This method can be used to asses how novel therapeutic agents or strategies, influence or augment the cytotoxic T cell response during pancreatic cancer. This technique allows tumors to be rapidly digested and stimulated in vitro, allowing analysis of multiple parameters of the T cell response simultaneously, which can be easily adapted for other applications. To begin, use forceps, transfer the tumor onto a petri dish.
Store five milliliters of digestion medium in a 50 milliliter tube on ice to prevent enzyme activity commencing. Take a small aliquot of this solution, to cover the tumor on the Petri dish. Use a sterile scalpel and forceps to cut the tumor into small pieces, roughly less than three millimeters in length.
Scrape the tumor pieces into the tube, and gently invert the tube until all pieces are submerged in digestion media. Transfer the tube onto a shaking device for 20 minutes at 37 degrees celsius to digest. Make sure all pieces of tumor are submerged, and not stuck to the edge of the tube.
Immediately after the digestion step, place the tube on ice to slow enzyme activity. Add EDTA to achieve a final concentration of 20 millimolar, and briefly vortex the sample to mix in order to further slow enzyme activity. Open the tube and rinse any tumor digest off the lid of the tube with fresh RPMI medium.
Then prepare a 70 micron strainer, place it on a 50 milliliter open tube on ice. Pre-wet the filter with medium. Resuspend the digested cells and wash the sides of the tube using a 25 milliliter or larger Stripette.
The wider opening of the Stripette allows the thick digest to pass easily. Transfer all of the digest onto the strainer, using the 25 milliliter Stripette. Mash up and down the tumor on top of the filter using a one milliliter syringe plunger.
Continuously wash cells through the strainer with RPMI. Make sure to wash with enough force to push the cells through. Continue washing until all cells have passed through the strainer.
Centrifuge the tube for five minutes at 300 times G, and four degrees Celsius. Carefully resuspend the cell pellet, and complete RPMI, and pass directly through another filter to remove any extracellular matrix, or large cell clumps that cannot be adequately resuspended. If no stimulation is required, immediately stain the isolated cells for flow cytometry analysis, or resuspend them in freezing medium of 10%DMSO and FBS, and store at minus 80 degrees Celsius.
To perform stimulation, first count the cells. Dilute the cells in complete medium to achieve a concentration of two times ten to the six per 100 microliters. Plate 100 microliters of cells in each well of a U-bottomed 96 well plate.
Add 100 microliters of complete medium, containing a 2X preparation of PMA, and Ionomycin. Place the plate in an incubator at 37 degrees celsius, but 5%Carbon Dioxide for one hour. Then, at 20 microliters of a 10X preparation of Brefeldin A, and Monensin, and complete media to achieve a final concentration of 1.06 micromolar, and 2.0 micromolar, respectively.
Put the plate back into the incubator for another four hours. After injecting 1000 TB32048 cells into the pancreas, orthotopic tumors take approximately 30 days to develop. After digestion and stimulation of the harvested orthotopic tumors, a fluorescence minus one was performed to determine background fluorescence for gating.
And Brefeldin A Monensin only control was performed to determine basal production of cytokines. For interferon-gamma incubation with Brefeldin A and Monensin, resulted in no increase in interferon gamma over FMO control, in both spleen and tumor samples. However, the addition of PMA and ionomycin, increased the percentage of intracellular interferon gamma detectable in both splenic and tumor-derived CD4 positive and CD8 positive T cells.
Splenic CD4 positive and CD8 positive T cells, used as a positive control, have a relatively higher interferon gamma production, than tumor-infiltrating T cell subsets. Indicating immunosuppression occurs within the tumor. Using the same strategy for TNF alpha, a high percentage of splenic CD4 positive T cells were positive for intracellular TNF alpha.
Compared to tumor-infiltrating CD4 positive T cells. Splenic and tumor-infiltrating CD8 positive T cells, produced similar levels of TNF alpha. Make sure the tumor is sufficiently chopped, and that all the pieces are submerged in digestion media.
When mashing the tumor, be sure to be gentle, and wash cells through thoroughly. The tumor digestion protocol can be combined with flow-assisted cell sorting, to purify individual immune cell subsets for additional, functional, or gene expression analysis. We have used this method to characterize how B cells shape the immune response during pancreatic cancer, generating orthotopic tumors in both B cell knockout and B cell-depleted mice.
