Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

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

1. Preparation for Tumor Transplantation

1. Animal Housing
   1. Obtain generic C57BL/6 mice from a commercial breeding vendor.
   2. House mice (5) in individual ventilated cages at the following conditions: Temperature: 20–26 °C; humidity 30–70%; lighting cycle: 12 h light and 12 h dark.
   3. Use corn cob bedding that is changed weekly.
   4. For diet, provide irradiation sterilized dry granule food during the entire study period.
   5. For water, provide animals free access to sterile drinking water.
2. Donor tumor fragment preparation
   1. Begin by monitoring body weight (BW), via weighing using a balance, and tumor volume (TV), by caliper measurement, of tumor-bearing donor mice.
   2. When TV reaches 500–1,200 mm³, euthanize the animal in a biohazard hood as per protocol.
   3. Sterilize the skin around the tumor using iodophor swabs.
   4. Surgically remove the tumor as per protocol.
   5. Inspect visually and by palpation for the presence of palpable tumors before termination.
   6. For termination, first euthanize mice as per approved protocol, and open the abdomen and visually examine the pancreas for tumors.
   7. Cut off the tumor by post mortal surgery and add it to cold PBS. Place all animals in a clean, uncharged, translucent euthanasia chamber with a special lid with a port for delivery of the carbon dioxide to be used.
   8. Discharge gas into the chamber at a flow rate that produces rapid unconsciousness with minimal distress to the animal. The optimum flow rate for CO₂ should be around 2–2.5 L/min.
   9. Visually observe each animal during the euthanasia procedure to assure all animals receive adequate gas concentrations and do not regain consciousness during the euthanize procedure.
   10. Maintain gas flow for at least 1 min after apparent clinical death to minimize the possibility that an animal may recover (if apneic but not dead).
   11. Remove adjacent non-tumor tissues. Cleaned tumor can be photographed quickly.
       Put the tumor tissues into RPMI-1640 and keep on ice before dissociation.
       NOTE: Animal carcasses are bagged and stored in the appropriate freezer to await removal.
   12. Place the tumor in a Petri dish containing 20 mL of PBS (pre-chill the media or buffer to 4 °C prior to euthanizing animals).
   13. If there is contaminating blood, transfer the tumor into another Petri dish and wash the tumor with PBS.
   14. Cut the tumor in half, removing any extra skin, vessels, calcification and/or necrosis.
   15. Choose only intact pieces of tumor and place them into a sterile 50 mL centrifuge tube and add 20 mL of PBS then transport the tube to a separate animal room for pharmacology studies.

2. Pancreatic orthotopic implantation

1. Anesthesia and Analgesia
   1. Use a 2 mL ketamine injection and 0.42 mL xylazine injection (20 mg/mL) mixed in 5.91 sterile injection water or saline at a dose volume of 0.06–0.1 mL/20-25 g body weight.
      NOTE: According to animal welfare, analgesia is necessary both pre and post operation. 0.05–0.1 mg buprenorphine /kg, SC. The first dose is pre operation and then dosed 3 times every 4 hours post operation continually.
2. Surgical operation for orthotopic implantation
   1. Anesthetize mice via intramuscular injection (IM). Anesthetize the recipient animal with 5% isoflurane, which is maintained by a nose cone at 1%. The animal will begin to relax, losing their righting reflex and eventually become immobile. At this depth of anesthesia they can easily be roused by painful stimuli; allow anesthesia to deepen until such responses to pain are absent.
   2. After the animals are fully anesthetized, fix the mice on an experiment board in the right lateral position.
   3. Keep the mice in the right lateral position. Disinfect the skin around the spleen with iodine then de-iodinate with 75% ethyl alcohol.
   4. Find the medium point of the spleen and make a 1 cm vertical incision on abdomen to expose the spleen.
   5. Draw out a part of pancreas tissue under the spleen gently with flat-tip tweezers, and suture a mouse homograft tumor piece from seed mouse on the pancreas of recipient mouse by 9-0 Absorbable surgical suture.
   6. Close the abdomen with a 6-0 silk suture by double seam. Achieve homeostasis by compression.
   7. After finishing tumor implantation, if neither bleeding nor tumor tissue leakage occurs, keep the animals in a warm cage.
   8. Monitor the animal until it regains sufficient consciousness to maintain sternal recumbency; return the animal to the animal room after full recovery from the anesthesia. Monitor the tumor bearing mice by palpating the abdomen near the spleen and select out the mice bearing orthotopic tumors.

3. Tumor-Bearing Mice Health Monitoring

1. Check the water and food consumption daily.
2. Examine the mouse appearance for an ungroomed hair coat, lumps, thinness, abnormal breath or ascites.
3. Palpate the abdomen to check if there are spontaneous tumors on the liver or spleen.
4. Weigh mice weekly using a balance.
5. If any of the following clinical signs are observed, the mice are sacrificed for sample collection and necropsy: BW loss >20%; impaired mobility (not able to eat or drink); unable to move normally due to significant ascites and enlarged abdomen; effort in respiration.