The overall goal of this procedure is to prepare an orthotopic mouse model of pancreatic cancer that uses in vivo bioluminescence imaging to non-invasively monitor tumor development. This is accomplished by first resus suspending luciferase tagged cancer cells in Matrigel. The second step is to perform a laparotomy to open the abdominal cavity and to locate and secure the pancreas.
Next, a suspension of tumor cells in matrigel is injected into the pancreas. The final step is to replace the pancreas and use sutures to close the abdominal cavity. Ultimately, bioluminescence imaging is used to monitor tumor progression and track response to therapeutic interventions.
The main advantage of this model over in vitro based cell assays or subcutaneous models of pancreatic cancer is that it really enables us to investigate the interactions between the pancreatic tumor cells and the pancreatic microenvironment. The kinetics of disease progression in this model are highly reproducible and occur in a short timeframe, making this model useful for the examination of novel therapeutics in pancreatic cancer, Which really demonstration of this model is critical. As injection of tumor cells has to be performed carefully to prevent leakage To begin this procedure culture.
Pancreatic cancer cells that have been transduced to express luciferase as is described in the written protocol until they are 70%confluent, then harvest the cells with 0.05%tripsin EDTA, and count them using a hemo cytometer, ensure that viability is greater than 90%Next, pellet the cells by gently centrifuging at 200 times gravity for five minutes following centrifugation, remove the supernatant and resuspend the pancreatic cancer cells at 20 million cells per milliliter. In a three to two mixture of matrigel and chilled PBS, place the mixture on ice. Then using a 0.3 milliliter 29 gauge insulin syringe.
Draw up the prepared matrigel cell suspension into the syringe. To begin this procedure, anesthetize the mouse using two to 3%inhaled isoflurane. After one to two minutes, check the depth of anesthesia by gently pinching the toe of the mouse to check for a lack of pedal reflex.
Next, apply sufficient lubricant to each eye in order to prevent desiccation during surgery. Then position the mouse on its back on a 37 degree Celsius heating pad, and gently turn the mouse to raise the left side of the abdomen. Once correctly oriented, prepare the mouse's abdomen for surgery.
Using sterile surgical instruments, make a 1.5 centimeter incision through the skin approximately two 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 for 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 resus. Suspend 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 four aut suture with a round needle using a continuous stitch. Next, close the external skin with a non-absorbable monofilament. Six aut 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 seven 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 two to 3%inhaled isof fluorine, and place lubricant on the eyes to prevent drying dying. Once anesthetized, inject 150 milligrams per kilogram of Lucifer via tail vein injection and allow one to two minutes for the luciferian 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 Illumina two imaging system. Then capture white light and bioluminescence images using the imaging software as previously described in other JOV 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. The use of luciferase tagged cell lines provides a unique way to measure tumor growth.Non-invasively. Shown here is a mouse 10 days after injection with a relatively small tumor, 31 days after injection where the tumor has enlarged and five days after the tumor was resected.
The bioluminescence seen in this image shows recurrence of the pancreatic tumor five days after resection, as well as metastasis to the nearby liver. The graph shown here show similar growth rates for the matrigel cell injection method. Described in this video compared another method where pancreatic tumor pieces are transplanted landed.
Similar growth kinetics can be seen for both the pan one and capin one. Cell lines following sacrifice, H and d staining has been performed to show the invasive nature of the pancreatic cancer cells into the surrounding pancreatic tissue shown here denoted by the yellow stars as well as metastases in other organs, including the liver shown here. Once mastered, this methods can be done in about 10 minutes.
In addition, bio cent visualization of metastatic and recurrent disease progression makes this model clinically relevant as currently most cancer therapeutics are palliative.
