The overall goal of this procedure is to observe the effect of immune therapy given in the perioperative period on reducing metastasis and a mouse model of tumor and surgical stress. This is accomplished by first injecting four T one tumor cells in the mouse memory fat pad in order to establish a primary tumor. On day 13 or one day prior to cancer surgery, immune therapy is administered, which is hypothesized to attenuate postoperative metastatic disease on day 14, post tumor cell implantation.
The primary tumor is resected, followed by additional surgical stress in the form of abdominal nephrectomy and a subset of mice. The final step is to sacrifice and quantify the number of metastatic lung tumor nodules in all mice. Ultimately, this mouse model of spontaneous metastases and surgical stress is used to show the beneficial effect of perioperative immune therapy.
This method can help answer key questions in the field of surgical oncology, such as how major surgery impacts on the immune system and as a result on the formation of metastatic disease in the postoperative period, and how immune therapies can be used to attenuate this effect. Demonstrating this procedure will be Christiano 10 dusa, a research technician and Dr.Rebecca s laboratory To set up the tumor cell culture. Begin by placing unmodified 41 tumor cells in complete DMEM in 10 centimeter tissue culture plates and incubating them at 37 degrees Celsius 5%CO2 in a tissue culture incubator.
Split the cultures two to three times per week as needed so that the cells do not exceed 80%confluence. Within a month of setting up the cell culture, begin harvesting the tumor cells by aspirating the culture medium from the tissue culture plate. Add 10 milliliters of one X sterile PBS containing two milliliters of EDTA and incubate the plate at 37 degrees Celsius, 5%CO2 for five to seven minutes.
Then after removing the PBS solution from the plate, rinse the plate two to three times with PBS and transfer the cells to a 50 milliliter conical tube. Next, centrifuge the cells in a benchtop centrifuge at 500 Gs and four degrees Celsius for five minutes. After aspirating the supernatants resuspend the cell pellet and serum free DMEM.
Next, use a cell counter to determine the cell concentration and dilute the cells with serum free medium to one times 10 to the six cells per 500 microliters and place the cells on ice one hour prior to surgery. Inject a mouse subcutaneously with 0.05 milligrams per kilogram buprenorphine for pain management. After anesthetizing the mouse with 2.5%ISO fluorine, pinch the foot pad to ensure an adequate level of anesthesia.
Then load a 30 gauge half inch ultra fine syringe with precisely 50 microliters of the tumor cell suspension. Tap the side of the syringe to dislodge air bubbles. Continuing the ISO fluorine anesthesia, place the mouse ventral side up on the surgical table.
Now introduce the needle horizontally and directly into the fourth memory fat pad and slowly dispense the syringe volume. It should take about one minute to finish the injection. Use a cotton swab to clean any leakage after the injection.
Allow the mouse to recover from the anesthesia prior to returning it to its cage. Continue pain management by administering 0.05 milligrams per kilogram buprenorphine subcutaneously every eight hours for two days at 13 days postum cell injection. Use an external caliper to measure the size of the primary tumor.
Measure the greatest longitudinal diameter or length and the greatest transverse diameter or width with a caliper. The modified ellipsoidal formula is used to calculate the tumor volume as one half of the length times width squared. At this time, assuming the primary tumor measures approximately one cubic centimeter, the perioperative therapy reagent of choice, which in this case is oncolytic virus in one x, sterile PBS has been prepared.
Load a 27 gauge half inch insulin syringe with precisely 100 microliters of the oncolytic virus therapy. Remove all air bubbles from the syringe. Next place a mouse into a restrainer, leaving the tail accessible.
Using warm tap water, gently heat the tail. To visualize the lateral tail veins, select the lateral tail vein and inject it with the oncolytic virus. If the needle is appropriately inserted into a lateral vein, no resistance should be felt when depressing the syringe.
When finished, return the mouse to its cage overnight at 14 days Postum cell injection. Treat the mouse subcutaneously with 0.05 milligrams per kilogram buprenorphine one hour before surgery for pain management, induce and maintain anesthesia using 2.5%ISO fluorine. After making a one to two centimeter skin incision, gently remove the entire primary four T one tumor from the mammary fat pad, the tumor and any attached debris should come out in one piece.
Save the tumor in Formin for later immunohistochemistry analysis. Following excision of the tumor, close the incision with two to three nine millimeter staples. The next step is to perform a major surgery in a subset of the mice.
