A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

The overall goal of this experimental protocol is to rapidly evaluate multiple treatment conditions affecting perineural invasion in head and neck squamous cancer with very high assay success rates and reproducibility. This method can help answer key questions in the field of head and neck oncology, such as what are the factors that influence head and neck cancer cell perineural invasion. The main advantage of this technique is that a large number of assays can be rapidly and reproducibly performed from one mouse in a relatively short period of time.

Before beginning the dorsal root ganglion, or DRG, dissection procedure, add fresh room temperature medium to at least 40 wells of a V-bottom 96-well plate. Then, use a pair of fine scissors to make a longitudinal incision along the spinal column from the root of the tail to the head of the mouse. And transversely divide the tissue below the sacral spine, dissecting both sides of the spine, all the way up to the skull base.

Transfer the specimen under the microscope, and observe the cervical end of the spinal column at low power. The white spinal cord is apparent in the middle of a bony ring, which is surrounded by variable amounts of perispinal muscles and soft tissue. Using microscopic spring scissors, split the dorsal, or superior aspect, of the first two to three vertebral bodies.

Then, use the spring action of the scissors to open this initial bony cut, to ensure that the vertebral body dissection occurs at the midline. Continue in small increments toward the sacral spine, bisecting the spine with identical cuts on the ventral, or inferior, aspect of the vertebral bodies. Placing one hemispine aside, transfer the other half of the spine face down on a sterile plate with the spinal cord in place.

And use two 18-gauge needles to secure both ends of the hemispine onto the polystyrene dissecting platform. Next, starting at the narrower, cervical end, gently peel back the spinal cord from about four vertebral levels. In the region where the sensory nerves insert into the DRG, gently grasp the surrounding fascia with microscopic forceps, and use microscopic spring scissors to trim this fascia and other nerve tissue to free the DRG.

Under increased magnification, use the spring scissors to cut the peripheral nerve as close to the DRG as possible to release the tissue, and trim the proximal branches. Then, trim the DRG of any stray nerve fibers or fascial attachments and place the isolated tissue into one well of the 96-well V-bottom plate. To prepare the semisolid matrix droplets, transfer one glass well-bottom plate from ice onto an ice block under the operating microscope.

Place the tip of a no more than 10 microliter pipette directly onto the glass at a 45-degree angle, and carefully dispense a 1.5-microliter droplet of matrix onto the glass. Slowly move the pipette tip away from the glass as the matrix becomes engaged with the plate. Deposit one droplet of matrix onto each of the four corners of the plate, and transfer the plate to a room temperature surface for about one minute.

When the matrix has slightly stiffened, return the plate to the ice block, and use closed microscopic forceps to scoop the DRG gently with the left hand. Using the right hand, carefully transfer the nerve tissue to the tip of a 21-gauge needle and use the needle to gently insert the DRG into the middle of the matrix droplet. The DRG should release easily into the matrix for central positioning with the needle.

After inserting a DRG piece into each of the four droplets, place the plate into a 37-degree Celsius incubator for three minutes. When all of the DRG have been embedded, hold the plates one at a time, at a slight angle and slowly add four milliliters of DMEM supplemented with 10%FBS to the plates, such that the medium only gradually comes into contact with the matrix DRG units. Return the plates to the incubator for another 48 to 72 hours.

When the neurites reach at least 75%of the way to the edge of the matrix, aspirate the medium from the wells without removing the matrix DRG units. Then, using a 200-microliter pipette, with a smooth trigger, place two drops of three times 10 to the fifth cells per milliliter of medium over each matrix DRG assay. And return the plates to the incubator.

After one hour, gently add four milliliters of DMEM with 10%FBS along the side wall of the glass-bottom plate, and return the plates to the incubator until their next microscopic evaluation. After their dissection and placement within the matrix droplets, the appearance of the assay should resemble the matrix-embedded DRG shown here. Note that the DRG is not perfectly round, but is centered within the matrix droplet, allowing for the outgrowth of neurites in 360 degrees.

Immediately after plating, the head and neck squamous cancer cells form a circumferential ring around the matrix, expanding along the neurites over the next two to three days. Cells plated onto blank control droplets containing matrix alone actively divide around the matrices, but do not enter or extend over the top. For quantification of the perineural invasion of the cancer cells, images of the assays are divided into four quadrants via one vertical and one horizontal line.

A series of points are then scored for each perineural invasion extension observed in each quadrant, allowing comparison of the cancer cell ingrowths along the neurites between different cancer cell lines. Once mastered, the dorsal root ganglia isolation technique can be complete in less than two hours if it's performed properly. While attempting this procedure, it's important to minimize excessive handling of the dorsal root ganglia as crush injury is the most common reason for assay failure.

Following this procedure, the mechanisms of perineural invasion can be studied, potentially leading to the development of targeted therapies against this type of soft tissue invasion. After watching this video, you should have a good understanding of how to dissect dorsal root ganglia from a mouse and set up an assay for examining perineural invasion.