An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8⁺ T Cells against Cancer Cells

1. Preparation of target cells that express red fluorescent protein in their nuclei

1. Obtain a target mouse cancer cell line from an appropriate source.
   NOTE: In this protocol, a highly metastatic derivative of E0771 mouse mammary tumor cells (E0771-LG) is used. Parental E0771 cells originate from C57BL/6 mice.
2. Thaw and maintain a vial of E0771-LG cells with Dulbecco's Modified Eagles Medium (DMEM) including 10% (v/v) fetal bovine serum (FBS) in a cell culture incubator at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: It should be confirmed that cells are negative for mycoplasma. To this end, culture E0771 cells (or cells to be tested) for 2-3 days as described above (in the absence of antibiotics and antimycotics), and collect 500 μL of culture medium. Centrifuge the medium at 12,419 x g for 60 s to eliminate cell debris and transfer the supernatant into new tubes. Determine mycoplasma contamination using a commercially available mycoplasma test kit (refer to Table of Materials) and/or PCR following the manufacturer's instructions.
3. Seed 5 x 10³ E0771-LG cells per well into a 12-well plate, and culture the cells with 10% (v/v) FBS-DMEM overnight in an incubator at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: If the proliferation rate of target cells is low (population doubling time greater than 36 h), the number of cells can be increased to 1 x 10⁴.
4. Replace the medium with 1 mL of 10% (v/v) FBS-DMEM including 10 μg/mL polybrene and add 25 μL of lentiviral particles (1 x 10⁶ TU/mL) encoding a nuclear restricted red fluorescent protein (mKate2, refer to Table of Materials).
5. Culture the cells for 24 h in an incubator at 37 °C, 95% humidity, and 5% CO₂.
6. Replace the medium with 10% (v/v) FBS-DMEM and culture the cells for 24-48 h in an incubator at 37 °C, 95% humidity, and 5% CO₂.
7. Replace the medium with 10% (v/v) FBS-DMEM including 1 μg/mL puromycin when cells start to express a red fluorescent protein, and culture the cells until they are 80-90% confluent.
   NOTE: The concentration of puromycin will be different between target cell types and should be optimized using un-transfected cells.
8. Subculture the surviving cells for 1-3 passages with 10% (v/v) FBS-DMEM including 1 μg/mL puromycin, and cryopreserve stocks in a liquid nitrogen vapor phase storage system until use.

2. Isolation of suppressor cells from the tumors in mice

NOTE: In this protocol, suppressor cells (i.e., metastasis-associated macrophages (MAMs) and monocytic-myeloid-derived suppressor cells (M-MDSCs)) are isolated from the lung containing metastatic tumors established by E0771-LG cells. Conditions for tissue dissociation and cell sorting should be optimized to isolate the cells from different tissues.

1. Inject 1 x 10⁶ cancer cells (E0771-LG) into the tail vein of syngeneic (C57BL/6), female, 7-10 week old mice.
2. After 14 days, isolate the lung containing metastatic tumors and prepare single-cell suspensions from the perfused lungs via enzymatic digestion as previously described.
   NOTE: In this protocol, four mice are injected with cancer cells and their metastatic lungs are combined to obtain sufficient suppressor cells.
3. Incubate the single cell suspensions with anti-mouse CD16/CD32 antibody for 30 min on ice, and stained with fluorescent antibodies to CD45, F4/80, CD11b, Ly6C, and Ly6G (refer to Table of Materials) for another 30 min.
4. Wash the stained cells once with 1 mL of PBS containing 2% (w/v) bovine serum albumin (BSA), and re-suspend the cell pellet with 500–1000 μL of PBS containing 2% (w/v) BSA.
5. Add 3 μM of DAPI, and sort M-MDSCs (DAPI^–CD45⁺F4/80⁺Ly6G^–CD11b^highLy6C^high) and MAMs (DAPI^–CD45⁺F4/80⁺Ly6G^–CD11b^highLy6C^low) using a cell sorter (Figure 1).
   NOTE: The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). The purity of the sorted cells is measured via flow cytometry with an expected purity of more than 90%.
6. Resuspend the sorted cells with 400 μL of DMEM containing 20% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 2 mM L-glutamine, 1% (v/v) non-essential amino acid, 1 mM sodium pyruvate, and 50 nM 2-mercaptoethanol (called enriched-DMEM, E-DMEM).
7. Count the number of live cells using the Trypan blue exclusion method and adjust to 2 x 10⁶ cells/mL with E-DMEM.
8. Keep the cells on ice until use.

