The overall goal of this procedure is to generate spontaneous metastatic renal cell carcinoma that is clinically relevant. This is accomplished by first preparing reka Luke cells for implantation. The second step is to perform an orthotopic kidney tumor cell implantation.
Next, after the tumor has grown, the primary tumor is removed by performing a nephrectomy. The final step is to monitor post nephrectomy spontaneous metastasis progression and assess disease distribution in various locations. Ultimately, this protocol allows monitoring of spontaneous metastatic renal cell carcinoma disease and offers a guideline for standardizing the assessment of the efficacy of experimental therapeutics.
The main advantage of this technique over existing methods such as ectopic, often superficial or subcutaneous tumor cell implantation methods, is that it recapitulates renal cell carcinoma or RCC as it presents in patients, which includes the ability of tumor cells to spread to the rest of the body. The implications of this technique extend toward the treatment of renal cell carcinoma because it is the systematic dis metastatic disease that is the really the primary cause of mortality in patients. Mouse models typically do not treat spontaneous metastatic disease that can sometimes follow nephrectomy.
Though this method can provide insight into spontaneous metastatic RCC disease. It can also be applied to other cancer types where primary tumor removal is typical and the main cause of mortality is port surgical metastatic disease recurrence. This includes models of breast and melanoma among others.
Generally individuals new to this method will struggle because of first, the difficulty in finding the kidney and ensuring the tumor cells remain localized upon implantation and do not leak. And second, the ne effectiveness is performed efficiently and allows for post-surgical recovery. Prior to orthotopic implantation grow mouse luciferase tagged reca cells or reka Luke cells as a monolayer to 75%co fluency following trypsin, ization and resus suspension in 5%FBS containing media centrifuge, the cells repeating three times with a wash in PBS then resuspend the cells in serum free media to a concentration of five times 10 to the fourth renco Luke per five microliters of serum free media.
After anesthetizing a bowel sea mouse with two to 3%isof fluorine pinch a foot to check for sufficient anesthetization after applying vet ointment to the eyes to prevent drying, place the mouse in right lateral recumbent and shave the left side between the four and hind limbs. Then apply alcohol and iodine along the dorsal lumbar area. Use surgical scissors to make a one centimeter skin incision in a longitudinal direction between the last rib and the hip joint.
Then use blunt dissection scissors to loosen the connective tissue under the skin. Next, use surgical scissors to make a 0.5 centimeter incision in a longitudinal direction in the abdominal wall. Now use curved forceps to gently push down around the open wound.
This exteriorize the kidney and allows for gentle immobilization of the organ prior to injection. Load a Hamilton syringe with five microliters of the prepared cell mixture for a superficial subcapsular implantation. Hold the needle parallel to the longitudinally oriented kidney with the beveled edge up and insert the needle under the kidney capsule, but above the parenchyma.
Once the needle is in place, inject all of the cells until a small white bubble forms. Slowly remove the needle from the capsule and immediately use a sterile cotton tipped applicator to block the injection site for an internal subcapsular implantation. Starting from the side of the kidney opposite to the desired implantation site.
Hold the needle with the beveled edge up and in. Insert it through the interior of the kidney until the needle is visible, but not puncturing the subcapsular space on the far side. Inject the cells until a white bubble forms and swab the injection site to prevent any leakage.
Gently return the kidney to the body cavity. Close the abdominal wall using absorbable 5.0 Vicryl suture. Once complete, hold the skin layer together and place two to three wound clips prior to recovery.
Administer 500 microliters of 0.9%sodium chloride and 100 microliters of buprenorphine subcutaneously. 26 to 30 days post implantation, anesthetize the animal and expose the kidney as before. Then use forceps to gently remove the connecting fat tissue from the coddle end of the kidney and remove the adrenal gland from the cranial end.
Use forceps to gently grasp the kidney and then slide an absorbable 5.0 Vicryl suture around the ureter, renal artery and renal vein. Next, slowly tie a double knot after clamping the threads with hemostats. Use scissors to cut above the knot and remove the kidney.
Once the kidney is removed carefully check for any bleeding from the tide, artery and cauterize if necessary. After suturing the abdominal body wall using 5.0 Vicryl, use wound clips to close the skin. Monitor the animal daily for signs of distress, bleeding, or limited mobility.
After euthanizing the animal, use surgical scissors to make an incision beginning above the urethra and continuing up the thoracic cavity to the lower mandible. Use blunt ended scissors to separate the skin from the muscle of the abdominal wall. Next, cut the skin along the arms and legs and slowly peel the skin away.
Closely examine the skin fascia and the abdominal wall for nodules. Then examine the superficial lymph nodes. Make an incision in the abdominal wall and cut along the left and right lateral sides up to the diaphragm.
With one horizontal cut. Remove the front portion of the abdominal wall to expose the viscera. Examine the undisturbed appearance of the viscera for grossly notable tumors.
Also note the contents of the stomach and intestines for evidence of food or hemorrhaging. Visually score each organ for tumor presence or absence working from a checklist of 22 sites, scores are tabulated for the animals in a particular group to compare disease distribution. Now carefully remove the liver and wash it with PBS.
Then examine it for gross tumors or abnormalities and score it as before. Then proceed to remove and score the spleen, the kidney, and the pancreas. Next, inspect the abdominal lymph nodes, including the lumbar sacral, renal, and mesenteric lymph nodes.
After assessing the diaphragm for evidence of metastatic spread, remove the diaphragm and make two lateral incisions in the thoracic cavity, removing the frontal portion to expose the lungs and heart, gently lift the esophagus and cut the trachea above the heart. After removing the lungs and heart, assess the tissues for nodules. Next, use forceps to squeeze the heart.
A lack of springing consistency may indicate the presence of a tumor. Lastly, use surgical scissors to cut linearly through the skull. Gently remove small pieces of cranium until the whole brain is exposed.
A visual scoring system was used to assess disease, distribution and severity of disease in five different groups of animals. These groups showed different extent and severity of disease. An additional measurement consisting of bioluminescent imaging immediately following sacrifice was used to quantitatively assess micro and macro metastatic lesions.
Once mastered both implantation, nephrectomy or necropsy can be done in five to 10 minutes per animal if it is done properly. To examine the whether treatment responses would differ between types of metastatic tumor cells. Cells from metastatic renal cell carcinoma.
Patients are currently being used to implant directly into animals to assess local and metastatic disease growth After its development. This technique paved the way for researchers in the field of RCC to explore clinically relevant disease progression and responses to therapy. In particular, whether those responses differ depending on primary localized disease or spontaneous metastatic growth.
Don't forget that working with tiny animals that are undergoing multiple surgeries require strict adherence to institutional animal protocol guidelines, and sterile conditions. Animal comfort and recovery are critical variables to properly perform with this procedure.
