This study investigates neural invasion, a mechanism of cancer metastasis where neurotrophic cancer cells grow around nerves. The research utilizes an in vitro model involving murine dorsal root ganglia (DRG) to observe the interactions between cancer cells and nerve projections.
Neural invasion is the mechanism of cancer metastasis wherein neurotrophic cancer cells demonstrate an intrinsic ability to grow in and around nerves. To observe this phenomenon in vitro, first, take an isolated murine dorsal root ganglion or DRG - a cluster of cell bodies of sensory neurons.
Place the DRG in an ice-cold medium to retain its viability. Now, transfer the DRG into a drop of chilled extracellular matrix or ECM, which serves as a three-dimensional scaffold around the DRG. Next, seed the neurotrophic cancer cells into the ECM at an appropriate distance around the DRG.
Allow the ECM to solidify in order to embed the DRG and cancer cells within. Finally, add a suitable growth medium to the ECM and incubate. The nutritive growth media and the ECM proteins provide an optimum microenvironment for the explants.
During incubation, the reciprocal interaction between the explants promotes DRG to sprout small nerve projections or neurites that outgrow radially towards the cancer cells. Simultaneously, cancer cells migrate unidirectionally away from their colonies and invade the newly formed neurites.
Working in a laminar flow hood, place a 35-millimeter glass-bottom Petri dish on a paper grid on ice. Using a pre-cooled pipette tip, dispense approximately 10 microliters of growth factor-depleted ECM at the center of the grid. While viewing the dish through a stereomicroscope under 2X 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 in 40 microliters of ECM on ice.
Once again, view the grid through the stereomicroscope, 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 2 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.
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