The overall goal of this procedure is to develop a mouse orthotopic model of fluorescent pancreatic cancer that can be monitored non-invasively in a live animal. This model allows a non-invasive study of the effects of a specific drug of interest on a primary tumor and its metastasis in a live animal. The main advantage of this technique is that the effects of the treatments can be easily observed in situ.
Begin by thawing an aliquot of high-concentration Matrigel according to the manufacturer's instructions. Next, wash each flask of cells with five milliliters of DPBS, and harvest the cultures with trypsin. After adding RPMI medium, transfer the resulting cell suspensions into 15-milliliter tubes and spin down the cells.
At the end of the centrifugation, transfer the cells to a bio-safety cabinet and re-suspend the pellets in 10 milliliters of fresh DPBS with gentle pipetting. After counting, centrifuge the cells again and dilute the cell suspensions to a concentration of three times 10 to the sixth cells per 50 microliters of ice-cold, serum-free medium and Matrigel, a high-concentration, basement-matrix membrane. Then, gently vortex the samples and place them on ice.
To implant the cells, first apply ointment to an anesthetized mouse's eyes and place the animal on a heating pad covered with a sterile drape. Then, gently disinfect the flank of the animal with three iodine scrubs, followed by three 70%ethanol rinses. After confirming a lack of response to toe pinch, load approximately 200 microliters of the prepared cells into a pre-cooled one-milliliter TB syringe equipped with an 18-gauge needle.
Replace the 18-gauge needle with a 27-gauge needle and place the cells back on ice. Then, locate the general area of the spleen in the upper left quadrant of the abdomen. Using forceps, pinch the skin above the spleen and make an approximately one-centimeter incision to create a pocket.
Then, pinch the smooth muscle on top of the spleen and cut through the tissue to access the peritoneal cavity. Next, gently grab the caudal end of the spleen and pull it out of the body cavity. Using a wet, sterile, cotton swab, spread the pancreas attached to the end of the spleen to locate the pancreatic tail.
Deliver 50 microliters of the cells into the pancreatic tail, then slowly rotate the needle out of the pancreas. A successful implantation will look like a superficial bubble without any leaks. Return the organs to the peritoneal cavity and enclose everything with the muscle and skin.
Then, use a 6-0 suture to close the incision. Afterwards, inject the animals with ketoprofen and monitor them until they are fully recovered. At the appropriate experimental time point, use a commercial small-animal image system to image the tumor growth in the live animal under anesthesia and select the appropriate excitation and emission filters.
Next, obtain an initial image using white light only. Keeping the animal in the same position, switch to the GFP filters and acquire a second image. To analyze the tumor growth, superimpose the white and fluorescent images to assess the fluorescent area and intensity of the tumor cells.
The detection of a green fluorescent signal between two and three weeks post-implantation provides a visual cue for confirming the presence of a developing pancreatic cancer tumor, as the animals that do not develop tumors do not exhibit a GFP signal. Further, this method allows non-invasive monitoring of the tumor growth progression, with an increase in the GFP signal observed over time as the tumor size increases. Metastasis of the tumor to specific organs can be confirmed upon removal of the tissues of interest for further ex vivo fluorescent imaging.
Once mastered, this technique can be completed in six to eight minutes and can be used to study the effects of different chemotherapeutic agents on pancreatic cancer and its metastasis. While attempting this procedure, it's important to keep the Matrigel in ice, or else the gel will be hard to inject. Also, it is important to remember to use aseptic techniques and to monitor all the anesthesia levels at all times.
After watching this video, you should have a good understanding on how to produce an orthotopic model of pancreatic cancer in mice. Don't forget that working with human cancer cell lines can be hazardous, and that precautions such as wearing protective equipment and properly disposing biological waste, should be taken at all times.