3. Isolation of CD8⁺ T cells from the spleen of mice

1. Isolate the spleen from a mouse that is syngeneic to the target cancer cell line (i.e., C57BL/6 mice in this protocol) as follows:
   1. Euthanize the animal by CO₂ inhalation.
   2. Place the animal on a clean dissection board and wipe the skin with 70% (v/v) ethanol.
   3. Cut the abdominal skin using scissors to expose the spleen
   4. Isolate the spleen, which is located inferior to the stomach, and place it into a tube containing 5 mL of ice-cold PBS.
2. Using the inner plunger of a sterile 5 mL syringe, grind the spleen on a 100 μm cell strainer set on a 50 mL tube.
3. Pass the cells through the filter using a total of 10 mL of PBS.
4. Centrifuge the cell suspension at 337 x g for 5 min, and aspirate the supernatant.
5. Resuspend the cell pellet in 1 mL of PBS containing 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% (w/v) BSA (running buffer) and filter through a 40 μm cell strainer.
6. Count the live cell number and adjust to 1 x 10⁸ cells/mL using the running buffer.
   NOTE: Keep a small aliquot of cells as a pre-enrichment sample for a purity check.
7. Enrich for CD8⁺ T cells using a negative selection kit and a magnetic sorter (refer to Table of Materials).
   1. Transfer 1 x 10⁸ (1 mL) of the splenocyte cells to a 5 mL polystyrene round-bottom tube.
   2. Add 50 μL of biotinylated antibodies, and incubate at room temperature for 10 min.
   3. Add 125 μL of streptavidin-conjugated magnetic beads, and incubate at room temperature for 5 min.
   4. Add 1.325 mL of running buffer, and gently mix by pipetting.
   5. Place the tube into the magnet, and incubate at room temperature for 2.5 min.
   6. Pick up the magnet, and pour the enriched cell suspension into a new tube.
8. Resuspend the enriched cells with 200 μL of E-DMEM (i.e., DMEM containing 20% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 2 mM L-glutamine, 1% (v/v) non-essential amino acid, 1 mM sodium pyruvate, and 50 nM 2-mercaptoethanol).
9. Count the number of live cells and adjust to 2 x 10⁶ cells/mL with E-DMEM. Keep the cells at 37 °C in a CO₂ incubator until use.
   NOTE: Keep a small aliquot of cells as a post-enrichment sample for a purity check.
10. Determine the purity of CD8⁺ T cells by flow cytometry as follows:
    1. Take 1 x 10⁴ cells from pre-enrichment (step 3.6) or post-enrichment (step 3.9) samples and adjust the total volume of each to 100 μL using a running buffer.
    2. Incubate the single cell suspensions with anti-mouse CD16/CD32 antibody for 30 min on ice, and stain with fluorescent antibodies to CD45, CD3, CD4, and CD8 (refer to Table of Materials) for another 30 min.
    3. Wash the stained cells with 500 μL of PBS containing 2% (w/v) BSA, and re-suspend the cell pellet with 500–1000 μL of PBS containing 2% (w/v) BSA.
11. Add 3 μM of 4',6-diamidino-2-phenylindole (DAPI), and determine the percentage of CD3⁺CD4^–CD8⁺ cells in the total CD45⁺ cell population.

4. Activation and expansion of the isolated CD8⁺ T cells

1. Aliquot 1 x 10⁵ cells per 50 μL CD8⁺ T cells (prepared in step 3.9) into wells of a U-bottom 96-well plate.
2. Add 1 x 10⁵ cells per 50 μL suppressor cells (prepared in step 2.7) or 50 μL of E-DMEM into the wells.
3. Prepare activation medium that consists of E-DMEM, 4 x 10⁴ U/mL colony-stimulating factor 1 (CSF-1), 240 U/mL interleukin-2 (IL-2), 8 μg/mL anti-mouse CD3ε antibody, and 16 μg/mL anti-mouse CD28 antibody.
   NOTE: CSF-1 is not required for T cell activation, but is essential for survival of suppressor cells in this protocol (i.e., MAMs and M-MDSCs). Thus, it is retained in the co-culture of T cells with target cancer cells to maintain consistency in culture conditions. Since CSF-1 is found in nano-molar concentrations in all tissues and is required for monocyte/macrophage viability in vivo, this is a physiological context for these cells.
4. Add 50 μL of activation medium (step 4.3) and 50 μL of E-DMEM with or without reagents to be tested.
5. Place the plate into an incubator at 37 °C, 95% humidity, and 5% CO_2, and culture the cells for 4 days.

