Fluorescent Orthotopic Mouse Model: Implanting GFP Expressing Cancer Cells into Mouse for Assessing Tumor Progression In Vivo

- Cancer cells expressing green fluorescent protein or GFP enable monitoring tumor growth and metastasis by non-invasive optical imaging. We usually implant these cells into an orthotopic model, which allows the recreation of the tumor microenvironment and mimics the interaction of tumor cells with their surroundings.

Begin by placing an anesthetized mouse on a warm heating pad. Now, insert its nose into a nose mask to supply oxygen and isoflurane, an anesthetic. Using a scissor, make a small incision and cut the tissue. Gently pull out the spleen along with the pancreas and locate the pancreatic tail.

Load a syringe with fluorescent cancer cells suspension and gently inject it into the pancreatic tail. A superficial bubble appears at the injected site confirming successful implantation. Place the organs back into the body cavity and use a sterile suture to close the incision. Now, return the mouse to a cage and monitor it until recovery.

After the desired duration, place the mouse in an imaging chamber and take images. The light beam hits the cancer cells and excites the GFP, which, in turn, produces a green fluorescence. In the following protocol, we will perform surgical implantation of GFP-expressing pancreatic cancer cells in an orthotopic murine mouse model.

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 1 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 1-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.