Name: high-content screening assay for the identification of antibody-dependent cellular cytotoxicity modifying compounds
Uploaded: 2023-08-18T14:30:00
Description: Watch this Scientific Journal Video about High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds at JoVE.com

LV received funding from National Research, Development and Innovation Office grants GINOP-2.3.2-15-2016-00010 TUMORDNS&#34;, GINOP-2.3.2-15-2016-00048-STAYALIVE and OTKA K132193, K147482. CD16.176V.NK-92 cells were obtained from Dr. Kerry S. Campbell (Fox Chase Center, Philapedlphia, PA, on behalf of Brink Biologics, lnc. San Diego, CA), are protected by patents worldwide, and were licensed by Nantkwest, lnc. Authors are thankful to Gy&#246;rgy Vereb and &#193;rp&#225;d Sz&#246;&#337;r for their help with the use of the NK-92 cell line and for technical advice.

NOTE: Key steps of the assay workflow are presented in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64485/64485fig02.jpg" /> 
Figure 2: Workflow of the ADCC screen. JIMT-1-EGFP target cells seeded into 96 well HCS plates are treated with drugs of the compound library. In turn, unstained NK (effector) cells and trastuzumab are added, and the plate ...

<ol>
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L., Kamen, L., Song, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thaw-and-use+target+cells+pre-labeled+with+calcein+AM+for+antibody-dependent+cell-mediated+cytotoxicity+assays.">Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.</a> Journal of Immunological Methods. 447, 37-46 (2017).</li><li>Lee-MacAry, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+flow+cytometric+cell-mediated+cytotoxicity+assay+using+the+fluorophores+PKH-26+and+TO-PRO-3+iodide.">Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.</a> Journal of Immunological Methods. 252 (1-2), 83-92 (2001).</li><li>Tanito, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+natural+killer+cell-mediated+cell+killing+assay+based+on+the+leakage+of+an+endogenous+enzyme+or+a+pre-loaded+fluorophore.">Comparative evaluation of natural killer cell-mediated cell killing assay based on the leakage of an endogenous enzyme or a pre-loaded fluorophore.</a> Analytical Science. 37 (11), 1571-1575 (2021).</li><li>Lin, S., Schorpp, K., Rothenaigner, I., Hadian, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+high-content+screening+in+drug+discovery.">Image-based high-content screening in drug discovery.</a> Drug Discovery Today. 25 (8), 1348-1361 (2020).</li><li>Guti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multitargeted+receptor+tyrosine+kinase+inhibitor+sunitinib+induces+resistance+of+HER2+positive+breast+cancer+cells+to+trastuzumab-mediated+ADCC.">The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC.</a> Cancer Immunology, Immunotherapy. 71 (9), 2151-2168 (2022).</li><li>Li, F., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+on+NK+cells+and+ADCC:+A+promising+immunotherapy+approach+in+targeted+therapy+for+HER2-positive+breast+cancer.">Focusing on NK cells and ADCC: A promising immunotherapy approach in targeted therapy for HER2-positive breast cancer.</a> Frontiers in Immunology. 13, 1083462 (2022).</li><li>Perussia, B., Loza, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+antibody-dependent+cell-mediated+cytotoxicity+(ADCC)+and+reverse+ADCC+(redirected+cytotoxicity)+in+human+natural+killer+cells.">Assays for antibody-dependent cell-mediated cytotoxicity (ADCC) and reverse ADCC (redirected cytotoxicity) in human natural killer cells.</a> Methods in Molecular Biology. 121, 179-192 (2000).</li><li>Garcia-Alonso, S., Ocana, A., Pandiella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trastuzumab+emtansine:+Mechanisms+of+action+and+resistance,+clinical+progress,+and+beyond.">Trastuzumab emtansine: Mechanisms of action and resistance, clinical progress, and beyond.</a> Trends in Cancer. 6 (2), 130-146 (2020).</li></ol>

The ADCC reaction has been described a relatively long time ago. Key molecular events of the process have also been described19. Methods for measuring ADCC range from the gold standard radioactive chromium release assay, cytoplasmic enzyme release assays to several fluorescence-based flow cytometry or microplate assays20. However, a common limitation of these assays is that they are not amenable to high-throughput applications. Previously, we developed an image-based HCS as...

