Name: High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds (Video) | JoVE Journal
Uploaded: 2023-08-18T14:30:00.000+00:00
Duration: 13 min 59 s
Description: Learn high-content screening to identify compounds modulating antibody-dependent cellular cytotoxicity in breast cancer using NK cells and trastuzumab. This automated method enables rapid drug discovery for enhanced immunotherapy.

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5 minutes

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="ADCC diagram; cell incubation, trastuzumab, NK and JIMT-1 cells, high-content analysis." class="xfigimg" src="/files/ftp_upload/64485/64485fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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 is imaged at 0 timepoint and after 3 h of incubation. ADCC evaluation is based on the change in the number of viable (surface adherent) target cells. <a href="https://www.jove.com/files/ftp_upload/64485/64485fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Coating of the HCS plate
<ol>
	<li>Coat the 96 well high-content screening (HSC) plates with 50 µL/well JIMT-1 medium (DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS), 0.3 U/mL insulin (100 IU/mL, Humulin R, and 1% penicillin-streptomycin).</li>
	<li>Place the plate into a CO2 incubator for 1 h. 
	​NOTE: Coating is crucial for attaching JIMT-1 cells to the glass surface of the plate.</li>
</ol>
2. Seeding of JIMT-1 Enhanced Green Fluorescent Protein (EGFP) cells
NOTE: EGFP-expressing JIMT-1 cells were generated in our previous work18, and the cells were cultured in T25 tissue culture flasks in JIMT-1 media (see composition in step 1.1).
<ol>
	<li>Wash the cells with 2 mL of sterile PBS.</li>
	<li>Add 1 mL of trypsin-EDTA to the flask and put the flask back into a CO2 incubator for 10 min.</li>
	<li>After the incubation, tap the flask to check if JIMT-1 cells are detached.</li>
	<li>Stop the digestion with 2 mL of JIMT-1 media and collect the cell suspension into a 15 mL tube.</li>
	<li>Count the cells with 0.4% trypan blue (80 µL of the dye + 20 µL of the cell suspension) in a Bürker chamber and adjust the cell number to 133, 000 cells/mL.</li>
	<li>Aspirate the coating medium from the 96 well plate (step 1.2).</li>
	<li>Pipette 75 µL of the cell suspension to each well of the HCS plates (see Table of Materials).</li>
	<li>Allow cells to attach during an overnight incubation at 37 °C in a CO2 incubator.</li>
</ol>
3. Pre-treatment of JIMT-1 EGFP cells with the compound library
<ol>
	<li>Aspirate the medium from the JIMT-1 cells and add 50 µL/well fresh JIMT-1 medium to the wells. Transfer the plate to the high-throughput screening laboratory. 
	NOTE: Using a liquid handling robot makes the addition of compound libraries more efficient and reproducible.</li>
	<li>Transfer the test compounds from the compound library plate to the assay plate with a pin tool calibrated to a 25 nL volume. Perform this four times. Four rounds of transfer give a final volume of 100 nL (and a 20 µM final concentration).</li>
	<li>Between each step, wash the pin tool, first with 50% DMSO and then with 70% ethanol.</li>
	<li>Incubate the plates for 1 h in a CO2 incubator at 37 °C.</li>
</ol>
4. Starting the ADCC assay by adding the effector cells
NOTE: CD16.176V.NK92 cells (hereafter referred to as NK92 cells) were cultured in α-MEM supplemented with 20% FBS, 1% MEM-NEAA, 1% Na-pyruvate, 1% glutamine, 1% penicillin-streptomycin and 100 IU/mL IL-2.
<ol>
	<li>Count NK92 cells with trypan blue (80 µL of the dye + 20 µL of the cell suspension). Adjust the cell number to 400,000 cells/mL.</li>
	<li>Centrifuge 4 mL of the cell suspension at 150 x g for 3 min at room temperature.</li>
	<li>Prepare the ADCC medium by adding 20 µg/mL anti-HER2 antibody (trastuzumab) to the JIMT-1 medium.</li>
	<li>Resuspend the NK cell pellet in 5 mL ADCC medium.</li>
	<li>Pipette 20,000 NK cells in 50 µL of ADCC medium to the target JIMT-1 cells. The final volume is 100 µL, and the final trastuzumab concentration is 10 µg/mL.</li>
	<li>Place the assay plate into the high-content analysis equipment with a built-in incubator set at 37 °C.</li>
</ol>
5. Imaging
NOTE: The plates should be imaged at two time points, first, immediately after the addition of the effector cells to the target cells and second, at 3 h after the addition of NK cells. For imaging, the high-content analyzer and its software or suitable alternatives can be used (see Table of materials).
<ol>
	<li>Select Plate type (96-well cell carrier ultra) from the list of plates.</li>
	<li>Select the Two peak autofocus if the assay is carried out in plates.</li>
	<li>Use 10x objective in non-confocal mode.</li>
	<li>Select Binning 2 to double the signal to noise ratio.</li>
	<li>Take brightfield images at 650-760 nm and fluorescent images of the EGFP-transduced JIMT-1 cells at 488 nm (excitation) and 500-550 nm (emission) wavelengths.</li>
	<li>Select the number of fields and the number of timepoints for the imaging.</li>
</ol>
6. Image analysis
NOTE: To analyze the ADCC efficiency, the viable JIMT-1 cells are counted. Target cells killed by ADCC detach from the surface and move away from the focal plane of the microscope. Therefore, the difference between the number of viable cells at the beginning and at the end of the ADCC reaction corresponds to target cells eliminated by ADCC. To show how to build up the evaluation sequence, a control ADCC well is shown in the video.
<ol>
	<li>Use the Find cells module to detect regions on the image that correspond to cells. 
	NOTE: Each cell is detected as a region on the image with a higher fluorescence intensity than its surrounding.</li>
	<li>Select cells using the built-in M algorithm with a minimum of 80 µm in diameter.</li>
	<li>Set Splitting sensitivity, which parcels out a large object into smaller objects, to 0.5.</li>
	<li>Set the Common threshold (the lowest level of pixel intensity) to 0.</li>
	<li>Exclude the detection of background area with high EGFP fluorescence intensity in two steps.
	<ol>
		<li>First, use the Calculate Intensity Properties function to determine the EGFP fluorescence intensity in the previously selected Cells region.</li>
		<li>Set the minimum and maximum intensity threshold using the Select population option.</li>
	</ol>
	</li>
</ol>

