Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

The overall goal of this procedure is to determine whether a candidate therapeutic agent effectively inhibits the growth of M-P-N-S-T cells grafted into the sciatic nerve of immunodeficient mice. This is accomplished by first removing the tumor cells from a tissue flask, washing them and resus suspending them at the proper concentration. The second step of the procedure is to surgically expose the sciatic nerve and then inject the nerve with 5, 000 cells.

The next step is to determine the success of the grafting using in vivo, light-based imaging randomizing the CIN to study groups and to beginning therapy with the candidate therapeutic agent. The final step of the procedure is to collect the tumors 30 days post grafting and analyze the effects the candidate therapeutic agent had on the tumor. Ultimately, results can be obtained that show whether the therapeutic agent decreased tumor growth through methods such as comparison of tumor weights, looking at relative rates of proliferation via immuno staining for KI 67 and assessing the levels of tumor cell death in control and treated mice with tunnel assays.

The main advantage of this technique over existing methods like subcutaneous grafting, is that it models the normal microenvironment, which better models M-P-N-S-T growth demonstrating this technique will be Amy Turk, a technician from my laboratory. Non nude immunodeficient mice are prepared the day before grafting. Use clippers to shave the hair from an anesthetized mouse working on a heating pad to maintain body temperature.

Use a chemical depilatory agent to remove any remaining hair. Once all of the hair is removed, place the mouse onto a heating pad in an isolation cage without bedding until fully recovered. On the day of grafting centrifuge the cells at 3000 RPM for five minutes.

Remove the snat carefully and resuspend the cell pellet in DMEM 10 without pur mycin to a final concentration of five times 10 to the third cells per three microliters. Keep the cells on ice until ready to use. To begin, place an anesthetized animal onto a heating pad and apply ophthalmic ointment to each eye.

Clip an ear tag with a unique identifier number to the right ear of each mouse. For ease of identification throughout the study, place the animal on its stomach and spread its hind legs. Cover the mouse with a sterile drape and expose a surgical window where the grafting will occur.

Apply Betadine to the surgical site with a cotton tipped applicator starting in the center of the flank and gradually spiraling outward to the edges of the surgical window. 70%ethanol is then applied to the surgical site in the same manner. Consecutive applications of Betadine followed by ethanol are repeated twice more.

Remove the cells from ice and either gently vortex or flick the tube to resuspend the settled cells. Use a pipette to remove 3.2 microliters of the cell suspension and eject it onto a piece of paraform. Pull three microliters of the cell suspension into a 10 microliter Hamilton syringe.

Equipped with a 33 gauge needle. Hold the cells and the cell containing syringe on ice until ready to use. Use a number four scalpel handle, equipped with a number 22 scalpel blade to make an incision through the skin of the flank just below and parallel to the femur.

Open the skin incision with the surgical clamp to expose the underlying muscle. Use a pair of sharp tipped scissors to blunt dissect through the fascia running along the length of the leg and expose the sciatic nerve that lies beneath a white linear structure about the thickness of a thick thread working under a microscope. Use a pair of sharp tipped curved forceps to carefully dissect under the nerve loosening it from the underlying muscle.

Leave the forceps in place to keep the nerve both elevated and isolated carefully Insert the needle of the Hamilton syringe into the nerve trying to maintain the angle of injection as parallel with the nerve as possible, so as not to puncture through to the underside. Once the needle is properly positioned in the nerve, relax the tension produced by the forceps and slowly inject the cells over the course of 45 to 60 seconds. A slow injection is essential to prevent a backwash of tumor cells resulting in their loss.

After all cells have been successfully injected, slowly withdraw the needle to minimize loss of injected material. After the injection is complete, remove the surgical clamp and the forceps. Then carefully place the nerve back into its original location.

Close the incision with vet bond surgical glue, holding it together with forceps for approximately 20 seconds to allow the glue to dry. Place the animal into a heated cage free of bedding until it is fully recovered. Monitor the health of the mice.

Daily mice are removed from the study. If the tumor burden exceeds 10%of the animal's normal body weight or weight loss exceeds 20%To determine the success of the procedure, perform bioluminescence imaging one and three days post grafting. Inject a mouse with 2.5 milligrams of Lucifer and place the anesthetized animal on the heated imaging platform.

Light emission from tumor expressed firefly luciferase is detected 10 minutes after injection of the substrate. Using software from Xeno Gen set image acquisition times to a range of one second to 10 minutes. Next, take black and white photographs of the mice Pseudocolor.

Scaling of bioluminescence is overlaid with these images to provide a measure of light emission intensity. To be eligible for inclusion in a preclinical trial cohort, mice must have a detectable bioluminescence signal, both one and three days post grafting, and the intensity of the signal must increase between days one and three. Shown here is a typical progressive increase in bioluminescence observed one to 18 days post grafting in a properly established orthotopic xenograft in a nude mouse.

Note the similarity in the signals detected within the region of interest of different mice. Re-imaging 10 days and 18 days post grafting shows that bioluminescent signals progressively increase at the graft site in individual mice. Quantification of the bioluminescent signals observed one to 24 days after grafting shows that although these signals progressively increase tumor growth markedly accelerates in the later stages of the study period.

Given the aggressive growth of M pns ts, the grafted tumor cells often breach the normal barriers of the nerve and invade adjacent tissues. This photo micrograph of the graft site demonstrates tumor growth and focal invasion into adjacent skeletal muscle Once mastered, this technique can be performed in one mouse in 10 minutes when done properly.