In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Four- to six-week-old female C57BL/CJ mice (Harlan, Jerusalem, Israel) were used in the experiment according to the Association for Assessment and Accreditation of Laboratory Animal Care specifications. All experimental procedures were done in accordance with Institutional Animal Care and Use Committee and the Department of Agriculture regulations.

1. Harvesting the Spinal Cord

1. Euthanize the mouse using a CO₂ chamber. Avoid cervical dislocation as it might cause damage to the ganglion roots due to shear forces. From here on, perform all steps under sterile conditions.
2. Soak animal down with 70% ethanol. This step is important for sterilization and for preventing the hair from dragging through the inner organs.
3. Leave the mouse to dry from ethanol. Prevent any contact of ethanol with the inner organs due to neurotoxicity.
4. After drying the mouse, position it using pins in prone position (face down). Place the pins on each hindlimb and forelimb.
5. Using forceps, make a midline incision extending craniocaudally from the posterior neck to the lumbar spine. Next, raise dermal flaps bilaterally using forceps and expose subcutaneous tissue.
6. Palpate mouse skull until reaching the craniocervical junction. Using scissors perpendicular to the animal, cut through the cervical muscles and spine at the craniocervical junction (the 7^th cervical vertebrae), using heavy forceps. Dissect the spine caudally and excise the spine with heavy forceps until reaching the lumbar level (hind limbs level).
7. Perform a complete caudal transection at the lumbar spine level (the 5^th lumbar vertebrae) using heavy forceps.
8. Roll the mouse over (face up). There is no need to cover the incision since the DRG is extracted from lower vertebral levels and not the cervical.
9. Cut down at the midline from the neck to the abdomen, retract the skin laterally and then cut to open the peritoneum.
10. Remove the inner organs inside the peritoneum (liver, spleen, pancreas, stomach, and intestine) en bloc. Next, remove the retroperitoneal organs (i.e., kidneys and pancreas). Cranially, open the chest wall.
11. Using forceps and a surgical blade, cut the ribs, leaving 5 mm of ribs bilaterally away from the vertebral column. At this point, the vertebral column is separated from the rest of the body.
12. Wash the vertebral column from blood with cold (4 °C) fresh phosphate buffered saline (PBS) twice.

2. Isolating the Dorsal Root Ganglia (DRG)

1. Use a stereomicroscope with 4X magnification.
2. Position the spine on a non-adherent non absorbing platform (Telfa/nylon). Position the spine face up at the same cranio-caudal orientation.
3. Remove any spinal muscles and connective tissue. Use the ribs as a landmark for nerves as they leave the spine. Ribs also protect the nerves from the surgical tools. The DRG are located at the cervical, thoracic, and lumbar sites of the vertebrae.
4. Next, using scissors cut the vertebral body in the midline and expose the spinal cord and DRG roots by gentle lateral retraction of the vertebral body.
5. In the intercostal space, follow the peripheral nerve medially along the rib lateral to the DRG.
6. Identify the DRG. It looks like the yolk of 'fried egg' lying on the nerve.
7. Cut the intercostal nerve distal to the DRG leaving 2-3 mm of nerve distal to the ganglia, to be used for retraction. This "tail" will be removed after harvesting.
8. Grasp the nerve using forceps. Pinching the DRG body itself will cause neural cell damage and should be avoided.
9. Approach the DRG proximally along its attachment to the spinal cord, via its anterior and posterior roots.
10. Apply gentle retraction to the DRG by pulling the efferent nerve (intercostal nerve stump) laterally, and cut the anterior and posterior roots close to the DRG.
11. Keep the DRG in a 35 mm Petri dish filled with fresh, ice cold DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% Sodium pyruvate.

3. Implantation in the Petri Dish

1. Perform the next steps on ice using pre-cooled pipette tips to prevent ECM solidification.
2. Under a stereomicroscope at 2-4X magnification, place a 35 mm glass bottom petri dish on a paper grid.
   IMPORTANT: Work on ice in order to keep the ECM in liquid state. Create a 1 mm diameter spot (approximately 20 µl) of growth factor-depleted ECM at the center of the grid.
3. Under direct visualization, place the DRG at the center of the ECM spot close to the bottom of the dish.
   Note: The cancer cells used in this protocol are murine pancreatic cancer cells (KPC) established from freshly isolated tumor specimens from KPC mice as described elsewhere¹⁹.
4. Harvest 40,000 cancer cells from confluent cultures, wash them once with PBS, and resuspend them in 40 µl ECM on ice.
5. Under direct visualization of the grid using a stereomicroscope measure 500 µm at each direction from the DRG. At this point slowly seed 10,000 cells/10 µl ECM. Use a 2-10 µl precooled tip to inject the cells close to the dish bottom, avoiding their spread in the matrix or detachment of the matrix. (Figure 1).
6. Leave the dish within a laminar flow hood for 10-15 min to solidify. Avoid ECM drying.
7. Slowly add DMEM, prepared as previously mentioned, against the side wall of the plate. Add enough medium to cover the ECM (about 2 ml).
8. Put the dish back to the incubator at 37 °C.
9. Replace the medium with fresh medium on the following day.
10. Replace the medium every 2 days.

4. Data Acquisition, Time-lapse Videomicroscopy

1. On day 7 after the implantation, take the dish for time lapse microscopy. Note that certain cells might require acquisition earlier due to faster migration.
2. During the live imaging, place the dish in a closed environment at a mean temperature of 37 ±0.1 °C and 5% CO₂.
3. Record the cells by using a charge coupled device camera placed on the microscope.
4. Take digitized images every 10 min for up to 72 hr to follow cell locomotion from three to six different cells.
5. Replace medium after 24 hr.
6. Perform data acquisition and simple analysis using the microscope software.

5. Data Analysis

1. For more advanced applications (i.e., cell tracking in 2D or 3D) use specialized software (such as Imaris 4D). Note: This software determines the xy coordinates of a cell at any given time, along with the distance from origin, and speed of motion.
2. Calculate the mean velocity by measuring the travel distance between subsequent positions in 10 min intervals. Calculate the Forward Migration Index (FMI) which is the distance of the cell front from the neurite and describes the unidirectional movement of the cells toward the DRG.