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, OTKA K132193 and K147482. This project has received funding from the HUN-REN Hungarian Research Network. CD16.176V.NK-92 cells were obtained from Dr. Kerry S. Campbell (Fox Chase Center, Philadelphia, PA, on behalf of Brink Biologics, lnc. San Diego, CA), are protected by patents worldwide, and were licensed by Nantkwest, lnc. (www.nantkwest.com). 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 the TR-F(ab&#8217;)2, and for technical advice.

1. Setting up the JIMT-1-enhanced fluorescent protein (EGFP) spheroid model
<ol>
	<li>To form a U-shaped cell-repellent bottom, coat the 96-well plate with 0.5% agarose-PBS solution (30 &#181;L/well). Incubate the plate at room temperature for approximately 30-45 min.</li>
	<li>Wash the JIMT-1-EGFP cells twice with 2 mL of sterile PBS (generation of JIMT1-EGFP cell line was reported in a previous publication17). Use T25 tissue culture flasks and JIMT-1 media (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) for the cell culture.</li>
	<li>Add 2 mL of trypsin-EDTA to the flask and incubate it for 10 min in a CO2 incubator.</li>
	<li>Tap the flask to check if JIMT-1 cells detached after the incubation time.</li>
	<li>Use 2 mL of JIMT-1 medium to stop the digestion and collect the cell suspension into a 15 mL tube.</li>
	<li>Count the cells in a B&#252;rker chamber with 0.4% trypan blue (80 &#181;L of the dye + 20 &#181;L of the cell suspension) and adjust the cell number to 20,000 cells/mL.</li>
	<li>Pipette 100 &#181;L of the cell suspension (2000 cells/well) to each well of the 96-well plate (pre-coated with 0.5% agarose-PBS solution as indicated in 1.1).</li>
	<li>Allow cells to clump together during a 3-day incubation time at 37 &#176;C in a CO2 incubator.</li>
	<li>Regularly check the size and shape of spheroids with an inverted microscope.</li>
</ol>
2. Coating of the HCS plate
NOTE: To prevent the attachment of JIMT-1-EGFP spheroids to the glass surface of the plate, coating the High-Content Screening (HCS) plate is crucial (otherwise high-content analysis would not be possible).
<ol>
	<li>On day 3 after induction of spheroids, coat the 96-well high-content screening plate with Pluronic-F127 (0.5% in DMSO, 50 &#181;L/well) and incubate the plate for 45 min at room temperature.</li>
	<li>Aspirate the coating solution and wash the wells twice with DMEM/F-12-serum free medium (100 &#181;L/well).</li>
</ol>
3. Transfer of spheroids to the HCS plate
<ol>
	<li>Using a 1 mL pipette, transfer the spheroids in triplicates to the glass bottom 96-well HCS plate.</li>
</ol>
4. Pre-treatment of JIMT-1 EGFP spheroids with test compounds
<ol>
	<li>Add the test compound (e.g., Sunitinib diluted in DMSO at a concentration of 40 &#181;M) by pipetting 10 &#181;L/well and add 10 &#181;L of fresh JIMT-1 medium to the control (CTL) wells.</li>
	<li>Incubate the plate for 1 h in a CO2 incubator at 37 &#176;C.</li>
</ol>
5. Induction of ADCC by adding the effector cells
NOTE: CD16.176V.NK92 cells (hereinafter referred to as NK cells) were cultured in &#945;-MEM supplemented with 20% FBS, 1% MEM-NEAA, 1% Na-pyruvate, 1% glutamine, 1% penicillin-streptomycin and 100 IU/mL of IL-2.
<ol>
	<li>Collect the NK cells in the flask into a 15 mL tube. Count the cells with trypan blue (80 &#181;L of the dye + 20 &#181;L of the cell suspension) and adjust the cell density to 20:1 effector-to-target (E:T) ratio (40,000 NK cells/well).</li>
	<li>Stain the NK cells with 10 &#181;M Cell Tracker Blue (CTB, 1 &#181;L in 1 mL of &#945;-MEM NK medium) and place them into a CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Centrifuge the NK cells at 150 x g for 3 min at room temperature twice to wash the excess of the dye with 1 mL of &#945;-MEM medium.</li>
	<li>Resuspend the NK cell pellet in 1 mL of fresh JIMT-1 medium.</li>
	<li>Add the stained NK cells along with the anti-HER2 antibody (Ab) (Trastuzumab, dissolved in sterile distilled H2O) to the target JIMT-1 spheroids by pipetting 55 &#181;L/well of CTB-stained NK cells and 55 &#181;L/well of 10 &#181;g/mL Ab in JIMT-1 medium (total volume of treatment added for the ADCC is 110 &#181;L/well and the final Sunitinib concentration is 20 &#181;M). 
