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

To begin, place an anesthetized mouse on a surgical bed and inject buprenorphine, an analgesic to relieve pain, subcutaneously into the shoulder. Next, insert the mouse nose into a nose mask to provide a continuous supply of oxygen and anesthetic, such as isoflurane to the animal. Using some lab tape, immobilize the limbs and sterilize the chest skin with chlorhexidine and iodine, two disinfectants, and ethanol.

Now use scissors to make an incision in the middle of the anterior chest wall and invert the skin with forceps to expose the white mammary fat pad. Carefully inject the cancer cell suspension expressing a bioluminescent protein into the mammary fat pad. Wait for 5 seconds before pulling out the needle to prevent backflow. Gently release the fat pad and use sterile sutures to stitch and close the incision.

The injected cancer cells inside the mammary fat pad divide rapidly to form a tumor. Return the animal to a cage after the procedure. Closely monitor the animal until it recovers from anesthesia and starts moving freely.

In the following protocol, we will implant mammary adenocarcinoma cells expressing luciferase into an adult mouse's mammary fat pad.

Begin by confirming a lack of response to toe pinch in an anesthetized female mouse and taping the animal's limbs to a surgical board. Using sterile cotton swabs, sterilize the surgical area with chlorhexidine, iodine, and 75% ethanol, and make a five-millimeter skin incision in the middle of the anterior chest wall. Next, lift the right side of the skin adjacent to the incision and use sterile microdissection scissors to detach the skin from the chest wall.

Invert the skin to expose the right number two mammary fat pad and use a one-milliliter insulin syringe equipped with a 28.5 gauge needle to carefully inject 20 microliters of cancer cell suspension through the wound into the fat pad. Hold the needle within the fat pad for five seconds to allow the gelatinous cancer cell protein mixture to solidify before removing the needle, and closing the incision with sterile 5-0 non-absorbable sutures. Then, place the animal in a clean cage with monitoring until full recumbency.