Translationally-Relevant Tumor Resection Model for Murine Preclinical Models of Oral Squamous Cell Carcinoma

This work focuses on the study of oral cancers, establishing a practical preclinical model for orthotopic tumor resection and recurrence. Surgery is a primary treatment modality for a majority of patients with oral cancer. This protocol allows us to investigate that.

To begin, obtain a culture flask containing 80%confluent Mach Two oral cancer cells. Remove the culture media from the flask. Wash the attached cells with magnesium and calcium-free PBS to remove remaining serum.

Add a sufficient volume to fully cover the surface of the vessel containing the cells, then aspirate. Add 0.05%trypsin into the culture flask to sufficiently cover the bottom surface. Incubate the flask at 37 degrees Celsius with 5%carbon dioxide for approximately 5 minutes to allow cell detachment.

When cell detachment is seen, quench the trypsin by adding at least two times the volume of complete media. Mix gently to stop enzymatic activity. Transfer the cell suspension to a centrifuge tube and centrifuge at 350G for 5 minutes at room temperature.

Discard the supernatant after centrifugation. Re-suspend the cell pellet in cold PBS. Remove an aliquot of the suspension for cell counting.

Using additional PBS, adjust the cell suspension to the optimized concentration for inoculation. Keep the prepared cell suspension on ice until ready for use. Mix the cell suspension thoroughly to ensure even cell distribution.

Collect the suspension into a 0.3 milliliter insulin syringe. Hold the anesthetized mouse with one hand from the posterior neck, so the anterior side faces the handler and the head is angled upward. Maintain the mouth in an open and accessible position.

Carefully insert the syringe into the open mouth at a downward angle toward the left lower mandibular vestibule. Advance the needle through the lower buccal mucosa. Deliver 30 microliters of the cell suspension into the target region.

Hold the syringe in place for 5 seconds, then slowly withdraw it to prevent tracking of cells. Ensure the mouse returns to normal physical and behavioral condition following inoculation. As tumors develop, monitor the weight of the anesthetized mouse using a balance and assess tumor growth with digital calipers two times per week.

Select a clean procedure room with a closable door and limited traffic. Place a new cage containing clean dry bedding and a paper towel nest partially on an electric warmer to prewarm it. Warm a bead sterilizer to an operating temperature of 250-265 degrees Celsius.

Turn on a water heated surgical pad and set it to approximately 39 degrees Celsius. Apply a small amount of hair removal cream to the tumor resection site on an anesthetized mouse using a cotton tip applicator. Ensure sufficient margins to keep hair out of the incision area.

After 10 seconds, wipe away hair using dry, clean gauze. Remove any residual cream with gauze or cotton swabs dampened with water. Place the anesthetized mouse on the heating pad in a supine position with the anterior side facing upward.

Position the nose just inside the nose cone. Now apply a sufficient amount of sterile lubricant ointment to each eye to prevent drying during the procedure. Using a clean cotton tip applicator, swab the resection site with 70%isopropyl alcohol followed by 10%iodine, and repeat the sequence three times.

Remove the iodine after the last pass with 70%isopropyl alcohol. Wearing sterile gloves and appropriate personal protective equipment, prepare a sterile field of at least 18 inches wide by 26 inches long. Lay out all required sterile tools and materials.

Cover the mouse with a sterile drape. Using sterile scissors, cut an access hole above the resection site. With a new surgical scalpel, create a submandibular incision just below the tumor along the left jawline.

Using Hartmann hemostats, gently widen the incision opening toward the tumor. Dab any blood with sterile gauze as needed. Locate the tumor and hold it in place using forceps.

An assistant in sterile PPE may hold the mouse or its skin in place during this procedure. Using surgical scissors, snip around the base of the tumor to resect all visible tissue. Reposition the skin over the resected tumor site, then close the incision with 4-6 interrupted sutures using 4/0 long lasting Vicryl sutures.

After closing the incision, gently transfer the mouse to the prewarmed cage and monitor it for return of consciousness and mobility until full recovery. Immediately provide softened food by placing water soaped pellets in an accessible container within the cage. If no complications arise, resume the mouse's normal solid diet at the next routine feeding.

Using the Mach Two cell line, tumors were established in and resected from the murine buccal mucosa of the oral cavity. Tumor growth kinetics were characterized prior to resection. Following tumor resection, a delay in tumor growth was observed in the days immediately after surgery, as evidenced by reduced tumor area shortly after resection compared with unresected tumors.

A minor extension in overall survival was observed in the resected group compared with the unresected control group, although this difference was not statistically significant. Mice in the unresected group first reached euthanasia criteria by day 12 post-surgery, whereas mice in the resected group did not meet euthanasia criteria until day 19. The most important consideration in using this protocol is consistency in tumor inoculation and engraftment.

Beyond this procedure, we can process the resected tumors for analysis by flow cytometry or immunohistochemistry. Future studies can build on this work by combining with additional treatment. You can also look into the investigation of the tumor immune microenvironment in relation to the resection and reoccurrence.