<ol><li>Gupta, S. L., Basu, S., Soni, V., Jaiswal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunotherapy%3A+an+alternative+promising+therapeutic+approach+against+cancers%5BTitle%5D)+AND+%22Molecular+Biology+Reports%22%5BJournal%5D)">Immunotherapy: an alternative promising therapeutic approach against cancers.</a> Molecular Biology Reports. 49 (10), 9903-9913 (2022).</li>
<li>Moretti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+past%2C+present%2C+and+future+of+non-viral+CAR+T+cells%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">The past, present, and future of non-viral CAR T cells.</a> Frontiers in Immunology. 13, 867013(2022).</li>
<li>June, C. H., O'Connor, R. S., Kawalekar, O. U., Ghassemi, S., Milone, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR+T+cell+immunotherapy+for+human+cancer%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">CAR T cell immunotherapy for human cancer.</a> Science. 359 (6382), 1361-1365 (2018).</li>
<li>Ross, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+HER-2+receptor+and+breast+cancer%3A+ten+years+of+targeted+anti-HER-2+therapy+and+personalized+medicine%5BTitle%5D)+AND+%22Oncologist%22%5BJournal%5D)">The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine.</a> Oncologist. 14 (4), 320-368 (2009).</li>
<li>Shitara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+and+development+of+trastuzumab+deruxtecan+and+safety+management+for+patients+with+HER2-positive+gastric+cancer%5BTitle%5D)+AND+%22Gastric+Cancer%22%5BJournal%5D)">Discovery and development of trastuzumab deruxtecan and safety management for patients with HER2-positive gastric cancer.</a> Gastric Cancer. 24 (4), 780-789 (2021).</li>
<li>Gianni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+and+safety+of+neoadjuvant+pertuzumab+and+trastuzumab+in+women+with+locally+advanced%2C+inflammatory%2C+or+early+HER2-positive+breast+cancer+%28NeoSphere%29%3A+a+randomised+multicentre%2C+open-label%2C+phase+2+trial%5BTitle%5D)+AND+%22Lancet+Oncology%22%5BJournal%5D)">Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial.</a> Lancet Oncology. 13 (1), 25-32 (2012).</li>
<li>Barok, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trastuzumab+causes+antibody-dependent+cellular+cytotoxicity-mediated+growth+inhibition+of+submacroscopic+JIMT-1+breast+cancer+xenografts+despite+intrinsic+drug+resistance%5BTitle%5D)+AND+%22Molecular+and+Cancer+Therapy%22%5BJournal%5D)">Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance.</a> Molecular and Cancer Therapy. 6 (7), 2065-2072 (2007).</li>
<li>Gauthier, M., Laroye, C., Bensoussan, D., Boura, C., Decot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+Killer+cells+and+monoclonal+antibodies%3A+Two+partners+for+successful+antibody+dependent+cytotoxicity+against+tumor+cells%5BTitle%5D)+AND+%22Crit+Rev+Oncol+Hematol%22%5BJournal%5D)">Natural Killer cells and monoclonal antibodies: Two partners for successful antibody dependent cytotoxicity against tumor cells.</a> Crit Rev Oncol Hematol. 160, 103261(2021).</li>
<li>Gruijs, M., Sewnath, C. A. N., van Egmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+exploitation+of+neutrophils+to+fight+cancer%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">Therapeutic exploitation of neutrophils to fight cancer.</a> Semin Immunol. 57, 101581(2021).</li>
<li>Mando, P., Rivero, S. G., Rizzo, M. M., Pinkasz, M., Levy, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+ADCC%3A+A+different+approach+to+HER2+breast+cancer+in+the+immunotherapy+era%5BTitle%5D)+AND+%22Breast%22%5BJournal%5D)">Targeting ADCC: A different approach to HER2 breast cancer in the immunotherapy era.</a> Breast. 60, 15-25 (2021).</li>
<li>van der Haar Avila, I., Marmol, P., Kiessling, R., Pico de Coana, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+antibody-dependent+cell-mediated+cytotoxicity+by+chromium+release+assay%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Evaluating antibody-dependent cell-mediated cytotoxicity by chromium release assay.</a> Methods in Molecular Biology. 1913, 167-179 (2019).</li>
