This protocol allows simple, faster, and reliable monitoring and characterization of each step of the cancer immunity cycle, and thus led to the identification of the mechanisms responsible for keeping the balance between cancer immune surveillance and immune evasion. The interaction and coevolution of cancer and immune cells in the tumor microenvironment have emerged as crucial factors determining therapeutic outcomes. Currently, several technologies are advancing research in our field, including flow cytometry, single-cell transcriptomics, special transcriptomics, proteomics, tumor-on-a-chip models, and in vivo models.
Our protocol offers significant advantages over other techniques, as it provides a simple, fast, reliable, and low-cost method for monitoring and characterizing each step of the cancer immunity cycle. In the future, we want to study the dynamics of the tumor microenvironment under immunogenic and immune therapies. To begin, plate three times 10 to the power of six MCA 205 fibrosarcoma cells in 10 milliliters of complete growth medium in a 100-millimeters Petri dish.
Irradiate cells with ultraviolet light and incubate at 37 degrees Celsius with 5%carbon dioxide for at least six hours to let them die. Wash in vitro-differentiated dendritic cells, or DCs, twice at 1, 100 G for four minutes in a complete growth medium. Count the bone marrow-derived MDDCs using the trypan blue dye exclusion test.
Centrifuge the irradiated cancer cells at 1, 100 G for five minutes. Set the co-culture of MDDCs with irradiated apoptotic cells at a 2:1 ratio in a 12-well plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the cells twice at 1, 100 G for five minutes in 10 milliliters of complete growth medium in 15-milliliter tubes.
For cell surface staining, resuspend the bone marrow-derived DCs in 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees Celsius in the dark. After washing the cells with FACS buffer, add 100 microliters of PBS containing vitality fixable near-infrared dye for 30 minutes. Wash the cells once with PBS before resuspending them in the FACS buffer.
Identify the cells of interest based on their FSCA and SSCA properties. Then, plot FSCA and FSCH to exclude cell doublets and clumps from the analysis. Plot 780-to 60-nanometer emission band pass filter area and SSCA to remove dead cells.
Using 575-to 26-and 530-to 30-nanometer emission band pass filters, detect cell positivity for CD11c marker and PKH67 fluorescent cell linker in two-parameter density plots After counting, culture the CD8 T-cells with bone marrow-derived DCS that had taken up apoptotic MCA 205 cells at a 2:5:1 ratio at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Add EdU to the co-culture medium at 10-micromolar final concentration and incubate for 16 to 20 hours. Centrifuge the CD8 cross prime twice at 1, 100 G for five minutes and stain for surface markers in cold FACS buffer for 20 minutes at four degrees Celsius in the dark.
After washing the cells twice in FACS buffer, resuspend them in 100 microliters of PBS containing the vitality fixable aqua dye for 30 minutes. Wash the cell suspensions once with three milliliters of 1%BSA in PBS in flow tubes. Add 100 microliters of 4%paraformaldehyde for 15 minutes for cell fixation.
Incubate the cells in 100 microliters of saponin-based permeabilization and wash reagent for 15 minutes. Add 500 microliters of the reaction cocktail and incubate for 30 minutes in the dark. After washing, resuspend the cells in 100 microliters of saponin-based permeabilization and wash reagent.
Identify the cells of interest based on their FSCA and SSCA properties. Then, plot FSCA and FSCH to exclude cell doublets and clumps from the analysis. Plot 525-to 50-nanometer emission band pass filter area and SSCA to remove dead cells.
Using 530-to 30-and 575-to 26-nanometer emission band pass filter density plots, detect cells'positivity for CD8a and CD3. Analyze cells for Alexa fluor 647 EdU incorporation in a single-parameter histogram using a 660-to 620-nanometer band pass emission filter. CD11c positive dendritic cells effectively engulfed apoptotic MCA 205 cancer cells at 37 degrees Celsius, demonstrating temperature-dependent phagocytosis in early cancer immunity cycle stages.
After phagocytosis, dendritic cells showed increased levels of CD86 and MHC2-II, along with reduced levels of PDL1. The clonal expansion of CD8+T-cells, once activated by mature dendritic cells, was evident with approximately 20%proliferation rate. Using tumor-on-chip models, the chemotactic response of these T-cells towards cancer cell-released alarmins was observed.
After recovering CD8 cross prime from co-culture, centrifuge them at 1, 100 G for 10 minutes. in 10 milliliters of complete growth medium. Resuspend one times 10 to the power of seven total cells in 100 microliters of dead cell removal microbeads and incubate for 15 minutes at room temperature.
Place separation columns in the magnetic separator and rinse them with binding buffer. Transfer cell suspensions into separation columns and collect the flow-through. After washing the column, collect the unlabeled cells that pass through and combine them with previously collected flow-through.
Centrifuge enriched live CD8 cross primed at 1, 100 G for five minutes in a complete growth medium. After counting, culture CD8 cross prime cells with fresh MCA 205 cells at a 2:1 ratio in 12-well plates and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Before recovering CD8 effector, Add monensin and brefeldin to the cancer immune cell co-cultures for six hours.
Then, recover CD8 effector and centrifuge twice at 1, 100 G for five minutes. Resuspend cells in three different primary antibody mixes prepared in cold FACS buffer and incubate for 20 minutes at four degrees Celsius, protecting from light. Add 100 microliters of fixation solution to the cell palate from mix C and incubate for 20 minutes at four degrees Celsius in the dark.
Resuspend the cells in a cold wash buffer and incubate for 30 minutes at four degrees Celsius. After washing cells twice in FACS buffer, add 100 microliters of PBS containing the vitality fixable aqua dye to cell pellets for 30 minutes. Identify cells of interest based on their FSCA and SSCA properties.
Then, plot FSCA and FSCH to exclude cell doublets and clumps from the analysis. Plot 525-to 50-nanometer emission band pass filter and SSCA to remove dead cells. Using 660-to 20-and 780-to 60-nanometer emission band pass filters, detect cell positivity for CD8a and CD3 in two-parameter density plots.
Further analyze cells for CD44, CD25, and CD69 markers for CD137 marker and for interferon gamma positive and granzyme B for mix A, B, and C, respectively. For tumor-killing assay, supplement the co-culture medium with a real-time cell death quantification dye. After warming the plate to 37 degrees Celsius in the live cell analysis system, perform data scanning every two hours for up to 72 hours.
Centrifuge the cells twice at 1, 000 G for five minutes. Stain cells for surface markers with 20 microliters of cold FACS buffer and incubate for 20 minutes at four degrees Celsius in the dark. Then, add the vitality dye propidium iodide to stain the cells for a gating strategy.
Identify the cells of interest based on their FSCA and SSCA properties. Then, plot FSCA and FSCH to exclude cell doublets and clumps from the analysis. Use a 450-to 50-nanometer emission band pass filter to detect cancer cells negative for CD45.
In a single-parameter histogram, using a 610-to 620-nanometer band pass emission filter, analyze cells for propidium iodide incorporation as mean fluorescent intensity. Through multi-parameter flow cytometry, the surface expression levels of the early activation marker CD69, late activation markers CD44 and CD25, membrane expression of CD137, and CD107a tumor reactivity markers were enhanced. The intracellular levels of cytotoxic molecules granzyme B and interferon gamma positive were progressively and significantly increased, reaching a peak of expression at 72 hours of co-culture.
Cognate co-cultured MCA 205 cells underwent significant levels of CD8 effector-mediated death.
