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

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

1. Preparation of cell lines for injection

NOTE: Use a volume of 20 µL with 100,000 cells for each injection. Use basement membrane matrix (BMM) to prevent leakage and ensure standardized injection. To ensure reproducible results, conduct cell line authentication assays (e.g., via STR profiling) at regular intervals.

1. Grow all colorectal cancer cell lines under standard culturing conditions (37 °C, 5% CO₂) and prepare them on the day of surgery.
2. Harvest the cells according to standard cell culture protocols, count the cells (e.g., in a coulter counter or a hemocytometer), and calculate the required amount for all injections depending on the numbers of animals to be injected.
3. Prepare 3-5x of the needed volume to account for pipetting losses and the dead volume of the injection syringe.
4. Centrifuge (5 min at 1,500 g) and wash the cell suspension twice with PBS.
5. Resuspend the cells in BMM at a concentration of 5 x 10⁶ cells/mL and keep them on ice.

2.  Orthotopic Mouse Model

1. Preparation of recipient animals for surgery
   NOTE: Use 6-8 week-old NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NOD scid gamma, NSG) mice as recipients. NSG is among the most immunocompromised mice, lacking mature B, T, and NK cells and multiple other immune defects. They reliably grow tumors even if low cell numbers are injected and are highly prone to distant metastases. NSG mice are excellent breeders and can be kept in conventional specific pathogen-free (SPF) units.
   1. Before the first incision, inject 0.05 mg/kg of buprenorphine subcutaneously.
   2. Use sevoflurane at 3-3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.
   3. Cover the eyes of the anesthetized mice with ophthalmic ointment to avoid desiccation of the cornea.
   4. Restrain the mice in a supine position on a small table.
   5. Shave the abdomen with an electric shaver (depilatory cream can be used alternatively) and disinfect with at least 3 times chlorhexidine/iodine and 70% alcohol.
   6. Cover the surgical field with sterile drapes.
      NOTE: The use of perioperative antibiotics is optional and subject to institutional guidelines.
2. Midline laparotomy and exposure of the cecum
   1. Use scissors (scalpels can be used alternatively) to make a small midline incision (3 – 5 mm) of the skin on the lower abdomen. Pick up the abdominal wall musculature with forceps and carefully incise it with scissors, opening the abdominal cavity.
   2. Identify and carefully exteriorize the cecum with atraumatic forceps. Position the blind-ending pouch of the cecum on the abdomen, pointing cranially.
   3. Once exteriorized, keep the cecum moist using warm saline swabs at all times.
3. Orthotopic injection, closure of the abdomen, and postoperative recovery
   1. For intracecal injection, use a standard 1 mL syringe with a 30 G cannula. Mount this syringe on a microinjection pump, which is, in turn, mounted on a micromanipulator.
   2. Carefully grasp the tip of the cecum with atraumatic forceps and gently smoothen it by stroking it downward with a second set of forceps moistened with warm saline.
   3. Position the cannula directly above the cecum.
   4. Perform the following steps under visual control with a binocular surgical microscope.
   5. Carefully grasp the cecum with two atraumatic forceps at both ends of the exteriorized part of the cecum, slightly stretch it and then slowly pull it over the cannula, which is positioned parallel and directly above. It is crucial not to perforate the entire bowel wall (thus injecting the cells into the lumen) as well as not to perforate the serosa beyond the initial point of penetration, as this would lead to leakage and peritoneal dissemination.
      1. Move the bowel towards the cannula, not the cannula towards the bowel. Hold and stretch the bowel between two forceps. The surgeon's hands must be resting on a surface to reduce tremor.
   6. Inject the cells between the serosa (seen as a very thin, translucent lining above the intramural blood vessels) and the muscularis. The cannula must therefore be visually placed above the blood vessels and underneath the thin translucent membrane.
   7. Use a footswitch to start the injection to reduce tremor while the cannula is inside the bowel wall.
   8. Once the cannula is in position, start the injection. Use a duration of 20 s, resulting in 1 µL/s setting on the control unit of the pump.
      NOTE: The injection into or near a damaged blood vessel leads to direct intravascular dissemination and distant metastasis and should therefore be avoided.
   9. After completion of the injection, carefully remove the cannula by pulling it backward.
   10. Place a dry swab under the cecum, and then thoroughly rinse the cecum with distilled water to lyse leaked cells and thus prevent artificial peritoneal dissemination.
4. Closure of the abdomen and postoperative recovery
   1. After rinsing, gently return the cecum to the abdominal cavity.
   2. Close the abdominal wall with 6-0 rapidly absorbable running sutures.
   3. Close the skin with surgical wound clips.
   4. Place the mouse on a heating map set to 38 °C until fully recovered from the anesthesia.
   5. Closely observe the postoperative condition of the mice for the following 48 h. In case of distress, treat with 0.05 mg/kg buprenorphine every 12 h.
   6. Monitor the mice at least once daily for signs of distress due to tumor growth.