This article discusses the process of perineural invasion (PNI) in cancer, highlighting an in vitro model to study this phenomenon. The method involves embedding dorsal root ganglia in a semi-solid extracellular matrix to observe cancer cell interactions with neurites.
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.
To prepare the semi-solid 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 1 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 21G 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 degrees Celsius incubator for 3 minutes. When all of the DRG have been embedded, hold the plates one at a time at a slight angle and slowly add 4 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 3 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 4 milliliters of DMEM with 10% FBS along the sidewall of the glass-bottom plate and return the plates to the incubator until their next microscopic evaluation.
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