5. Setup of co-culture of target cells with pre-activated CD8⁺ T cells

1. Add 30 μL of 1:100 diluted growth factor-reduced soluble basement membrane matrix (Table of Materials) into the wells of flat bottom 96-well plate suitable for microscopy, and incubate at 37 °C in a CO₂ incubator for at least 1 h.
2. Prepare target cells (i.e., E0771-LG cells expressing nuclear-restricted red fluorescent protein).
   1. Incubate the target cells with 1 mL of 0.05% trypsin/EDTA at room temperature for 1 min, and harvest the cells by gentle pipetting.
   2. Add 9 mL of DMEM including 10% (v/v) FBS, and centrifuge the cell suspension at 337 x g for 5 min.
   3. Re-suspend the cells with 500 μL of E-DMEM, and count the number of live cells.
      NOTE: Before counting the target cells, filter the cells through a 40μm cell strainer to produce a single-cell suspension.
   4. Adjust the density to 2 x 10⁴ cells/mL (= 1 x 10³ cells per 50 μL) by adding E-DMEM, and keep the cells on ice.
3. Prepare effector cells (i.e., pre-activated CD8⁺ T cells).
   1. Resuspend the cells in a well of a 96-well plate (step 4.5) thoroughly by pipetting, and transfer the floating CD8⁺ T cells into a new 1.5 mL tube.
      NOTE: MAMs and M-MDSCs tightly adhere to the well and are not detached by the pipetting.
   2. To collect the remaining cells, add 200 μL of PBS into the well and transfer it into the tube in step 5.3.1.
   3. Wash the cells with 1 mL of E-DMEM once (centrifuge at 337 x g) for 5 min, aspirate and discard supernatant), and resuspend them with 100 μL of E-DMEM.
   4. Count the number of live cells (using the trypan blue exclusion method), adjust the density to 1.6 x 105 cells/mL (= 4 x 10³ cells/25 μL), and keep the cells on ice.
4. Aspirate the basement membrane matrix from each well of the plate in step 5.1.
5. Add 1 x 10³ cells/50 μL of target cells (step 5.2.4) into each well and mix well.
   NOTE: To avoid edge effects due to evaporation of the medium, only the inner 60 wells of the 96-well plate should be used for analysis.
6. Add 25 μL of E-DMEM including 4 x 10³ U/mL IL-2 and 10 μM fluorogenic caspase-3 substrate (refer to Table of Materials).
7. Add 4 x 10³ cells/25 μL of CD8⁺ T cells (step 5.3.4) into appropriate wells and mix well.
   NOTE: The presence of too many cells in a well makes the analysis more difficult. In this model, a 4:1 effector: target ratio (total cell number 5 x 10³ cells/well) was optimal, but an 8:1 ratio was suboptimal. Wells containing the following four controls are necessary to aid with data analysis: target cells at the density used for the co-culture wells (1 x 10³ cells/well) in medium with and without caspase-3 substrate, effector cells (1 x 10³ cells/well) in medium with and without caspase-3 substrate.
8. Add 200 μL of PBS or sterile water into all empty wells (particularly wells on the periphery of the plate) to reduce evaporation of medium from experimental wells.
9. Set the plate into a time-lapse fluorescence microscope that is maintained at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: Shake the plate in a cross-pattern to distribute all cells evenly, and then allow the plate to remain at room temperature on a flat surface for 10-20 min before transferring to the incubator.

6. Imaging of the cells

NOTE: Detailed image acquisition settings will vary with the microscope and fluorophores used; the following general acquisition parameters should be employed for optimal results.

1. Using an appropriate autofocus routine on the microscope, acquire images covering at least 25% of the total surface area in each experimental well of the 96-well plate.
2. Set the microscope to acquire images in phase contrast as well as a fluorescent channel suitable for the nuclear-restricted red fluorescent protein (mKate2) and a fluorescent channel suitable for the green fluorogenic activated caspase-3 substrate (with excitation at 488 nm) (Figure 2).
3. Capture images in experimental wells in phase contrast and the 2 fluorescent channels every 1 to 3 h for at least 72 h.