1 Introduction

Programmed death ligand-1 (PD-L1, B7-H1, or CD274) is a glycoprotein expressed on the surface of antigen-presenting cells , as well as different types of cancer cells. The tumor -expressed PD-L1 binds to PD-1 on cytotoxic T cells, resulting in the inhibition of T cell-mediated antitumor responses and immune escape [1,2,3]. In addition, the tumor-expressed PD-L1 has tumor intrinsic effects that include increased cancer cell proliferation , survival, and mTOR signaling [4,5,6,7]. While the inhibition of T cell-mediated antitumor responses is mediated by the surface PD-L1, the intrinsic functions of PD-L1 might be partly mediated by the intracellular PD-L1.

The PD-L1 expression in cancer cells is induced by interferon-γ (IFNγ) through the JAK-STAT-IRF signaling [8, 9]. In addition, recent studies have shown that the PD-L1 expression is regulated by epigenetic mechanisms, and that inhibition of histone deacetylases (HDAC ) increases PD-L1 expression in cancer cells, suggesting that HDAC inhibition might increase effectiveness of immune checkpoint inhibitors in cancer treatment [10,11,12,13,14,15,16,17]. This is particularly important in solid tumors, such as ovarian cancer (OC), where HDAC inhibitors and immune checkpoint inhibitors, as single agents, have produced disappointing results.

Ovarian cancer is the leading cause of death from gynecologic cancer in the United States, with a high morbidity and low survival rates [18,19,20]. Recent studies have shown that IFNγ induces PD-L1 expression in OC cells, resulting in their increased proliferation and tumor growth [21,22,23]. Here, we analyzed whether the surface PD-L1 expression is induced also by the FDA-approved class I HDAC inhibitor romidepsin. Using flow cytometry (FC) analysis of the surface PD-L1 expression in SKOV3 cells, our data show that romidepsin increases the PD-L1 surface expression in OC cells by about 100%. These results suggest that HDAC inhibition might provide a useful strategy to modulate the PD-L1 levels on OC cells. The protocol below describes an FC analysis of the surface PD-L1 expression in IFNγ- and romidepsin-treated OC cells. However, it can also be modified for the analysis of PD-L1 in other types of cancer cells.

2 Materials

2.1 Cell Culture

  1. 1.

    SKOV3 cells (American Type Culture Collection).

  2. 2.

    RPMI complete medium: RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM l-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate.

  3. 3.

    Phosphate-buffered saline (PBS), pH 7.4.

  4. 4.

    Interferon gamma (IFNγ) stock solution: Dissolve IFNγ in sterile PBS to a final concentration of 50 μg/mL. Aliquot, and store at −80 °C (see Note 1).

  5. 5.

    Romidepsin stock solution: Dissolve romidepsin in sterile DMSO to a final concentration of 10 μM. Aliquot, and store at −80 °C (see Note 1).

  6. 6.

    0.25% Trypsin-EDTA solution.

  7. 7.

    T-75 flasks.

  8. 8.

    T-25 flasks.

  9. 9.

    Trypan Blue solution.

  10. 10.

    Hemocytometer.

  11. 11.

    1.5 mL Microcentrifuge tubes.

  12. 12.

    15 mL Centrifuge tubes.

2.2 Cell Preparation for Flow Cytometry

  1. 1.

    HBSS (Hank’s Balanced Salt Solution), without calcium, magnesium, or phenol red.

  2. 2.

    Accutase™ cell detachment solution.

  3. 3.

    RPMI complete medium: RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 2 mM l-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate.

  4. 4.

    Incubation buffer: Dissolve 0.5% bovine serum albumin (BSA) in 100 mL of HBSS and filter. Prepare fresh and store at 4 °C.

  5. 5.

    Human-specific rabbit monoclonal PD-L1 IgG antibody for FC (see Note 2).

  6. 6.

    Control IgG.

  7. 7.

    Anti-rabbit IgG Alexa 488 conjugate secondary antibody for FC.

  8. 8.

    1.5 mL Microcentrifuge tubes.

  9. 9.

    15 mL Centrifuge tubes.

  10. 10.

    Slides and coverslips.

3 Methods

The protocol below describes FC analysis of PD-L1 expressed on the surface of OC cells, using antibody that specifically recognizes the extracellular domain of PD-L1. Using this approach, our data show that IFNγ induces the surface PD-L1 expression in SKOV3 cells approximately 28-fold (Fig. 1), and HDAC class 1 inhibition by romidepsin approximately two-fold (Fig. 2). The protocol can be easily modified and used for analysis of surface PD-L1 in other cells.

