This study investigates the surgical debulking of subcutaneous tumors in mice and the subsequent application of anti-cytotoxic protein antibodies. The approach aims to enhance T cell activation and improve therapeutic outcomes in tumor treatment.
Begin with an anesthetized mouse bearing a subcutaneous tumor.
In this tumor microenvironment, T-regulatory cells express an anti-cytotoxic protein that strongly interacts with CD80 on antigen-presenting cells or APCs, preventing conventional T cell interaction.
Disinfect the surgical area and make a skin incision near the tumor.
Open the wound to expose the tumor and trim it partially, leaving remnants of the tumor tissue.
Close the wound and allow the mouse to recover.
Post-surgery, take a syringe filled with antibodies specific to the anti-cytotoxic protein and intraperitoneally inject them into the anesthetized mouse for an adjuvant or additional therapy.
These antibodies travel to the tumor site and bind to anti-cytotoxic proteins on T-regulatory cells, blocking their function.
This enables APCs to interact with T cells, activating them to release cytotoxic molecules.
These molecules kill remaining tumor cells, potentially reducing tumor regrowth, suggesting effective adjuvant therapy.
Begin this surgery by swabbing the surgical area with chlorhexidine. Using forceps and a pair of scissors, make a centimeter straight incision along the dorsal side, 3 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 debulked 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, 5 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, 3 to 4 clips are applied, with 2-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 adjuvant 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.
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