	NOTE: For the selection of Ab concentration and E:T ratio, we relied on our previous publication17. Preliminary experiments were performed with different E:T ratios and concentrations of Trastuzumab to assess which was the most effective in inducing ADCC in spheroids (Supplementary Figure S1).</li>
	<li>Add 110 &#181;L/well of fresh JIMT-1 medium to control (CTL) wells.</li>
	<li>Incubate the plate for 24 h in a CO2 incubator at 37 &#176;C. 
	NOTE: As NK92 cells are known to exert non-specific cytotoxic functions, for control use JIMT-1 cells co-incubated with both NK cells alone and with an isotype control or a F(ab&#39;)2 Ab. Here F(ab&#39;)2-Trastuzumab (TR-F(ab&#39;)2) was used as a negative CTL. TR-F(ab&#39;)2 fragment was prepared as reported by T&#243;th et al.19 and added in the same volume as Trastuzumab along with NK cells as described in 5.5. Concentration was adjusted to 6.6 &#181;g/mL corresponding to an equimolar concentration with Trastuzumab23.</li>
</ol>
6. Annexin V-647 staining of spheroids
<ol>
	<li>To measure apoptotic cell death, stain the spheroids with Annexin V-Alexa Fluor 647 conjugate for 1 h in JIMT-1 medium (1:100) by pipetting 50 &#181;L/well.</li>
</ol>
7. Imaging
NOTE: The plate is imaged at 24 h after the addition of the NK effector cells to the target cells. For imaging, a High-Content Analyzer and an image analysis software are used.
<ol>
	<li>Select the type of microplate from the list of plates using the Plate Type option. Use 96-well High-Content Screening (HCS) plate.</li>
	<li>Select Two Peak autofocus as the assay is carried out in plates with 10x objective with 0.3 numerical aperture (NA), using the Autofocus and Objective options, respectively. Choose confocal mode with the Opt. Mode option and apply Binning 2 using the Binning option.</li>
	<li>For imaging spheroids, select the appropriate channels using the Channel Selection option. To detect the EGFP-transduced JIMT-1 cells choose EGFP (integration time 200 ms, laser power 50%, stack height 2.0 &#181;m; ex: 488 nm em: 500-550 nm), and to detect apoptotic cells use Alexa 647 (integration time 100 ms, laser power 50%, stack height 10.0 &#181;m; ex: 640 nm em: 650-760 nm). To visualize the NK cells within the spheroids, choose the following channel: DAPI (integration time 100 ms, laser power 50%, stack height 2.0 &#181;m; ex: 405 nm em: 435-480 nm).</li>
	<li>In Layout Selection option, select Z-stacks since cells in the spheroids tend to be on different focal planes of the microscope. 10 planes (with distance of 10 &#181;m) are sufficient to cover the whole spheroid region. Set the values of the first plane and last plane at 0 &#181;m and 90 &#181;m, respectively. 
	NOTE: Before starting the measurement, sample images can be taken with the snapshot function in order to check the correct settings.</li>
	<li>Set the number of wells and fields for imaging using the Define Layout option.</li>
</ol>
8. High-content analysis (HCA)
NOTE: Analyze the images with the Harmony software or export images for analysis using a preferred third-party software. For the analysis of ADCC efficiency, the fluorescence intensity of Annexin V 647 is measured. Target cells killed by ADCC appear in the peripheral&#160;area of the spheroids. Therefore, the Annexin V positive cells are measured in this spheroid &#34;ring&#34;. In order to validate this method, analyses were performed with different parameters to assess which was the most reliable and producing the best results (Supplementary Figure S2). An ADCC well is shown in the video in order to demonstrate step-by-step the image analysis process.
<ol>
	<li>Identify the spheroids by the EGFP fluorescence of JIMT-1 cells using Find Texture Regions option and filter them out by size (&#62; 25,000 &#181;m2).</li>
	<li>Remove border objects by Select Population option.</li>
	<li>Since Annexin V positive (apoptotic) cells appear on the periphery of spheroids, measure Annexin intensity in this apoptotic &#39;Ring&#39;, selected by the Select Region option (outer border -90%) to determine apoptotic cell death.</li>
	<li>Express Annexin V intensity values as mean intensity.</li>
</ol>

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Authors declared no conflicts of interests.

