Intracecal Tumor Cell Injection: A Technique to Deliver Cancer Cells for Establishing Orthotopic Colorectal Cancer Mouse Model

- Intracecal orthotopic tumor injection delivers tumor cells matching the cecal tissue histotype directly into the cecum of the recipient animal. To begin, prepare an anesthetized mouse for surgery. Incise its abdominal wall to exteriorize the cecum. Scrape the cecal wall to diminish any intestinal peristaltic movement. Align a syringe containing colorectal cancer cell or CRC suspension parallelly over the cecum.

Next, stretch the cecum to remove any folds. Gently pull the cecum over the cannula such that the tip perforates just the thin, translucent, outer intestinal membrane, or serosa. Next, inject the cancer cell suspension into the space between the serosa and inner muscular layer. Ensure complete delivery of the cell suspension and retract the cannula. Return the cecum to the abdominal cavity and suture the surgical incision. Allow the mouse to recover.

Within days of injection, an orthotopic tumor develops at the site of injection or metastasizes to distant tissues or organs. In the following protocol, we will demonstrate an intracecal colorectal cancer cell injection technique to develop an orthotopic CRC mouse model.

- Begin by using atraumatic forceps to carefully exteriorize the cecum of a six to eight-week-old immunocompromised mouse, placing the blind end of the cecum on the abdomen so that the pouch points cranially. For the intracecal injection, mount a standard 1-milliliter syringe equipped with a 30 gauge cannula and loaded with the tumor cells onto a microinjection pump that is mounted on a micromanipulator. Next, use the atraumatic forceps to carefully grasp the tip of the cecum, and use a second forceps moistened with warm saline to gently smooth the cecum with downward strokes.

Now, move the animal under a binocular surgical microscope, and with the cannula directly parallel to and above the cecum, use two atraumatic forceps to carefully stretch the tissue at both ends of the exteriorized end of the cecum. Slowly pull the cecum over the cannula, taking care not to perforate the entire bowel wall or the serosa beyond the initial point of penetration, and place the cannula above the blood vessels and under the thin translucent membrane inside the bowel wall. When the cannula is in place, use a footswitch to inject 20 microliters of cells over a period of 20 seconds between the thin translucent serosa lining above the intramural blood vessels and the muscularis.

When all of the cells have been injected, carefully remove the cannula and place a dry swab under the cecum. To prevent artificial peritoneal dissemination, lyse any leaked cells with a thorough distilled water rinse and gently return the cecum to the abdominal cavity. Close the abdominal wall with 6-0 rapidly absorbable running sutures and the skin with surgical wound clips. Then, place the mouse on a heating mat set at 38 degrees Celsius with monitoring until full recumbency.