Fig. 1
figure 1

Flow cytometry analysis of surface PD-L1 expression in IFNγ-treated OC cells. (a) Histogram of PD-L1 surface expression in IFNγ-treated SKOV3 cells, illustrating the fluorescence intensity (horizontal axis) against the number of events detected (vertical axis). Cells incubated with isotype-matched control IgG are plotted in green; untreated cells are plotted in blue, and IFNγ (50 ng/mL, 48 h)-treated cells are plotted in red. (b) Relative fold surface expression of PD-L1 in untreated cells vs. IFNγ-treated cells

Full size image
Fig. 2
figure 2

Flow cytometry analysis of surface PD-L1 expression in romidepsin-treated OC cells. (a) Histogram of PD-L1 surface expression in romidepsin-treated SKOV3 cells, illustrating the fluorescence intensity (horizontal axis) against the number of events detected (vertical axis). Cells incubated with isotype-matched control IgG are plotted in green; untreated cells are plotted in blue, and romidepsin (10 nM, 48 h)-treated cells are plotted in red. (b) Relative fold surface expression of PD-L1 in untreated cells vs. romidepsin-treated cells

Full size image

3.1 Cell Culture

  1. 1.

    Grow SKOV3 cells in a T-75 flask containing RPMI complete medium until they reach about 80% confluence.

  2. 2.

    Discard the medium and wash cells with 7 mL of PBS. Add 4 mL of pre-warmed 0.25% trypsin-EDTA solution to the flask, and incubate at 37 °C till the cells detach (see Note 3).

  3. 3.

    Add an equal volume of RPMI medium to neutralize the trypsin.

  4. 4.

    Collect cells in a 15 mL centrifuge tube and centrifuge at 300 × g at 4 °C for 5 min. Discard supernatant, and resuspend cells in 5 mL of RPMI medium (see Note 4).

  5. 5.

    For cell counting, transfer 50 μL of the above cell suspension into a 1.5 mL centrifuge tube, add 50 μL of PBS, and 100 μL of trypan blue solution. Mix thoroughly by pipetting.

  6. 6.

    Add 10 μL of the above cell mixture into each chamber of the hemocytometer.

  7. 7.

    Count the number of viable cells and calculate the total cell concentration using the formula: Cell concentration = average cell count in four squares × 4 × 104 cells/mL.

  8. 8.

    Dilute the cell suspension from step 4 to a final concentration of 0.5 × 106 cells/mL using fresh RPMI medium.

  9. 9.

    Add 800 μL of the cell suspension to a T-25 flask containing 5 mL of RPMI complete medium, so that each flask contains about 4 × 105 cells (see Note 5).

  10. 10.

    Allow cells to attach to the flask for 24 h, aspirate the medium, and add 5 mL of fresh RPMI complete medium to each flask.

  11. 11.

    Set up the experimental protocol for cell-surface PD-L1 detection in IFNγ- and romidepsin-treated cells as follows:

    1. (a)

      No Antibody.

    2. (b)

      Control IgG and secondary antibody.

    3. (c)

      PD-L1 primary antibody and secondary antibody in untreated cells.

    4. (d)

      PD-L1 primary antibody and secondary antibody in IFNγ-treated cells.

    5. (e)

      PD-L1 primary antibody and secondary antibody in romidepsin-treated cells.

  12. 12.

    Incubate cells for 48 h with IFNγ (final concentration 50 ng/mL), romidepsin (10 nM), or with an equal volume of sterile vehicle solution at 37 °C in a 5% humidified CO2 incubator (see Note 6).

3.2 Cell Preparation for Flow Cytometry

  1. 1.

    Discard tissue culture medium and wash cells with sterile, room-temperature HBSS. Remove liquid by aspiration (see Note 7).

  2. 2.

    Add 3 mL of pre-warmed Accutase™ Cell Detachment Solution into each T-25 flask to cover the cells (see Note 8).

  3. 3.

    Incubate at 37 °C for 5 min, monitor cells under microscope every 2–3 min until more than 80% cells are detached (see Note 9).

  4. 4.

    Add 4 mL of RPMI complete medium to neutralize Accutase, and pipette gently up and down multiple times to disperse any cell clumps (see Note 10).

  5. 5.

    Transfer the entire content to a new pre-labeled 15 mL centrifuge tube.

  6. 6.

    Confirm the presence of single cells by removing 10 μL of the above cell suspension, placing it on a slide, and observing under microscope (see Note 11).

  7. 7.

    Centrifuge the cell suspension from step 5 at 300 × g for 5 min. Discard supernatant and collect the cell pellets. Gently resuspend the cell pellets in 8 mL of HBSS, and centrifuge again at 300 × g for 5 min. Discard the supernatant.

  8. 8.

    Wash the cell pellets in 5 mL of Incubation buffer. Resuspend the washed cell pellet in 1 mL of Incubation buffer, and transfer the cell suspension to a pre-labeled dark-colored Eppendorf tube (see Note 12).

