Medicine
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A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies
Chapters
Summary July 28th, 2020
In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.
Transcript
This protocol aims to mimic the clinical setting of residual disease after tumor debulking surgery, and therefore allows us to test new cancer therapies in the context of postoperative wound healing. The advantage of this technique is that it allows standardization of the amount of wound inflicted and the amount of tumor to remain. This technique is particularly important for preclinical studies in that identifying new treatments for cancers, that it relapses locally an example of which is soft tissue sarcoma.
This visual demonstration is critical to demonstrate the inversion technique, which allows us to expose the tumor attached to the skin side. Begin this procedure with the subcutaneous inoculation of prepared cancer cells in mice, as described in the text protocol. On day 12, post inoculation, when tumors have reached a size of approximately 50 square millimeters, dose mice with 100 microliters of buprenorphine subcutaneously in the scruff of the neck, 30 minutes prior to surgery.
Set up the surgical area with the heat pad covered with bench coat and set up a nose cone for anesthesia. Arrange clean surgical equipment so that they are within easy reach, including chlorhexadine, swab, gauze, eye gel, two curved forceps, scissors, clip applicator, clip remover, and clip refills. Warm the heating chamber to 37 degrees Celsius and set up another heat pad for recovery.
Note that this procedure requires two people. An assistant must monitor the breathing of the mouse closely during the procedure. After anesthetizing the mouse as described in the text protocol, perform a pinch test and corneal reflex test to ensure that the mouse is fully anesthetized before commencing surgery.
Cover the mouse's eyes with a small amount of ophthalmic gel to avoid eye dryness. Begin this surgery by swabbing the surgical area with chlorhexadine. Using forceps and a pair of scissors make a one centimeter straight incision along the dorsal side, three millimeters away from the tumor.
Do not cut the skin directly covering the tumor as it will not heal well. Avoid using a scalpel to cut the tumor and use tweezers to scoop the pieces. Using tweezers, pull away the fascia and subcutaneous fatty tissue between the tumor and peritoneum.
The subcutaneous tumor is normally attached to the skin side. Open the wound by gently holding the skin on the tumor bearing side using tweezers and invert the tumor so that it is visible outside. Using a pair of scissors, cut away the tumor capsule from the half to remove, starting from the base of the tumor closest to the opening.
For 50%debulk surgery, cut across the middle of the tumor. Using curved forceps scoop up the section of the tumor to be removed. Scoop up any remnants from the debulk area.
For 75%debulk, start by performing a 50%tumor debulk then cut in half the remaining 50%of tumor. Scoop up 25%of the tumor using curved forceps. To close the surgical site, place the remaining tumor back underneath the skin and using forceps, pull the skin flaps together and line up the skin along the wound.
Hold the skin together five millimeters from the edge of the wound and use surgical clips to close the wound starting on the side, closest to the forceps. Apply as many clips as needed to ensure no underlying tissue is exposed. Generally, three to four clips are applied with two millimeter gaps between clips.
Allow the mice to recover by putting them into the warm heating chamber. Place the mouse's cage on the heat pad. Monitor the mice in the heating chamber until they have recovered from the anesthetic.
Then, put the mice back into the cage, leave the cage on the heat pad for a further 10 minutes until the mice have become more active. Give the mice wet and soft food. Monitor the mice one hour after surgery for recovery.
Monitor the mice again at the end of the day and the following morning and ensure clips remain in place. Treat mice perioperatively with advent or neoadjuvant therapy at any given time, depending on the treatment of interest. For example, treat mice with one dose of 100 micrograms of anti-CTLA-4 intraperitoneally on day 15 after inoculation.
Alternatively, treat the mice with three doses of 200 micrograms of anti-PD-1 intraperitoneally on days, 15, 17, and 19 after inoculation. The incomplete surgical resection of 50 square millimeter tumors, indicated here by the dotted line results in 100%reproducible regrowth of the tumors in the absence of adjuvant immunotherapy. Therefore, tumor growth to a size of 50 square millimeters is an ideal size for partial debulk.
The model was then used to test adjuvant immunotherapies using antibodies against checkpoint modules, cytotoxic T lymphocyte associated protein four and programmed death receptor one. Treatment of mice with anti-CTLA-4, resulted in a cure rate of 80%Whereas treatment of mice with anti-PD-1 resulted in a cure rate of 25%The response with anti-PD-1 provides an opportunity to test it novel combinations, to further improve the response rate. It is important that the remaining tumor stays attached to the skin, otherwise the blood supply will be cut off and the tumor will become the necrotic.
Following this procedure, tumors can be analyzed by a variety of methods, such as flow cytometry in order to assess how surgery affects tumor infiltrating cells. This mouse model has allowed us to explore how wound healing impacts cancer immunotherapies.
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