The overall goal of this procedure is to image resident tumor infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slicers using confocal microscopy. This method can help answer key questions in tumor immunology, such as the nature of the elements that positively and negatively regulate T cell migration within human tumors. The main advantage of this technique is that it allows us to monitor the daily behavior of resident CD8 T cells on a preserved human tumor environment.

We first an idea for this method when we established the method in Myrin lymph nodes to track T cells, plant them onto slices, a protocol originally published in JoVE in 2011. To begin, add one gram of low melting point agarose in a container, and then add 20 milliliters of sterile calcium and magnesium free PBS. Microwave the solution to completely dissolve the agarose.

Cool this 5%agarose solution in an incubator at 37 degrees Celsius for five minutes. In the tissue culture hood, add 1.1 milliliters of complete RPMI-1640 medium into each well of a six well sub well subculture plate. Then, using a pair of sterile forceps, place a sterile 30 millimeter cell culture insert into each well.

Incubate the plate at 37 degrees Celsius for 30 minutes until later use. Next, place the tumor biopsy in a 10 centimeter plastic dish, and using a sharp, sterile blade, cut the tumor into a small cube shaped fragment that is approximately five by five millimeters. Now, take out the prepared agarose solution from the incubator.

Using a pair of sterile forceps, carefully transfer the tumor fragments to a tissue wipe to drain out excess medium, then pour the agarose into a 35 millimeter plastic dish and insert the tumor fragments so as to position them at the bottom of the plastic dish. Leave the agarose gel on ice for five minutes to solidify. Once the agarose solidifies, invert the plastic dish.

Using a spatula, release the entire gel. With a sharp blade, trim the excess agarose surrounding the tumor fragments, leaving three to five millimeters of gel around the tissue. Using a drop of non toxic tissue glue, mount the agarose block on the specimen disc of the vibratome.

Fill the vibratome buffer tray with sterile, ice cold PBS, and install the specimen disc containing the agarose block in the tray. Set the vibratome speed and frequency to the desired values. Once the vibratome setup is ready, take out the six well plate from the incubator.

Now start cutting the mounted block into 350 micron slices using the vibratome. With the help of fine forceps, careful ly collect the tumor slices as they are being cut, and place them flat over the cell culture inserts in the six well plate. Transfer one slice over each insert.

Next, using fine forceps, place pre-sterilized and pre-wet stainless steel washers on each slice. Make sure that the washers are well positioned on the agarose surrounding the tumor tissue. Leave the plate to 37 degrees Celsius for 10 minutes in a humidified 5%carbon dioxide incubator prior to immunostaining.

Dilute the required fluorescently labeled antibodies and the fluorescent nuclear dye, DAPI, to the required vinyl concentrations in Phenol Red free RPMI medium. Next, remove the culture plate from the incubator and, using a fine tip, aspirate out the liquid inside the stainless steel washers. Now, without touching the slice, add 40 microliters the diluted antibodies onto each tumor slice.

To allow for staining, incubate the plate at 37 degrees Celsius for 15 minutes. After the staining step is complete, using fine forceps, remove the washers. Then, gently take out the slices and dip them in Phenol Red free RMPI-1640 medium for 10 seconds.

Place the slices back onto the cell culture inserts and add a drop of Phenol Red free RMPI-1640 medium over each slice. Incubate the plate at 37 degrees Celsius for 10 minutes, and then proceed to imaging. Several hours prior to starting this experiment, set the temperature of the microscope heat chamber to 37 degrees Celsius, then prepare the microscope setup to constantly profuse tumor slices with oxygenated Phenol Red free RMPI medium and to aspirate the solute to a waste collection flask using peristaltic pump.

With the help of fine forceps, lift the stained slice out of the six well plate, and transfer it to a 35 millimeter plastic dish filled with Phenol Red free RMPI medium, the place a slice anchor over the slice to support it. Next, place this Petri dish on the imaging stage of the microscope. After connecting the inlet and outlet tips of the perfusion system, turn on the peristaltic pump to allow the oxygenated medium to go through the perfusion tubes.

Lower the water emersion objective to the slice, and focus on the top of the slice with bright field light, or with the appropriate wave length. Then, using appropriate filters, visualize and select a region of interest that contains all the florescently labeled cells of interest, namely CD8 T cells, the tumor eyelets, and the CD90 positive stromal cells. Setup an imaging session using appropriate laser intensity and exposure times to suit the brightness of the fluorescent signals observed.

Then setup the Z-stack thickness to image within the tumor slice. Next, select the Z-stack image time interval and the total recording time to capture images at specific time intervals. After capturing and exporting the fluorescent images, analyze CD8 T cell migration as described in the text protocol.

Shown here is a representative image of a stained human lung tumor slice. Note the distribution of green CD8 positive T cells, blue epithelial cell adhesion molecule stained tumor cells, and red CD90 or Thyrum positive stromal cells. In particular, note that CD8 T cells are more abundant in the stroma compared to the tumor eyelets.

Plotted here are trajectories of individual resident CD8 T cells stained green, and the red stromal and blue tumor cell compartments of the human lung tumor slice. Collagen detected using second harmonic generation and displayed in red, marks the stromal compartment. Although less abundant than tumor eyelets, CD8 T cells migrate more actively in this compartment compared to the stroma.

Once mastered, this technique can be done in usually four, five hours if it's performed properly. After this video, you should have a good understanding of how to get an immunostain humor tumor samples, in order to monitor resident CD8 T cells using a confocal microscope.