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.

high-content-screening-assay-for-identification-antibody-dependent

Research

JoVE Journal

Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...