The overall goal of this procedure is to establish mouse orthotopic renal tumors through the intrarenal implantation of tumor cell for the study of tumor growth and metastasis and the efficacy of anti-tumor therapies. This method can be used to answer key questions in the urologic oncology field such as how effective is a novel therapy in a pre-clinical model of kidney cancer. The main advantage of this technique is that intrarenal tumor implantation establishes a primary lung metastasizing renal tumor mimicking clinical renal cell carcinoma pathogenesis.
To prepare the Renca tumor cells first detach the cell culture with 0.25%Trypsin and HBSS after rinsing with PBS. Neutralize the reaction after five minutes with Complete RPMI Medium. Transfer the cells to a conical tube for centrifugation and re-suspend the pellet in fresh HBSS.
After counting, adjust the volume to a two times ten to the six cells per milliliter concentration in HBSS, and draw the cells into a one milliliter syringe equipped with an 18 gauge needle. Next, place a sterile surgical drape over a heating pad and confirm the appropriate level of sedation by toe pinch. Remove the hair from the left flank of the mouse and apply vet ointment to the eyes.
Then swab the implantation site with 5%povidone iodine antiseptic and use a sterile scalpel to make a single one to one and a half centimeter incision on the left flank, taking care not to penetrate the peritoneum. Using scissors, separate the dermis from the peritoneum, dabbing the incision site with sterile cotton gauze to remove any blood. Then grasp the head with the left hand and support the flank with the right hand to locate the spleen through the peritoneum.
Using one finger from the right hand, palpate the mouse from underneath. The kidney should become visible. Keeping a firm hold on the animal to provide tension across the peritoneum and kidney, have an assistant slowly deliver 0.1 milliliters of the Renca cells through the intact peritoneum into the center of the kidney.
Hold the needle in place for five to 10 seconds to reduce the backflow of the cells. Then, close the incision with a thin layer of cyanoacrylate tissue adhesive. At the experimental end point, use scissors to make a midline incision, starting at the mid-abdomen through the ribcage, neck, and salivary glands.
Using scissors and forceps, carefully remove the ribs without compromising the lung tissue, and remove the connective tissue to expose the trachea. Fill a three milliliter syringe equipped with an 18 gauge needle with 10%India Ink and PBS. Then use forceps to carefully thread a silk suture under the trachea and then tie a loose knot.
Next, carefully insert the needle into the trachea and slowly inflate the lungs with one to one and a half milliliters of the India Ink solution. Pull the knot tight around the trachea to inhibit the backflow of the ink, then remove the needle and carefully cut away the connective tissue to remove the inflated lungs. Fix the lungs in a 15 milliliter conical tube containing five milliliter of Fekete's solution in a chemical fume hood at room temperature.
After 24 to 48 hours, remove the intact de-stained lungs and carefully pull apart the lobes in a 35 by 10 millimeter petri dish. Then count the number of white tumor nodules under a dissecting microscope. A successful mouse intrarenal Renca cell implantation results in tumor development in the kidney and metastasis to the lungs with tumor-bearing animals demonstrating a robust tumor burden as measured by the increased weight of the tumor-implanted kidneys.
As observed, Renca tumors can be identified by immunohistochemical staining for Cytokeratin eight and 18. The implantation of a luciferase expressing Renca cell line allows tracking of the tumor burden in the kidney and metastasis to the lung by bioluminescent imaging as further confirmed by india link lung inflation. Once mastered, this technique can be completed in less than five minutes per mouse if performed properly.
While attempting this procedure it's important to remember to clearly identify the kidney through the peritoneum to ensure an accurate intrarenal tumor implantation. Following this procedure, other methods, like preclinical drug testing can be performed to answer additional questions, such as to what extent does novel drug effect the growth or metastasis of intrarenal tumors? After watching this video, you should have a good understanding of how to perform intrarenal tumor cell implantation for establishing orthotopic kidney tumors in the mouse.
Don't forget that working with live animals and surgical instruments can be hazardous and that precautions, such as following institutional animal handling guidelines and maintaining a sterile surgical area and instruments should always be taken while performing this procedure.
