Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Cancers of the head and neck region are becoming increasingly prevalent. Yet, there's limited understanding of the tumor microenvironment and the mechanisms of treatment resistance in this region. This technique can be used to recapitulate the native microenvironment of head/neck tumors in an accessible manner and produces clinical manifestations similar to those seen in humans.

When the tumor cell culture has reached 70%confluency, wash the cells three times with cold PBS per wash and attach the cells with enough 0.25%trypsin to cover the bottom surface of the culture flask. After three to four minutes in the cell culture incubator, confirm detachment under a light microscope and neutralize the trypsin with 12 milliliters of DMEM F12 medium, supplemented with fetal bovine serum. Transfer the cell suspension into a 50 milliliter conical tube and mix the cells three to four times by inversion.

Then collect the cells by centrifugation and re-suspend the pellet in serum and antibiotic-free DMEM at a one times 10 to the sixth tumor cells per 50 microliters of medium concentration on ice. Immediately before the injection, mix the cells at a one to one tumor cells suspension to basement membrane matrix ratio on ice. Load one 0.5 milliliter syringe equipped with a 23-gauge needle with 100 microliters of cells per recipient animal.

Place the syringes on ice and confirm a lack of response to toe pinch in an anesthetized mouse. Next, insert the needle into the right or left buccal region through the available open space on either side of the mouth keeping the syringe parallel to the buccal region while it is inside the oral cavity to facilitate injection of the full 100 microliter volume of the cell basement membrane matrix suspension over a period of five seconds. Keep the syringe in place for an additional five seconds to ensure that all of the material has been injected before withdrawing the syringe gently.

The tumors will become grossly visible in about a week. One week after the injection, use calipers to measure the length and width of each tumor to determine the tumor volume one to two times a week. And measure the weight of each animal to assess the effects of the tumor growth on feeding.

At the appropriate experimental endpoint, use sharp scissors and blunt forceps to make a skin incision through the midline in the neck. And insert the scissors gently under the skin covering the tumor to create air pockets by pushing the scissors across and into the skin. Once the skin is sufficiently detached from the tumor, excise the draining lymph nodes to avoid having the tumor tissue confounded by the presence of lymph tissue and cut through the borders of the tumor until the entire volume is detached.

To process the tumor for downstream analysis, cut the tumor into one to two millimeter sized pieces and place the pieces into a 50 milliliter conical tube containing collagenase three, DNAs one, and trypsin inhibitor. After incubating the tumor pieces at 37 degrees Celsius for 30 minutes, with shaking every 10 minutes, add 20 milliliters of HBSS to the tube and pass the suspension containing the tumor pieces through a 70 micrometer pour nylon strainer. Use a five milliliter syringe plunger to mash the tumor pieces in the strainer and add an additional 10 milliliters of HBSS through the strainer.

Spin down the cells by centrifugation. Re-suspend the pellet in two to three milliliters of red blood cell lysis buffer with rigorous pipetting, neutralizing the lysis with 20 milliliters of fresh HBSS after two minutes at room temperature. Then re-suspend the cells in an additional 20 milliliters of HBSS for another centrifugation and strain the cells through a 40 micrometer pour filter to remove any final debris from the tumor cell suspension.

LY2 tumors grow at a higher rate, compared to B4B8 tumors. And mice that exhibit jaw displacement rapidly develop weight loss, due to dysphagia. The median survival in LY2 mice is also less than half that observed for B4B8 tumor-bearing mice.

Magnetic resonance imaging of tumor-bearing mice shows well-demarcated tumors extending into the inner layer of the buccal mucosa, but not into the tongue or other nearby organs. Histologic examination indicates that all LY2 tumor-bearing mice develop poorly differentiated squamous cell carcinoma, while mice bearing B4B8 tumors develop moderately differentiated squamous cell carcinoma. All LY2 tumor bearing mice also have histologically confirmed necrosis, with the majority demonstrating moderate to severe necrosis.

In this representative experiment, a LY2 tumor was harvested and processed three weeks after buccal region tumor cell injection, as demonstrated. CD45-positive immune cells represented 7.3%of the total tumor cell population. CD11b-positive myeloid cells represented 37.8%of all of the CD45-positive cells, the majority of which were determined to be F480-positive macrophages, with small populations of neutrophils and myeloid-derived suppressor cells also observed.

T cells comprised 15.9%of the CD45-positive immune cell population, 53.4%of which were CD4-positive FoxP3-positive regulatory T cells. Natural killer cells comprised less than 2%of all all of the CD45-positive cells. It is important to practice injection in order to develop confidence and comfort in holding the needle and the recipient mouse and in exposing the mouth of the animal.

Once the procedure has been successfully performed, experiments involving the assessment of tumor response to therapy or assessment of the tumor intrinsic and microenvironmental factors can be performed. This technique has paved the way in designing an investigator-initiated clinical trial, assessing the effects of combining radiotherapy with anti-PL1 therapy in head and neck cancer patients.