Despite significant improvements in treating BC over the past several decades, patients still regularly develop medication resistance or experience negative side effects24. The high morbidity and mortality linked to BC demand a continuing investigation into the underlying molecular mechanisms, just as robust screening platforms to identify new molecules actionable for therapeutic development25. These strategies require cell culture-based translational assays. 3D tumor cultu...

Multicellular tumor spheroids (MCTS) are widely used three-dimensional (3D) models that form due to the tendency of adherent cells to aggregate and represent an important tool for gaining mechanistic insight into cancer cell biology. They can be generated from a broad range of cell types by numerous techniques, such as liquid-based and scaffold-based 3D cultures1. Their main advantage over monolayer 2D models is that they recapitulate the main features of in vivo tumors, namely structural organization and hypoxia, by mimicking the biological behavior of tumor cells, especially the mechanisms leading to therapeutic escape and drug resistance2. Thus, since MCTS can improve the predictability of toxicity and drug sensitivity, they are widely used to study cancers in 3D and could enhance the development of effective drugs for different types of cancer3.
To study any disease, there is a critical need for relevant and convenient models. Setting up models for cancer immunology studies is challenging because the immune system consists of multiple cell types. Each cell type has several subtypes and a broad spectrum of activation states. These different immune cell types interact with cancer cells and other tumor components, ultimately influencing the outcome of the disease. 2D in vitro cell culture methods fail to recapitulate these complex cellular interactions, as they lack translatability and are unable to predict the action of a drug at the system level (e.g., in tissues)4,5. Moreover, mouse models also have severe limitations due to the fundamental differences between the human and murine immune systems. 3D culture systems can, therefore, fill the current gaps in available models, providing an alternative method and improving our understanding of cancer immunology6. Specifically, spheroid models might be used for testing immunotherapies, mainly to assess the efficiency of drug screening and therapeutic antibodies for enhancing immune cell infiltration and anti-tumoral effects against the spheroid targets7. Furthermore, the potential of MCTS composed of cells in different metabolic and proliferative states to study the interactions between stroma cells (e.g., lymphocytes, macrophages, fibroblasts) and cancer cells and for the development of new anticancer strategies has been amply demonstrated8. Hence, there is a vital need to corroborate predictive and accurate platforms in order to boost the drug-testing process, taking into account the pathophysiology of the tumor microenvironment.
Breast cancer (BC) is the most frequent cancer diagnosed worldwide in women. The clinical classification of this heterogeneous disease is based on the presence of transmembrane receptors e.g., estrogen (ER) and progesterone (PR) receptors (collectively called hormone receptors, HR) along with the overexpression or amplification of the human epidermal growth factor receptor 2 (HER2) protein/oncogene. Based on the immunohistochemical expression of these receptors, four subtypes are commonly recognized: luminal A (HR+/HER2-), luminal B (HR+/HER2+), HER2-positive (HR-/HER2+) and triple-negative breast cancer (HR-/HER2-). The HER2+ group constitutes 10-15% of BC cases and is characterized by high HER2 expression with absence of ER and PR, having a worse prognosis compared to luminal tumors, and requiring specific drugs directed against the HER2/neu protein9.
BC development is a multi-step process, and an early diagnosis is essential for a successful treatment of the disease10. However, despite recently emerged personalized BC treatment options (e.g., endocrine and anti-HER2 antibody therapies), BC continues to challenge oncologists. Just like surgery, chemotherapy, and radiotherapy, these personalized therapies can also have severe adverse effects and patients can develop resistance to these agents, making it a long-term challenge to determine the best strategy11,12. Hence, improved understanding of the interplay between the tumor and its microenvironment is essential and expected to provide new directions for the development of novel treatments that are taking into account the specificities of the different BC subtypes13. A new wave of immunotherapies, such as antibody drug conjugates, adoptive T-cell therapies, vaccines and novel HER2-directed monoclonal antibodies (mAbs) are being studied in a broad population of patients with HER2-expressing tumors14.