<li>Broussas, M., Broyer, L., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+antibody-dependent+cell+cytotoxicity+using+lactate+dehydrogenase+%28LDH%29+measurement%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement.</a> Methods in Molecular Biology. 988, 305-317 (2013).</li>
<li>Toth, G., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitating+ADCC+against+adherent+cells%3A+Impedance-based+detection+is+superior+to+release%2C+membrane+permeability%2C+or+caspase+activation+assays+in+resolving+antibody+dose+response%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.</a> Cytometry A. 91 (10), 1021-1029 (2017).</li>
<li>Chung, S., Nguyen, V., Lin, Y. L., Kamen, L., Song, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thaw-and-use+target+cells+pre-labeled+with+calcein+AM+for+antibody-dependent+cell-mediated+cytotoxicity+assays%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+novel+flow+cytometric+cell-mediated+cytotoxicity+assay+using+the+fluorophores+PKH-26+and+TO-PRO-3+iodide%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">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/pubmed?term=((Comparative+evaluation+of+natural+killer+cell-mediated+cell+killing+assay+based+on+the+leakage+of+an+endogenous+enzyme+or+a+pre-loaded+fluorophore%5BTitle%5D)+AND+%22Analytical+Science%22%5BJournal%5D)">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/pubmed?term=((Image-based+high-content+screening+in+drug+discovery%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">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/pubmed?term=((The+multitargeted+receptor+tyrosine+kinase+inhibitor+sunitinib+induces+resistance+of+HER2+positive+breast+cancer+cells+to+trastuzumab-mediated+ADCC%5BTitle%5D)+AND+%22Cancer+Immunology%2C+Immunotherapy%22%5BJournal%5D)">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/pubmed?term=((Focusing+on+NK+cells+and+ADCC%3A+A+promising+immunotherapy+approach+in+targeted+therapy+for+HER2-positive+breast+cancer%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((Assays+for+antibody-dependent+cell-mediated+cytotoxicity+%28ADCC%29+and+reverse+ADCC+%28redirected+cytotoxicity%29+in+human+natural+killer+cells%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Trastuzumab+emtansine%3A+Mechanisms+of+action+and+resistance%2C+clinical+progress%2C+and+beyond%5BTitle%5D)+AND+%22Trends+in+Cancer%22%5BJournal%5D)">Trastuzumab emtansine: Mechanisms of action and resistance, clinical progress, and beyond.</a> Trends in Cancer. 6 (2), 130-146 (2020).</li>
</ol>

Authors report no conflict of interest.

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="NK cell and tumor cell interaction diagram with FcγR-Trastuzumab-HER2 binding process." class="xfigimg" src="/files/ftp_upload/64485/64485fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Effector and target cell interactions in ADCC. The cell surface Fcγ 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><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.&amp;nbsp;</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&amp;ndash;061</td><td>in NK medium</td></tr><tr><td>Harmony software&amp;nbsp;</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&amp;aacute;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&amp;ndash;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&amp;ndash;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>&amp;alpha;-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

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...

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 ...

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Cancer Research

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Learn high-content screening to identify compounds modulating antibody-dependent cellular cytotoxicity in breast cancer using NK cells and trastuzumab. This automated method enables rapid drug discovery for enhanced immunotherapy.

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

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