Begin with a ventral midline incision through the skin and subcutaneous layer. Then make a three to four centimeter incision at the linear elbow to access the peritoneum. Now, move the overlying bowels to one side to expose the left interior side of the abdomen.
Keep the intestines moist with a saline soaked sterile gauze. Next, using a blunt pair of surgical forceps, grasp the left kidney gently. Then use a 3.0 wax coated braided silk suture tied into a loop to ligate the hilum of the left kidney.
Secure the suture with three surgical knots. Now remove the left kidney with surgical scissors. Inspect the suture tie carefully to ensure that adequate hemostasis is achieved.
Next, close the subcutaneous layer with a continuous loop suture using five oh braided absorbable sutures. Then staple the skin layer using nine millimeter staples. The nephrectomy should take 10 minutes per animal.
When recovered, return the animal to its cage and continue pain management for two days by administering 0.05 milligrams per kilogram subcutaneous buprenorphine every eight hours at 28 days after the four T one tumor injection, euthanize the mouse and spray it with 70%ethanol. Begin the lung removal by using scissors to make an initial incision just below the rib cage. Cut through the skin and subcutaneous layer along the ventral midline of the chest cavity to expose the thorax.
Then make lateral incisions through the skin and tissue on each side up to the neck. Now, gently grasp the lungs while snipping away the connective tissue until the lungs are free. Cut through the trachea above the bifurcation, which will hold the left and right lobes together.
Next, dip the extracted lungs in cold PBS to remove any residual blood and place them in 10%Buffered formin finish by photographing the lungs for tumor metastases assessment and weighing them for tumor burden quantification. 14 days after the four T one tumor resection, the lungs were harvested and visualized for metastases shown here as the lung from a mouse that did not have an abdominal nephrectomy. Some metastasized tumors are visible.
In contrast, a surgical nephrectomy immediately after the tumor resection resulted in a visible increase in the number of pulmonary metastases. This photograph shows lungs from a mouse treated with oncolytic virus following tumor resection and nephrectomy shown here are lungs from a mouse treated with an influenza vaccine following tumor resection and nephrectomy Counting the lung tumor nodules in each of these categories shows a clear increase in the number of pulmonary metastases and mice with nephrectomy surgery as compared to those without surgery, or those with a preoperative therapeutic intervention. The lungs of the mice with nephrectomy surgery, but no preoperative therapy weigh significantly more than the other test categories.
Thus, the preoperative administration of replicating oncolytic virus and inactivated influenza vaccine significantly rescued the prometastatic effects of major surgery following tumor resection. These graphs show the number of lung tumors in mice with normal or intact numbers of NK cells and in mice whose NK cells were pharmacologically depleted using anasi ALO GM one. The NK depleted mice had a reduced therapeutic effect of perioperative oncolytic virus or influenza vaccine immunotherapies.
This suggests that the NK cells play a mediating role in preventing metastases post vaccine treatment. To further characterize NK cell function, ex vivo NK cell killing was assessed purify DX five positive cells from surgically stressed and untreated controls were cultured with yak one target cells. The black solid line indicates the NK cell killing ability from untreated mice and the black dotted line demonstrates NK cell killing ability from surgically stressed mice.
The decrease in percentage cytotoxicity shown by the downward shift from the black solid line to the black dotted line demonstrates a defect in NK cell cytotoxic function and mice following surgery. The gray solid lines indicate NK cell killing from mice that received immune therapy, either oncolytic virus or influenza vaccine, but no surgery. The gray dotted lines indicate NK cell killing ability from mice that received both perioperative immune therapy and surgery.
The decrease in percentage cytotoxicity shown by the downward shift from the gray solid line to the gray dotted line, demonstrates a defect in NK cell cytotoxic function and mice following surgery despite receiving perioperative immune therapy. Most importantly, the gray dotted lines are still much higher than the black dotted lines. This suggests that it may be possible to rescue surgery induced NK cell cytotoxic dysfunction with perioperative immune therapy.
After watching this video, you should have a good understanding of how to create an animal model of spontaneous metastases and surgical stress by establishing primary best tumors. Resecting the primary tumors inducing additional surgical stress by nephrectomy and assessing lung metastatic tumor nodules at endpoint. In addition, you should understand how this model can be used to study the suppressive effects of surgery on the innate immune system and the ability of immunotherapies to reverse this effect.
