Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice
Transcript
– Bioluminescence imaging allows the non-invasive tracking of cancer cells to study their distribution within animal tumor models. To generate a mouse model, take an anesthetized mouse and sterilize its abdominal area. Make a surgical incision to locate the spleen. Draw it out from the abdominal cavity and expose the underlying pancreas.
Next, inject a chilled suspension of transduced pancreatic cancer cells mixed in a suitable hydrogel matrix into the mouse's pancreas. These genetically modified cells express luciferase enzymes. Hold the syringe in place to allow the cell-matrix suspension to solidify, minimizing leakage.
Place the organs back in the abdominal cavity and suture the incision. Allow the mouse to recover. Once the tumor develops, anesthetize the mouse and inject a bioluminescent substrate solution into its tail vein. Allow the molecules to reach the tumor site containing luciferase-expressing cells.
Within the cells, luciferase enzymes catalyze the oxidation of substrate molecules, resulting in light emission. Place the mouse in a bioluminescent imaging chamber to capture images of the tumor to study tumor growth dynamics. In the following protocol, we will show the generation of a bioluminescent orthotopic pancreatic cancer mouse model by injecting luciferase-expressing pancreatic cancer cells.
– Using sterile surgical instruments, make a 1.5-centimeter incision through the skin approximately 2 millimeters left, lateral from the midline. Then, make a 1.5-centimeter incision in the underlying abdominal muscle. Next, locate the spleen using sterile forceps and gently remove it from the abdominal cavity. Secure the spleen along a sterile cotton bud to expose the underlying pancreas. Then, locate the tail of the pancreas adjacent to the spleen.
Resuspend the Matrigel cell suspension and inject 20 microliters into the tail of the pancreas. Following injection, hold the syringe in place for 30 to 60 seconds until the Matrigel has had time to solidify. This important step minimizes cell leakage.
After the needle is removed, inspect the site of injection to ensure no leakage occurred. Then, return the spleen and pancreas to the abdominal cavity. Close the abdominal musculature of the mouse with an absorbable, braided, 4-0 suture with a round needle using a continuous stitch.
Next, close the external skin with a non-absorbable, monofilament, 6-0 suture with a cutting needle using a continuous stitch and apply Betadine. Then remove the mouse from the inhaled anesthetic and inject between 0.05 and 0.1 milligrams per kilogram buprenorphine, subcutaneously, as a postoperative analgesic. Allow the mouse to recover in its cage placed on a 37 degree Celsius heating pad with free access to food and water.
Continue to monitor the mouse closely for the first few days after surgery. If the mouse demonstrates signs of pain, such as hunching or reduced mobility, buprenorphine may be given subcutaneously every 12 hours over a 36-hour period. After the surgical site heals in 7 to 10 days, anesthetize the mouse and remove the external sutures.
To begin bioluminescent tracking of the pancreatic cancer cells, anesthetize the mouse again using 2% to 3% inhaled isoflurane and place lubricant on the eyes to prevent drying. Once anesthetized, inject 150 milligrams per kilogram of D-luciferin via tail vein injection, and allow one to two minutes for the luciferin to make its way to the cells.
Place the mouse on its right side in the bioluminescent imaging system so that the tumor points towards the camera. Here we are using Lumina II imaging system. Then capture white light and bioluminescence images using the imaging software, as previously described in other JoVE articles. Once imaging is complete, remove the mouse from the inhaled anesthetic and allow it to recover in its home cage. Repeat these steps throughout the duration of tumor growth in order to investigate tumor growth kinetics.