Trastuzumab, for example, represents an efficient treatment modality for HER2+ BC. As part of its mode of action, Trastuzumab mediates fragment crystallizable gamma receptor (Fc&#947;R)-dependent activities. Fc&#947;Rs are distinguished by their affinity for the Fc fragment and the immune response they initiate. Activating Fc&#947;RIIIa (CD16A) on natural killer (NK) cells is crucial for mediating antibody-dependent cellular cytotoxicity (ADCC), while triggering Fc&#947;RIIa (CD32A) and Fc&#947;RIIIa on macrophages induces antibody-dependent cellular phagocytosis (ADCP)15. Studies on animal models showed that mice lacking Fc&#947;RI (CD64) and Fc&#947;RIII (CD16) receptors were unable to initiate protective immune responses against tumor-specific antigens, revealing that ADCC is likely a major mechanism of action for the mAb Trastuzumab16.
Since NK cells resort to tumor cell-bound Abs for cancer cell killing by ADCC, expression of Fc receptors is critical for an efficient treatment with Trastuzumab17 (Figure 1). Moreover, their action is efficiently balanced by a stimulation of activating and inhibitory receptors, e.g., Killer-cell immunoglobulin-like (KIR) receptors18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65922/65922fig01.jpg" /> 
Figure 1. Mechanism of ADCC in the context of an antitumor response. The Fc&#947; receptor of a natural killer (NK) cell recognizes the Fc region of an antibody, which had previously bound to a surface antigen on a cancer cell. This immunological synapse leads to the degranulation of the NK cell which releases cytotoxic mediators such as granzymes and perforin. These molecules contribute to pore formation in the cell membrane and activate apoptotic pathways causing programmed cell death of the target cell (image created with Biorender.com). <a href="https://www.jove.com/files/ftp_upload/65922/65922fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Immunotherapy&#160;development for HER2+ BC represents an evolving field. In this case, one should consider interactions between various components of the immune system. Furthermore, previous publications have extensively tested combination therapies involving all types of traditional, immune or cell therapies to identify synergizing combinations19.
Several 3D models of HER2+ BC have previously been used for drug discovery. For instance, Balalaeva et al. used SKBR-3 spheroids overexpressing HER2 to assess the cytotoxicity of the HER2-targeted immunotoxin 4D5scFv-PE4020. In another study, a 3D Matrigel-based HER2+ BC culture system was established to measure cell growth in response to Trastuzumab and endocrine agents21. These studies highlight the importance of tumor spheroid models of HER2 overexpressing cancer cells in representing an effective strategy to clinically improve therapeutic responses22.
Our group previously identified Sunitinib, a multitargeted tyrosine kinase inhibitor, as an inhibitor of Trastuzumab-dependent ADCC in JIMT-1 HER2+ BC cells in a 2D culture assay. The study revealed that Sunitinib induces autophagy and impairs NK cells killing function, downregulating HER2 expression and enhancing surface attachment of JIMT-1 cells17.
Here we established a novel 3D, spheroid ADCC model (NK.92.CD16+Trastuzumab+JIMT-1-EGFP cancer cells) to be used for high-throughput screening applications and, in order to validate the above-mentioned findings, Sunitinib was used as a model compound. First, we generated EGFP expressing JIMT-1 cells17 and grew spheroids from these cells. ADCC was induced by NK cells together with Trastuzumab, and spheroids were kept in culture in the presence or absence of test compounds for 24 h (Figure 2). Quantification of ADCC is based on the detection of apoptotic cancer cell death (Annexin V staining) using a High-Content Analysis system.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65922/65922fig02.jpg" /> 
Figure 2. ADCC in a 3D spheroid co-culture system. Our experimental settings are based on a 3D spheroid system that can more accurately model the in vivo microenvironment compared to 2D models. JIMT-1 EGFP breast cancer cells were seeded on a concave cell repellent bottom to form a round-shaped cellular cluster, called spheroid. ADCC was then initiated by adding NK92.CD16 natural killer cells (E:T ratio = 20:1) and an anti-HER2 monoclonal antibody, Trastuzumab. The experimental model has proved efficient and easily applicable for the identification of ADCC modifying test compounds (image created with Biorender.com). <a href="https://www.jove.com/files/ftp_upload/65922/65922fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We demonstrated that acquiring data in this manner can be done in real time and is statistically robust for the use in high-content screening in cancer drug discovery. Importantly, this model allows for an extended validation of a larger set of compounds, and it can be applied to several assays of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well glass bottom Cell Carrier Ultra microplates</td>
			<td>PerkinElmer, Waltham, MA, USA</td>
