Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

Perineural invasion or PNI refers to the migration of cancer cells along the nerves and their subsequent invasion into the perineurium - a suitable microenvironment for cancer growth and metastasis.

To mimic  PNI in vitro, begin by adding a drop of extracellular matrix or ECM suspension onto a culture dish such that it takes the shape of a hemisphere. Now, incubate the culture dish to reinforce the structural and functional features of the 3D scaffold.

Next, carefully place a pre-harvested dorsal root ganglion or DRG, a cluster of sensory neurons, at the center of the semisolid ECM droplet. Incubate to embed the DRG within the ECM droplet.

Thereafter, add a suitable growth media and incubate. The nutrient supplemented ECM favors the sprouting of DRG into small nerve projections called neurites that outgrow radially towards the circumference of the droplet.

Remove the spent medium to prep the ECM droplet for subsequent assay. Seed fluorescently stained adherent cancer cells over the droplet. Due to the hemispherical shape, cancer cells roll over and settle along the droplet periphery.

Incubate the culture dish to allow for cancer cell-neurite interaction. When viewed under the fluorescence microscope, the cancer cells can be seen tracking alongside the neurites and migrating towards the DRG, confirming perineural invasion.