Immunotherapy with anticancer antibodies, immune checkpoint inhibitors, or chimeric antigen receptor-expressing T (CAR-T) cells represents a powerful approach to cancer treatment1,2,3. Trastuzumab is a humanized monoclonal anti-HER-2 (human epidermal growth factor receptor 2) antibody used for treating HER-2 positive early stage or metastatic breast cancer, as well as HER-2 positive metastatic gastric cancer4,5,6. It primarily acts by inhibiting the proliferation stimulating effect of the epidermal growth factor4. It has been reported, however, that trastuzumab efficiently triggers cancer cell death even if the cancer cells have lost their responsiveness to HER-2 stimulation7. This paradoxical effect of the antibody is due to antibody-dependent cell-mediated cytotoxicity (ADCC)7. ADCC can be mediated by natural killer (NK) cells, granulocytes, and macrophages collectively known as the effector cells of ADCC8,9. If an antibody, such as trastuzumab, binds to tumor cells, then these effector cells use their Fc receptors to bind the constant (Fc) region of the antibody. The antibody bridges the tumor cells and the Fc receptor-bearing effector cells, triggering the release of their cytotoxic mediators10. Natural killer cells release the cytotoxic cargo of their granules containing perforin to generate pores in the target cell membrane and granzyme (triggering cell death signaling pathways) into the immune synapse leading to apoptosis of the cancer cells (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64485/64485fig01.jpg" /> 
Figure 1: Effector and target cell interactions in ADCC. The cell surface Fc&#947; receptor of the effector NK cell recognizes the Fc region of the anti-HER2 trastuzumab antibody specific for the HER2 molecule expressed on the surface of the tumor cell. Thus, the so-called immunological synapse is established between the two cells, inducing the directed exocytosis of cytotoxic granules of the effector cell. The released perforin and granzyme molecules eventually result in apoptosis of the target cell. <a href="https://www.jove.com/files/ftp_upload/64485/64485fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Several assays have previously been developed to quantify cytotoxicity, including ADCC. The gold standard is the radioactive chromium release method, where the target cells are labeled with radioactive 51Cr isotope, and ADCC is quantified by measuring radioactivity from the supernatant of lysed target cells11. Because of the obvious problems due to the strictly regulated handling, storage, and disposal of radioactive pharmacons and wastes, this method has become increasingly non-popular among life scientists. In addition, it is not amenable to high-throughput applications either. Measuring the activity of enzymes (e.g., lactate-dehydrogenase) released from the killed target cells can provide a non-radioactive alternative to the 51Cr assay12. These assays, however, fail to distinguish between target and effector cell deaths. Electric Cell-substrate Impedance Sensing (ECIS) proved suitable for the quantification of ADCC13, but the ECIS equipment is not available in most laboratories, and the technique is not compatible with high-throughput applications/screening. Fluorescently labeled cells represent a popular alternative in many cell biology assays and are often used in flow cytometry or plate reader-based applications14,15,16. However, these assays often contain washing steps or are otherwise incompatible with high-throughput applications (e.g., flow cytometry-based techniques). Some popular cytotoxicity assays, which in theory should be suitable for ADCC quantification, fail to reliably determine ADCC efficiency13. Recently, with the spreading of fluorescent confocal microscopy, image-based, high-content assays are becoming increasingly popular in various areas of life sciences17. On the one hand, cell imaging equipment are now rather ubiquitous, while, on the other hand, virtually endless morphological parameters can be gathered from the acquired images. Therefore, we set out to develop a high-content screening compatible ADCC assay and to demonstrate its suitability for compound library screening.
Here, we present an image-based ADCC assay and demonstrate how this assay can be used for High-Content Screening (HCS) to identify ADCC modulating compounds. The model is based on JIMT-1 breast carcinoma target cells, CD16.176V.NK-92 effector cells and the humanized monoclonal anti-HER2 antibody trastuzumab. With this method, it is possible to identify drugs that can enhance the tumor-killing action of NK cells or to gain insight into the mechanism of NK cell-mediated ADCC by identifying small molecules interfering with ADCC. We suggest that life scientists aiming to quantify cell-mediated cytotoxicity with special regard to ADCC may benefit from using this assay either for the discovery science or drug development. This assay may be an alternative if a laboratory has access to and some experience in fluorescent imaging and quantitative image analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-fluorouracil</td>
