High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Antibody-dependent cellular cytotoxicity, or ADCC for short, can be mediated by natural killer cells, which we refer to as NK cells, granulocytes and the macrophages. These are the effector cells of ADCC. If an antibody such as trastuzumab, which targets the epidermal growth factor receptor, HER2, binds to tumor cells, then these effector cells use their FcyR receptors to bind the constant FC region of the antibodies.

The antibody bridges the tumor cells and the FcyR receptor-bearing effector cells, triggering the release of their cytotoxic granules. This leads to apoptosis of the cancer cells. ADCC may be effective against tumors that are unresponsive to the anti-proliferative effects of trastuzumab.

To set up the ADCC system, we need to co-incubate the anti-HER2 positive JIMT-1 breast carcinoma cells with the NK-92 cell line in the presence of the anti-HER2 antibody trastuzumab. The JIMT-1 cell line we use for the assay stably expresses the green fluorescent protein. First, code the 96-well high-content screening plate with the JIMT medium.

Pipette 50 microliter of medium into each well of the plate. Place the plate into a CO2 incubator for one hour. Coating is crucial for the attachment of JIMT-1 cells to the glass surface of the plate.

During the coating step, trypsinize JIMT-1 cells. Wash the cells with two milliliters of sterile PBS. Then, add one milliliter of Trypsin-EDTA to the flask.

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 milliliters of JIMT-1 media and collect the cell suspension into a 15-milliliter tube.

Then, count the JIMT-1 cells with trypan blue in a blue cast chamber. Adjust the cell number to 133, 000 cells per ml. Aspirate the coating medium, and then add 75 microliter of cell suspension to each well.

Allow the cells to attach during an overnight incubation at 37 degrees Celsius in a CO2 incubator. The next day, aspirate the medium and add 50 microliter fresh JIMT-1 medium to the wells, and transfer the plate to the high-throughput screening laboratory. The robot is used to transfer the test compounds onto the high-content screening microplates.

With the robotic arm, the robot grabs the pin tool unit, which is calibrated to transfer 25 nanoliters. Transfer a total of 100 nanoliter of test compounds from the compound library plate to the assay plate in four steps. Make sure that the final concentration of test compounds is 20 micromolar.

Between each step, first, wash the pin tool in 50%DMSO, and then in 70%ethanol. Incubate the plate for one hour in a CO2 incubator at 37 degrees Celsius. Culture NK cells in T25 tissue culture flasks.

While the drug treatment of JIMT-1 cells is ongoing, Collect the NK cells from a T25 flask to a 15-milliliter centrifuge tube. Count the NK-92 cells with trypan blue in a blue count chamber. Adjust the cell number to 400, 000 cells per ml.

Centrifuge the volume of NK cell suspension that contains 400, 000 cells per ml at 150 g for three minutes at room temperature. Prepare the ADCC medium by adding 20 microgram per ml anti-HER2 antibody trastuzumab to JIMT-1 medium. Pipette 20, 000 NK cells in 50 microliter ADCC medium to the target JIMT-1 GFP cells.

The final volume is 100 microliter, and the final trastuzumab concentration is 10 microgram per ml. Place the assay plate into the high-content analyzing equipment, which has a built-in incubator set a 37 degrees Celsius. The plates are imaged at two time points.

First, we acquire images immediately after the addition of the effector cells to the target cells, and the second set of images is taken at three hours after the addition of NK cells. First, select the type of the microplate. Use 96-well high-content screening plates.

Select to pick out focus because the assay is carried out in plates with a 10x objective in non-confocal mode. Choose binning two to double the signal-to-noise ratio. Select the appropriate channels, exposure time, laser power, focus plane, and wavelength.

Take the brightfield images at 650-760 nanometers and detect the EGFP-transduced JIMT-1 cells with 488 nanometers excitation and 500-550 nanometers emission wavelength. Before starting the measurement, take sample images with the snapshot function in order to check the correct settings. Finally, set the number of fields and the number of time points for the imaging.

Choose, for example, nine fields and two time points. To analyze the ADCC efficiency, count the viable JIMT-1 cells in the control ADCC well. Use the fine cells module to detect regions on the image that correspond to cells.

Detect each cell as a region on the image that has a higher fluorescence intensity than its surrounding. Select the cells by using the built-in M algorithm with 80 micrometer as diameter. Set the splitting sensitivity setting, which parcels out a large object into smaller objects, to the value 0.5.

Adjust the common threshold, which is the lowest level of pixel intensity, to zero. Exclude the detection of background area with high EGFP fluorescence intensity in two steps. First, apply the calculate intensity properties function to determine the EGFP fluorescence intensity in the previously selected cells region.

Afterwards, set a minimum and maximum intensity threshold by using the select population option, so that we get the number of viable JIMT-1 GFP target cells at the end of ADCC. Calculate ADCC efficiency by dividing the cell number detected at after three hours by the cell number detected at time zero. We would like to demonstrate the practical application of our method with representative results.

We have set up a test plate with 16 compounds randomly taken off the shelves. We also included three compounds, colchicine, vincristine, and podophyllotoxin with expected ADCC inhibitory effect. DMSO was used as negative control.

Each drug was used in four repeats on the plate. The figure shows the result of the test screen. In the minus NK group, the toxicity of the drugs on JIMT-1 cells was tested.

In the plus NK group, NK cells and trastuzumab were added, which caused approximately 50%cytotoxicity. The expected ADCC inhibitor effect of the three test compounds could be detected with the assay. We have set up a co-culture assay system modeling the clinically relevant interaction between breast cancer cells, NK cells, and the therapeutic monoclonal antibody trastuzumab.

The assay is based on automated microscopy combined with image analysis and is suitable for screening chemical compound libraries to identify ADCC-modifying drugs. The procedure can potentially be used to identify adjuvant molecules that potentiate an antitumor response or to identify chemicals with an adverse effect.