Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

This protocol provides with clinical solid tumor immunotherapy model and a research platform to study the relationship between tumor cells and infiltrating immune cells. This cell-based tumor vaccine is convenient, straightforward to use, and can be combined with other therapeutics modalities such as checkpoint blockade, to achieve additive or synergistic effect that can result in more potent and high tumor immunity. Helping to demonstrate the procedure will be Ann Balancio, a research technician from my laboratory.

To begin, culture B16-F10 melanoma cells in IMDM, containing 10%heat inactive FBS, 2 millimolar glutamine, 1 millimolar sodium pyruvate, 1 millimolar MEM non-essential amino acids and 100 units per milliliter each of penicillin and streptomycin. Maintain the cell line at 37 degrees Celsius with 5%carbon dioxide. Seed and harvest the B16-F10 cells as described in the text manuscript.

Remove the culture medium and wash the flasks once with PBS. Aspirate PBS and add 5 milliliters of 0.25%trypsin-EDTA, followed by harshly tapping the rim of the culture flask. Add 15 milliliters of culture medium to neutralize the trypsin-EDTA and pour the contents of the flask into a 50 milliliters centrifuge tube.

Wash the dish surface with 10 milliliters of PBS and pour into the same 50 milliliters centrifuge tube. Centrifuge the cells for 5 minutes at 200 RCF. Discard the supernatant and break the cell pellet by finger tapping the bottom of the tube.

Add cold 10 milliliters of PBS and gently pipette the cell suspension. Then, manually count the cells using a hemocytometer. Keep the cells on ice before injection.

Intradermally implant 500, 000 cells B16-F10 tumor cells in 50 microliters of cold PBS in the left flank, using a 30 gauge needle. The dose of implanted B16-F10 tumor cells may need to be adjusted in a range of 50, 000 to 500, 000 cells for successful tumor development. After implementation, measure the tumor length and width three times a week using an electronic digital caliper.

Calculate the tumor volume and treat the mice with tumor vaccine when tumors have reached a size of two millimeters as described in the text manuscript. Using an x-ray irradiator set at 160 kilovolts and 25 milliamperes, irradiate cells at 150 gray doses of gamma rays. Count and check the cell viability by trypan blue staining before injection.

Intradermally inject the mice with 1 million irradiated B16-Flt3L cells in 50 microliters of cold PBS on the same flank as the original tumor implantation. Approximately one centimeter from the site of the primary tumor on days 3, 6, and 9 after the initial cell implantation. Mark the vaccine injection sites with a colored pen to distinguish them from the primary tumor.

If 50, 000 B16-F10 cells were initially implanted, it is recommended to perform vaccine treatment on days 8, 11 and 14. Surgically removed the tumor with the skin from each euthanized mouse and put it into a 24 well plate with 1 milliliter of 10%FBS RPMI 1640 medium. Cut the tumors into small pieces in two milliliters of digestion buffer and incubate for 25 minutes at 37 degrees Celsius.

Add 10 milliliters of 10%FBS RPMI 1640 medium to stop the digestion. Transfer the cells to a 40 micrometer cell strainer using a 25 milliliter serological pipette. Then use the plunger of a one milliliter syringe to grind the tissues.

Centrifuge the cells at 500 RCF for 5 minutes at 4 degrees Celsius. Resuspend the pellet in 5 milliliters of 40%density gradient specific medium in PBS diluted to 1x concentration. Add the cell suspension slowly on top of 5 milliliters of 80%density gradient specific medium containing PBS.

Centrifuge to the cells at 325 RCF with a low brake setting for 23 minutes at room temperature. After centrifugation, carefully collect the leukocytes layer at the interface between 40%and 80%density gradient specific medium, and pass it through a 40 micrometer cell strainer. Centrifuge the cells at 500 RCF for 5 minutes at 4 degrees Celsius.

Incubate the pellet in two milliliters of red blood cell lysis buffer for five minutes at room temperature. After incubation, add 10 milliliters of 10%FBS RPMI 1640 medium to quench the RBC lysis buffer. Centrifuge the cells at 400 RCF for 5 minutes at 4 degrees Celsius.

Resuspend the cells in 0.5 milliliters of 10%FBS RPMI 1640 medium, and count the total number of cells before use for further analysis. Collect the spleen or the draining lymph nodes as controls for the gating strategy of immune cell subsets by flow cytometry analysis. Follow the cell isolation method described in the text manuscript.

A visible black dot of the implanted B16-F10 cells was usually observed on the skin surface approximately three days after tumor implantation. Mice were treated with a tumor vaccine three, six and nine days after the tumor nodule had reached or exceeded the size of two millimeters. A significant tumor growth reduction was observed in the vaccinated mice group about two weeks after tumor implantation.

Cells collected from the leukocytes layer contained many tumor cells, making it difficult to readily define the lymphocyte population. Therefore, splenocyte were used in parallel, for proper gating of intratumoral immune cell subsets in flow cytometry analysis. The gating strategies of CD103 positive, CD11C positive DC, CD8 positive, CD4 positive, and Treg were plotted along with the compensation matrix.

Representative data of acquired accounts and frequency of each population was also provided. Intratumoral CD103 positive, CD11C positive DC from vaccinated mice, displayed a significantly elevated expression of the co-stimulatory ligand CD86. Vaccinated mice also displayed an increase in tumor infiltrating CD8 positive and CD4 positive, Foxp3 negative cells, as well as in CD8 positive granzyme B positive and interferon gamma positive CTLs.

Keep a sufficient distant between the primary tumor and the tumor vaccine. This physical separation is critical to avoid potential fusion between the two tumor implants.