The overall goal of this model is to recapitulate the cancerous neural microenvironment. This enables exploration of cancer cell neuron interactions. This method can help answer key questions in the tumor microenvironment field, such as interaction between tumor cell and neurons and the mutual influence of each set.
The main advantage of this technique is that it is reliable, easy to perform and address both cellular and cerebral factors in the neural niche. The implications of this technique extend towards therapeutic development and pharmacological targeting of key factors in the neural remodeling and invasion process throughout cancer development. After euthanizing the animal in a CO2 chamber, soak the fur with 70%ethanol and allow it to dry.
Pin the mouse in a prone position, and then make a midline incision extending craniocaudally from the posterior neck to the lumber spine. Next, use forceps to raise the dermal flaps bilaterally and expose the subcutaneous tissue. Palpate the mouse skull until the craniocervical junction is identified.
Use scissors angled perpendicular to the animal to cut through the cervical muscles and spine at the craniocervical junction. Then, dissect the spine caudally and use scissors to perform a complete caudal transection at the fifth lumbar vertebrae. Next, position the mouse in a supine position, and after making an incision along the midline from the neck to the abdomen, retract the skin laterally and then cut open the peritoneum.
Cranially, open the chest wall with scissors. After removing the peritoneal and retroperitoneal organs, use forceps and a surgical blade to cut the ribs, leaving approximately five millimeters of ribs protruding from the vertebral column. Remove the vertebral column from the rest of the body.
and wash twice with cold PBS. Position the spine facing up in the same craniocaudal orientation on a nonadherent absorbing platform under a stereo microscope equipped with 4X magnification. While looking through this stereo microscope, remove any spinal muscles and connective tissue.
Use the ribs as landmarks for nerves, as they leave the spine. The DRG are located at the cervical, thoracic, and lumbar vertebrae. Use scissors to cut the vertebral body at the midline, creating a window to the spinal cord, and then gently retract the vertebral body to expose the spinal cord and the DRG roots.
Next, using spring scissors, remove the upper rib to expose the DRG. Follow the peripheral nerve medially along the rib lateral to the DRG. Identify the DRG, which can be described as looking like the yolk of a fried egg lying on the nerve.
Cut the intercostal nerve distal to the DRG leaving two to three millimeters of nerve distal to the ganglia to be used for retraction. Carefully grasp the efferent nerve using forceps without pinching or damaging the DRG. Now apply gentle retraction to the DRG by pulling the nerve laterally.
Then cut the anterior and posterior roots close to the DRG. Transfer the isolated DRG to a 35-millimeter petri dish filled with ice-cold supplemented DMEM. Working in a laminar flow hood, place a 35-millimeter glass bottom petri dish on a paper grid on ice.
Using a precooled pipette tip, dispense approximately 10 microliters of growth-factor-depleted ECM at the center of the grid. While viewing the dish through a stereo microscope under 2 to 4X magnification, place the DRG at the center of the ECM close to the bottom of the dish. After harvesting and washing 40, 000 cancer cells of interest from confluent cultures resuspend the pellet and 40 microliters of ECM on ice.
Once again, view the grid through the stereo microscope, measure 500 microns from the DRG, and dispense 10 microliters of the cell suspension at this point. Repeat in all directions from the DRG. Leave the dish in the laminar flow hood for 10 to 15 minutes to solidify but not dry out.
Once the ECM has solidified, slowly add supplemented DMEM by pipetting against the sidewall of the plate. Add approximately two milliliters of media, enough to cover the ECM. The addition of DMEM should be done very slowly by pipetting against the sidewall of the plate to avoid detachment of the DRG from the plate bottom.
Set the plate in the tissue culture incubator, and follow the instructions in the written portion of the protocol to maintain the culture. This micrograph shows the DRG at the top of the field of view and cancer cells at the bottom of the field of view on day zero after seeding. Here, the same culture is shown seven days after seeding.
As indicated by the arrows, cancer cells migrate along the DRG neuron. This histogram shows the DRG nerve invasion index of different types of cancer cells. It can be seen that Kpc 989 and MiaPaCa cells have a higher nerve invasion index than QLL2 or NIH3T3 cells.
This coordinate graph depicts the migration path of a Kpc cancer cell in contact with the nerve shown in red and a QLL2 cell shown in purple. The X and Y coordinates are shown. This figure shows an analysis from distance of origin for migrating QLL2 cancer cells with axonal contact.
Here the distance of origin is shown for Kpc cancer cells. In each case, the direction of migration was towards the nerve ganglion. While attempting this procedure, it is important to remember to prepare more ganglia than required since the check rate is not 100%After it's development, the DRG motive paved the way for researchers in the field of cancer research to explore the neural niche within the tumor microenvironment.
After watching this video, you should have a good understanding of how to recognize anatomical landmarks such as the DRG in the mouse, extract the DRG, and finally, how to culture it in DCM. Coculturing of the DRG alongside with cancer cells is also presented.
