Establishing In Vitro Models of Dorsal Root Ganglia Culture: Complementary Approaches for Investigating Cancer-Nerve Crosstalk

Our research focuses on cancer-nerve crosstalk, using 3D dorsal root ganglia or DRG explants and 2D dissociated cultures to quantify neuro-guided tumor migration and to test molecular strategies to limit their interaction. With this protocol, we have established complementary 3D and 2D DRG cultures, optimized neurite growth, and demonstrated an increase in cancer cell invasion along the neurite extensions. We addressed the lack of reproducible models that capture both intricate cell-level interactions and intact nerve architecture by creating complementary 2D and 3D DRG cultures from a single mouse.

Our protocol yields paired 3D explants and 2D neuron cultures from a single mouse, preserving angular texture, enabling high-content imaging, reducing animal use, and allowing us to obtain tissue-level and single-cell data in one experiment. Our model opened questions on which DRG-created factors drive invasion, how neuron activity, age and genotype shape it, and whether targeting neurotrophic factor receptors could decrease neurite guided cancer spread. To begin, lay a euthanized mouse on its abdomen.

Shave the fur, focusing on the area over the spinal column to expose the dorsal surface. Spray 70%ethanol over the animal's back. Using paper towels, wipe from tail to skull to remove any loose fur and prevent contamination during dissection.

With a pair of sterilized scissors, make a straight incision along the dorsal midline from the tail to the skull. Using blunt forceps, retract the skin on both sides of the incision to expose the spinal column. Now, use a fresh pair of scissors and forceps to avoid contamination and make a horizontal incision at the tail end of the spinal column.

Then make two parallel longitudinal incisions on either side of the spinal column to release it. Use forceps to gently lift the spinal column and remove the surrounding connective tissue. To fully release the spinal column, make an incision at the top of the spinal column near the skull.

Transfer the spinal column to a pre-chilled 10-centimeter plate containing HBSS with penicillin streptomycin kept on ice. Next, use spring scissors to remove any remaining connective tissue from the spinal column and expose the vertebrae. Transfer the trimmed spinal column onto a microscope stage.

Carefully split the column along the sagittal plane using spring scissors. Using fine-tipped forceps, remove the spinal cord from the column to reveal the dorsal root ganglia along the roots of the spinal nerves. Cut the spinal nerves using spring scissors.

Then remove any surrounding connective tissue from the dorsal root ganglia. Using fine-tipped forceps, gently extract the dorsal root ganglia from the dorsal roots. For whole-mount culture, place the DRGs into a three-centimeter plate containing HBSS with penicillin streptomycin kept on ice.

Use pre-chilled pipette tips to dispense two microliters of chilled Matrigel into each well of an eight-well glass-bottom slide placed inside a 10-centimeter plate on ice. To prevent premature polymerization, pipette only one droplet at a time. Next, use fine-tipped forceps to place one dorsal root ganglion on a dry plate.

Swirl gently to remove excess HBSS. Then transfer the clean dorsal root ganglion into the Matrigel droplet. After transferring all ganglions into the droplets, incubate the glass-bottom slide in a humidified incubator at 37 degrees Celsius for five minutes to allow the Matrigel to polymerize.

Next, carefully add 200 microliters of warm supplemented culture media to each well. Incubate the slide at 37 degrees Celsius with 5%carbon dioxide. Use brightfield microscopy to monitor the dorsal root ganglia daily for neuronal outgrowth and viability.

Centrifuge a 15-milliliter conical tube containing pooled dorsal root ganglia at 200G for five minutes at room temperature. After pipetting out the HBSS, rinse the ganglia with pre-warmed supplemented media, and gently tap. Then centrifuge the tube for five minutes at 200G to pellet the DRGs.

Replace the media with one milliliter of papain solution. Mix gently and incubate at 37 degrees Celsius in a tissue culture incubator for 20 minutes, gently agitating every five minutes. Next, add 10 milliliters of supplemented media to neutralize the papain, and mix gently.

Centrifuge for five minutes at 400G to pellet the dorsal root ganglia. After pipetting out the supernatant, add one milliliter of collagenase type IV in Dispase II solution, and incubate again. Add 4.5 milliliters of supplemented media to neutralize the enzyme solution, mix gently and centrifuge.

After pipetting out the supernatant, add five milliliters of supplemented media. Then add Dnase I to reach a final concentration of 0.2 milligrams per milliliter. The dorsal root ganglia should now appear as a loose clump, indicating complete digestion.

For mechanical dissociation, use a 1, 000-microliter filter pipette tip to triturate the ganglia solution by pipetting up and down four to five times. Switch to a 200-microliter pipette tip and repeat trituration by pipetting four to five times. Filter the resulting cloudy suspension through a 70-micrometer cell strainer into a 50-milliliter conical tube.

Then gradually rinse the strainer with 10 milliliters of media to ensure complete transfer of cells. Centrifuge the filtered cell suspension for five minutes at 1, 000G to pellet the cells. Remove the media from the pellet, and resuspend the cell pellet in 500 microliters of supplemented media.

Remove any residual PBS from the wells of the eight-well glass-bottom slide. Distribute the dissociated dorsal root ganglia suspension into the collagen-coated wells, and incubate. Use brightfield microscopy to monitor the dorsal root ganglia-derived cells daily, assessing cell viability and neuronal outgrowth For co-culturing the whole mouse ganglia with cancer cells first aspirate the media from the whole-mount DRG cultures on the eight-well glass-bottom slide.

Using a pipette, gently add 200 microliters of the cancer cell suspension to the well containing the Matrigel droplet and the dorsal root ganglia. Incubate the co-culture slide in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide. Refresh the media daily by washing once with 200 microliters of warmed supplemented media, and add 300 microliters of fresh media.

For the co-culture of dissociated ganglia and the cancer cells, aspirate the media off the dissociated DRG cultures on the eight-well glass-bottom slide. Then gently distribute 200 microliters of the cancer cell suspension into each well before incubation. Establishment of DRG was less efficient in the absence of supplements and neurite extension was markedly reduced.

Whole-mount DRGs from older animals produced less profuse and more distinguishable neurite extension and minimal glial cells, facilitating visualization of nerve cancer cell interactions. The dissociation of DRG neurons using two enzymatic steps resulted in higher neuron recovery. Within 24 hours of culture, non-neuronal cells, such as schwan cells, macrophages and fibroblasts, became evident, while neurites sprouting from DRG-derived cells was observed and further increased after 72 hours.

Cancer cells seeded in Matrigel-coated transwell inserts invaded towards HEC293 cells, but not towards DRG neurons after 24 hours of incubation. Whole-mount DRG cultures immersed in a Matrigel droplet allowed neurite extension outward through the matrix towards the periphery. With careful positioning of the DRG centrally in the droplet, providing maximal neurite extension without overgrowing the borders.

Cancer cells settled down after 12 hours of co-culture around the border of the Matrigel droplet containing the DRG. After 72 hours of co-culture, cancer cells had invaded the Matrigel and moved toward the ganglion, with direct interaction between dorsal root ganglion cell extensions and cancer cells observed. Co-culture of dissociated DRG-derived cells with cancer cells allowed detailed visualization of individual cell interactions.