  9. 9.

    Centrifuge again at 300 × g for 5 min, collect the cell pellet, and add primary PD-L1 antibody diluted in the Incubation buffer; incubate on ice for 60 min (see Note 13).

  10. 10.

    Centrifuge at 300 × g for 5 min, remove the supernatant, and collect the cell pellet. Wash the cell pellet twice in 1.5 mL of Incubation buffer. During each wash, gently resuspend the cell pellet, mix by inverting, and incubate on ice for 10 min. Centrifuge each time at 300 × g for 5 min (see Note 14).

  11. 11.

    Resuspend the cell pellets in 100 μL of Incubation buffer containing secondary antibody, and incubate on ice for 60 min (see Note 15).

  12. 12.

    Wash the cell pellets twice with 1.5 mL of Incubation buffer as in step 10. Centrifuge each time at 300 × g for 5 min.

  13. 13.

    Resuspend cells in Incubation buffer to a concentration of 7 × 105 to 1 × 106 cells/mL, and analyze samples by flow cytometry (see Note 16).

3.3 Data Analysis

  1. 1.

    Acquire at least 40,000 events during sample acquisition. Here, guavaSoft 3.3 (Millipore) was used for data analysis (see Note 17).

  2. 2.

    PD-L1 expression can be quantified by measuring the increase in fluorescence intensity in treated samples compared with untreated cells (Figs. 1 and 2).

4 Notes

  1. 1.

    Aliquot IFNγ and romidepsin stock solutions in sterile microcentrifuge tubes and store at −80 °C to avoid repeated freeze thaw cycles that may decrease the biological activity.

  2. 2.

    For FC analysis of surface antigens, it is important to use antibody that recognizes the extracellular domain of the particular protein. Here, we used the Cell Signaling rabbit monoclonal antibody #86744 that recognizes the extracellular domain of PD-L1.

  3. 3.

    Thaw aliquots of 0.25% trypsin-EDTA solution by placing them in the incubator at 37 °C in a 5% CO2 humidified atmosphere. If trypsin is not pre-warmed at 37 °C, cells may not detach completely.

  4. 4.

    Resuspend cells by pipetting up and down, and by gently inverting the tube; avoid vortexing.

  5. 5.

    Cells must be plated so that each flask yields about 1 million cells after 48 h incubation. Plate one T-25 flask per condition. The cells must not be over-confluent since the surface PD-L1 expression might be reduced due to contact inhibition.

  6. 6.

    After adding IFN or romidepsin, gently rotate the plate in a circular motion to assure an equal distribution of the drug in the medium.

  7. 7.

    Wash with HBSS thoroughly 5–6 times to remove dead cells, and any leftover medium and serum.

  8. 8.

    Thaw aliquots of Accutase by placing them in the 37 °C incubator in a 5% CO2 humidified atmosphere.

  9. 9.

    If most cells are still attached after 5-min incubation, remove the original Accutase solution, add a fresh Accutase solution, and continue the incubation with periodical microscopic observation. The detachment times may vary for different cell types.

  10. 10.

    Pipette gently 6–7 times to disperse any cell clumps.

  11. 11.

    Approximately 80–90% of the cells must be single, and not clumped. The flow cytometry instrument will detect PD-L1 expressed only on single cells. Clumped cells will produce inaccurate results.

  12. 12.

    Use dark-colored Eppendorf tubes so that after addition of secondary antibody, the loss of fluorescence is minimized.

  13. 13.

    Pre-dilute the primary antibody with Incubation buffer to avoid a pipetting error. After addition of primary antibody, resuspend the cells gently to ensure that all the cells are exposed to the antibody and there are no clumps. Here we used PD-L1 antibody obtained from Cell Signaling # 86744 (1:100 dilution).

  14. 14.

    Incubation on ice between the washes ensures that the cells are thoroughly washed.

  15. 15.

    Minimize exposure of the secondary antibody to light. Pre-dilute the secondary antibody with Incubation buffer to avoid a pipetting error. Pipette gently and ensure that all cells are exposed to secondary antibody. The tubes must be kept on ice and in dark. Exposure to light would lead to loss of fluorescence. Here we used anti-rabbit IgG Alexa 488 conjugate obtained from Cell Signaling (# 4412) in a 1:750 dilution.

  16. 16.

    The cells must have a minimum concentration of 5 × 105 cells/mL, and a volume of 500 μL for appropriate detection. Analyze immediately after incubation. Vortex samples for 30 s just before FC detection to disperse any cell clumps.

  17. 17.

    Before acquiring the data, ensure that the capillary in the flow cytometer is clean and ready for sample analysis; follow the manufacturer’s instructions. If analyzing more than one sample, run a quick clean between the samples so that any debris deposited in the capillary do not interfere with the sample analysis.