad, and a pen or a computer for recording the papillomas, an inhaled anesthesia system or a mouse restrainer with an opening on the back top, and a carbon dioxide narcosis system for sacrificing mice.

2. Skin Papilloma Induction and Promotion

1. Shave the back skin and weigh the animal. Later, shave the skin whenever needed but not at the time of chemical exposure.
   NOTE: Be careful when shaving around the papillomas and avoid making any cuts on the skin. Weigh each animal every 2 weeks to notice any potential weight loss.
2. Apply 50 µg of DMBA in 200 µL of acetone topically on the shaved area using a pipette 48 h after shaving the fur. If needed, restrain the animal using light inhalation anesthesia or a mouse restrainer.
3. After 7 days, give the first TPA dose. Apply 5 µg of TPA in 200 µL of acetone topically with a pipette 2x a week, preferably Monday and Thursday or Tuesday and Friday.
4. Count, record, and photograph the papillomas every week. A palpable mass greater than 1 mm in diameter is considered a papilloma if it stays longer than 1 week. Mark each individual papilloma on a map and list its size every week—store digital photographs.