Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

One of the most effective targeted cancer therapy is based on antibodies such as the anti-HER2 antibody trastuzumab. The anti-cancer action of trastuzumab involves antibody-dependent cell-mediated cytotoxicity, ADCC, by natural killer or NK cells. Understanding mechanisms regulating the sensitivity of cancer cells to anti-growth factor receptor antibody therapy with special regard to ADCC might be crucial for the development of novel, more efficient treatment modalities.

3D models such as cancer cell spheroids proved superior in predicting immune responses of tumors to various anti-cancer therapies. Therefore, we set up a ADCC spheroid model using the NK92 natural killer cell line, GFP-expressing JIMT-1 breast carcinoma cells, and trastuzumab. We set up the ADCC assay system by inducing the anti-HER2-positive JIMT-1 breast carcinoma cells to form three-dimensional clusters called spheroids.

The JIMT-1 cell line used in the experiments stably expresses the green fluorescent protein. We start the experiment by preparing the 96-well plate for spheroids formation. In order to create a U-shaped and cell-repellent bottom, coat the 96-well plate with a solution of 0.5%agarose in PBS.

Incubate the plate at room temperature for approximately 30 to 45 minutes. In the meantime, trypsinization and count JIMT-1 cells for cell seeding. Wash the cells with two milliliter of sterile PBS.

Add two milliliter of trypsin-EDTA to the flask, and put the flask back into a CO2 incubator for 10 minutes. After the incubation, tap the flask to check if JIMT-1 cells have detached. Stop the digestion with two milliliter of JIMT-1 media, and collect the cell suspension into a 50-milliliter tube.

Count the cells with Trypan Blue in a Burker chamber, and adjust the cell number to 20, 000 cells per ml. Seed the cells into the 96-well plate by pipetting 100 microliters cell suspension to each well for spheroids formation. Allow the cells to clump together during the three-day incubation time at 37 degrees in a CO2 incubator.

Check the size and shape of spheroids regularly with an inverted microscope. On day three after spheroids induction, coat 96-well high-content screening plate with a solution of Pluronic F-127 in DMSO. Coating is crucial to prevent the attachment of JIMT-1 EGFP spheroids to the glass surface or the plate.

After an incubation period of 45 minutes at room temperature, aspirate the coating solution, and wash the wells twice with DMEM F12 serum-free media. Transfer the spheroids to the 96-well high-content screening plate in triplicates using a one-milliliter pipette. Add sunitinib to the wells at the 40-micromolar concentration in 10 microliters per well, and pipette fresh JIMT-1 media to the control wells in order to bring up the volume.

Incubate the plate for one hour in a CO2 incubator at 37 degrees. While the incubation of the pretreated spheroids is ongoing, collect the NK92 cells from the flask into a 15-milliliter tube. Count the cells with Trypan Blue in a Burker chamber, and adjust the cell number to reach an effector-to-target ratio of 20 to 1.

Stain the NK cells with a 10-micromolar solution of CellTracker Blue, and place them into a CO2 incubator for one hour at 37 degrees. To wash the excess of the dye, centrifuge the NK cells twice at 150 RCF for three minutes at room temperature. Then resuspend the cells in fresh JIMT-1 media.

Add the stained NK cells to the target JIMT-1 EGFP spheroids by pipetting 55 microliters per well along with anti-HER2 antibody trastuzumab. Dilute trastuzumab in JIMT-1 media to reach a concentration of 10 micrograms per ml. Add the 110 microliters of fresh JIMT-1 media to the control wells, and incubate the plate for 24 hours in a CO2 incubator at 37 degrees.

As a result, the total volume of treatment added for the ADCC is 110 microliters, and the final sunitinib concentration is 20 micromoles per liter. To detect and measure apoptotic cell death following the treatments, stain the spheroids with a Annexin V Alexa Fluor 647 conjugate for one hour in JIMT-1 media. After the experimental steps are finished, transfer the plate to the high-content analyzing device.

The plate is imaged 24 hours after the addition of the NK factors to the target cells. For imaging, we are using the high-content analyzer and an image analysis software. Select the type of the microplate from the list of plates using the Plate Type option.

Use 96-well high-content screening plate. Select two peak autofocus as the assays carried out in plates with 10x objective with 0.3 numerical aperture, using the autofocus and objective options respectively. Choose Confocal mode with the OptiMode option, and apply Binning 2 to using the Binning option.

For imaging spheroids, select the appropriate channels using the Channel Selection option. To detect the EGFP-transfused JIMT-1 cells, choose EGFP with 200-millisecond integration time, 50%laser power, two-micrometer stack height, 488-nanometer excitation, and 500, 550-nanometer emission wavelengths. And to detect apoptotic cells, use Alexa 647 with 100-millisecond integration time, 50%laser power, 10-micrometer stack height, 640-nanometer excitation, 650, 760-nanometer emission wavelengths.

To visualize the NK cells within the spheroids, choose the following channel, DAPI with 100-millisecond integration time, 50%laser power, two-micrometer stack height, 405-nanometer excitation, and 435, 418-nanometer emission wavelength. In Layout Selection option, select Z stacks since cells in the spheroids tend to be on different focal planes of the microscope. 10 planes with distance of 10 micrometer are sufficient to cover the whole spheroid region.

Set the values of the first plane and last plane at 0 micrometer and 90 micrometer, respectively. Before starting the measurement, sample images can be taken with the Snapshot function in order to check the correct settings. Set the number of wells and fields for imaging using the Defined Layout option.

Identify the spheroids by the EGFP fluorescence or JIMT-1 cells by using the Fine Texture Region option and filter them out by size. Remove border objects by using the Select Population module. Since the Annexin-positive cells appear on the peripheral spheroids, measure Annexin intensity in the apoptotic ring.

In the Select Region module, set outer border to minus 90. Express Annexin intensity values as mean intensity. As indicated by Annexin V staining, addition of NK cells and trastuzumab to the JIMT-1 cells caused cell death in the tumor spheroids.

Annexin-positive apoptotic cells emerged in the peripheral zone of the spheroids. In the absence of trastuzumab, however, NK cells did not trigger tumor cell apoptosis. To distinguish between the direct or ADCC-mediated effects of trastuzumab, trastuzumab-Fab2 lacking FC region binding capability was used in this study as a negative control.

Since trastuzumab-Fab2 was ineffective in this model, tumor cell death is likely to be mediated via ADCC. Moreover, pretreatment with sunitinib reduced Annexin V staining in the spheroids, confirming sunitinib-induced ADCC resistance and revealing the prevention of apoptotic cell death in JIMT-1 spheroids co-incubated with NK cells and trastuzumab. This data confirmed the cytoprotective effect of sunitinib in the ADCC model and urge caution concerning the potential combinations of ADCC-based immunotherapies and sunitinib.

In summary, our HCS assay might be suitable for the identification of ADCC-boosting compounds.