			<td>LLC 6055302</td>
			<td>for spheroids measurements</td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td>TPP</td>
			<td>92096</td>
			<td>for cell seeding</td>
		</tr>
		<tr>
			<td>&#945;-MEM medium</td>
			<td>Sigma</td>
			<td>M8042</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td>for spheroids seeding</td>
		</tr>
		<tr>
			<td>Annexin V-Alexa Fluo 647 conjugate</td>
			<td>Invitrogen-ThermoFisher Scientific</td>
			<td>A23204</td>
			<td>for apoptosis measurement with HCS</td>
		</tr>
		<tr>
			<td>CD16.176 V.NK-92 cells</td>
			<td>Dr. Kerry S. Campbell (the Fox Chase Cancer Center, Philadelphia, PA on behalf of Brink Biologics, Inc. San Diego, CA)</td>
			<td>ATCC CRL-2407</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Cell Tracker Blue</td>
			<td>Invitrogen-Thermo Scientific, Waltham, MA, USA)</td>
			<td>C2110</td>
			<td>for staining of NK cells</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td>in JIMT1-EGFP medium</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td>for coating HCS plate before transfering the spheroids</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>Glutamine</td>
			<td>Gibco</td>
			<td>35,050&#8211;061</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0.1</td>
			<td>GraphPad Software Inc., San Diego, CA, USA</td>
			<td></td>
			<td>for statistical analysis</td>
		</tr>
		<tr>
			<td>Harmony software</td>
			<td>PerkinElmer, Waltham, MA, USA</td>
			<td></td>
			<td>for HCA</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>Proleukin, Novartis Hung&#225;ria Kft., Budapest, Hungary</td>
			<td>PHC0026</td>
			<td>in NK medium</td>
		</tr>
		<tr>
			<td>Insulin (Humulin R)</td>
			<td>Eli Lilly</td>
			<td>HI0219</td>
			<td>JIMT-1-EGFP medium</td>
		</tr>
		<tr>
			<td>JIMT-1 breast cancer cells</td>
			<td></td>
			<td></td>
			<td>for cell culture</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>Opera Phenix High-Content Analysis equipment</td>
			<td>PerkinElmer, Waltham, MA, USA</td>
			<td>HH14001000</td>
			<td>for HCA</td>
		</tr>
		<tr>
			<td>PBS (Posphate buffered saline)</td>
			<td>Lonza</td>
			<td>BE17-517Q</td>
			<td>for washing the cells</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Biosera</td>
			<td>LM-A4118</td>
			<td>JIMT-1-EGFP and NK medium</td>
		</tr>
		<tr>
			<td>pLP-1, pLP-2, pLP-VSV-G, pWOXEGFP</td>
			<td>Invitrogen, (Prof. J&#243;zsef T<img alt="Equation 1" src="/files/ftp_upload/65922/65922eq01v2.jpg" style="margin: auto;" />zs&#233;r, University of Debrecen)</td>
			<td></td>
			<td>for JIMT-1-EGFP cell line</td>
		</tr>
		<tr>
			<td>Pluronic-F127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>for coating HCS plate before transfering the spheroids</td>
		</tr>
		<tr>
			<td>Sunitinib malate</td>
			<td>SigmaAldrich</td>
			<td>PZ0012</td>
			<td>for treatments</td>
		</tr>
		<tr>
			<td>Trastuzumab Ab (humanized anti-HER2 monoclonal antibody)</td>
			<td>Herzuma&#174;, EGIS Pharmaceuticals, Budapest, Hungary</td>
			<td>NDC-63459-303-43</td>
			<td>for treatments</td>
		</tr>
		<tr>
			<td>Trastuzumab-F(ab&#39;)2</td>
			<td>Gift from Prof. Gy&#246;rgy Vereb and &#193;rp&#225;d Sz&#246;<img alt="Equation 1" src="/files/ftp_upload/65922/65922eq01v2.jpg" style="margin: auto;" />r&#160;</td>
			<td>Department of Biophysics and Cell Biology, University of Debrecen</td>
			<td>for treatments</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4% solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1X in PBS</td>
			<td>Biosera</td>
			<td>LM-T1706</td>
			<td>for cells detachment</td>
		</tr>
	</tbody>
</table>

quantifying antibody-dependent cellular cytotoxicity in a tumor spheroid model: application for drug discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

EGFP expressing JIMT-1 cells were generated, and spheroids were grown from these cells. Sunitinib was used as a test compound as it was previously shown to affect the course of ADCC17. Spheroids were allowed to clump for 72 h. On day 3, 10 &#181;g/mL of Trastuzumab (or equimolar 6.6 &#181;g/mL TR-F(ab&#39;)219) and NK cells (20:1) were added to the spheroids in the presence or the absence of 20 &#181;M Sunitinib (1 h pre-treatment), for a total time of 24 h. JIMT...

Watch this Scientific Journal Video about Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery at JoVE.com

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

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

quantifying-antibody-dependent-cellular-cytotoxicity-tumor-spheroid

Research

JoVE Journal

Cancer Research

2.2K Views.  University of Debrecen. Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

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 ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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 ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

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 ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...