			<td>Applichem</td>
			<td>A7686</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>96-well Cell Carrier Ultra plate</td>
			<td>PerkinElmer</td>
			<td>LLC 6055302</td>
			<td></td>
		</tr>
		<tr>
			<td>Betulin</td>
			<td>Sigma</td>
			<td>B9757</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>CD16.176V.NK92 cells</td>
			<td>Nankwest Inc.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cerulenin</td>
			<td>ChemCruz</td>
			<td>sc-396822</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-200896</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Colchicine</td>
			<td>Sigma</td>
			<td>C9754</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Concanavalin-A</td>
			<td>Calbiochem</td>
			<td>234567</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td>in JIMT-1 EGFP medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma</td>
			<td>E1383</td>
			<td>E1383</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biosera</td>
			<td>FB-1090/500</td>
			<td>JIMT-1 EGFP and NK medium</td>
		</tr>
		<tr>
			<td>Fisetin</td>
			<td>Sigma</td>
			<td>F4043</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Freedom EVO liquid handling robot</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gallotannin</td>
			<td>Fluka Chemical Corp.</td>
			<td>16201</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>35,050&#8211;061</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Harmony software&#160;</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humanized anti-HER2 monoclonal antibody (Herzuma)</td>
			<td>EGIS Pharmaceuticals, Budapest Hungary</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Humulin R (insulin)</td>
			<td>Eli Lilly</td>
			<td>HI0219</td>
			<td>JIMT-1 EGFP medium</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>Novartis Hung&#225;ria Kft.</td>
			<td>PHC0026</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Isatin</td>
			<td>Sigma</td>
			<td>114618</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acids (MEM-NEAA)</td>
			<td>Gibco</td>
			<td>11,140&#8211;050</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td>Lonza</td>
			<td>BE13-115E</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>Sigma</td>
			<td>N5893</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>NQDI-1</td>
			<td>Sigma</td>
			<td>SML0185</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Opera Phenix High-Content Analysis equipment</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Biosera</td>
			<td>LM-A4118</td>
			<td>JIMT-1 EGFP and NK medium</td>
		</tr>
		<tr>
			<td>Pentoxyfilline</td>
			<td>Sigma</td>
			<td>P1784</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Lonza</td>
			<td>BE17-517Q</td>
			<td>to wash the cells</td>
		</tr>
		<tr>
			<td>Podophyllotoxin</td>
			<td>Sigma</td>
			<td>P4405</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Quercetin</td>
			<td>Sigma</td>
			<td>Q4951</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma</td>
			<td>T8406</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Sigma</td>
			<td>T2577</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4% solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Vincristine sulfate</td>
			<td>Sigma</td>
			<td>V0400000</td>
			<td>in compound library</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Sigma</td>
			<td>M8042</td>
			<td>in NK medium</td>
		</tr>
	</tbody>
</table>

high-content screening assay for the identification of antibody-dependent cellular cytotoxicity modifying compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

To demonstrate how the assay works in real life, we created a test library of 16 compounds selected randomly from the lab shelves (Figure 3). In addition, DMSO was also included as a negative control, and three microtubule polymerization inhibitor compounds (colchicine, vincristine, and podophyllotoxin) as positive controls. The latter were expected to inhibit ADCC by interfering with NK cell migration to the cancer cells and NK cell degranulation. All test compounds and DMSO were placed ont...

Watch this Scientific Journal Video about High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds at JoVE.com

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

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

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.

Research

JoVE Journal

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