Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

1. Immunostaining of resident CD8 T cells and tumor cells

1. Dilute the antibodies (final concentration 10 µg/mL) and the nuclear dye (final concentration 1 µg/mL) in RPMI-1640 media without phenol red.
   NOTE: Use fluorescently-coupled anti-CD8 (murine IgG, clone SK1) and anti-EpCAM (epithelial cell adhesion molecule) (murine IgG HEA-125) antibodies to label CD8 T lymphocytes and tumor epithelial cells, respectively. Use fluorescently-coupled anti-CD90/Thy1 (murine IgG, clone 5E10) antibody to label fibroblasts and activated endothelial cells. Use 4',6-diamidino-2-phenylindole (DAPI) to label the nucleus of cells with a compromised plasma membrane in order to assess tissue viability.
2. Remove the culture plates from the incubator and use a pipette to aspirate any liquid inside the stainless steel washers. Do not remove the 1.1 mL RPMI-1640 media placed under the cell culture inserts.
3. Without touching the slice and using a pipette tip, add 40 µL of the solution containing the antibodies onto each tumor slice.
4. Incubate the plate at 37 °C for 15 min to allow antibody staining.
5. With fine forceps, remove the washers, take out the slices, and dip them 10 s in RPMI-1640 media without phenol red. Place the slices back onto the cell culture inserts and add a drop of phenol red-free RPMI-1640 medium on each individual slice. Maintain the plate at 37 °C for 10 min. Proceed to imaging.

2. Imaging and analyzing resident CD8 T cell migration within human lung tumor slices

NOTE: The confocal microscope used in this protocol is an upright spinning disk equipped with a 25x water immersion objective (25x/0.95 N.A.).

1. Preparing microscope setup
   1. Set the temperature of the microscope heat-chamber at 37 °C a few hours before starting the imaging session.
   2. Set up a system to constantly perfuse tumor slices with oxygenated (5% CO₂, 95% O₂) phenol red-free RPMI medium (Figure 1). Use a peristaltic pump to flow the perfusion media into the imaging chamber and aspirate the solution to a waste collection flask.
   3. Run the peristaltic pump to allow the oxygenated solution to go through the perfusion tubes.
2. With fine forceps, take out the slice from the 6-well plate and transfer it to the 35-mm plastic dish filled with phenol red-free RPMI medium. Immobilize the slice with a 19.7 mm diameter slice anchor with 2 mm spaced threads. Place the Petri dish on the imaging stage of the microscope. Connect the inlet and outlet tips of the perfusion system. Turn on the perfusion system and set the media flow rate to 0.8 mL/min.
3. Lower the water-immersion objective to the slice. Focus on the top of the slice with bright field light. Using appropriate fluorescent light, select a region of interest that contains CD8 T cells, tumor islets (positive for EpCAM), and a stroma area (positive for CD90). Check the viability of cells within the slice by assessing DAPI staining at the cut surface and deeper into the tissue.
   NOTE: A healthy slice contains only a minority of DAPI-positive cells at several microns from the cut surface.
4. Set an imaging session with the following actions.
   1. Depending on the intensity of the fluorescent signal of the 3 fluorophores, set the exposure time between 50 to 800 ms and the laser intensity between 20 to 40%.
   2. Select the z stack thickness to image within the tumor slice.
      NOTE: Here, 10-12 optical planes spanning a total depth of 60-70 µm in the z dimension were captured.
   3. Select the start position at approximately 10 µm below the first labeled CD8 T cells.
   4. Define the time interval between each z-stack image between 10 to 30 s and the total recording time between 10 to 30 min.