Name: A Spheroid Killing Assay by CAR T Cells (Video) | JoVE Journal
Uploaded: 2018-12-12T13:00:00.000+00:00
Duration: 8 min 19 s
Description: Learn a spheroid killing assay to evaluate CAR T-cell cytotoxicity against 3D tumor models using real-time imaging. This method combines advanced imaging with sensitive immunological assays for immunotherapy research.

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5 minutes

This work was supported by the Norwegian Research Council under Grants #244388, #254817 and #284983; the Norwegian Cancer Society (#6829007); The Norwegian Health Region South East under Grant #17/00264-6 and #2016006.

1. Generation of Spheroids from Colorectal Cancer Cell Line
<ol>
	<li>Wash HCT 116 (stably transduced to express Cluster of Differentiation 19 (CD19) and Green Fluorescence Protein (GFP)) cell monolayers with phosphate buffered saline (PBS; 5 mL for a 25 cm2 or 10 mL for a 75 cm2 flask). Add trypsin (0.5 mL for a 25 cm2 or 1 mL for a 75 cm2 flask) and incubate cells at 37 &#176;C for 5 min.</li>
	<li>Check cell detachment under a microscope and neutralize cell dissociation enzyme with complete Roswell Park Memorial Institute 160 medium (RPMI 1640) (RPMI 1640 + 10% FCS + Gentamycin; 10 mL for a 25 cm2 or 20 mL for a 75 cm2 flask).</li>
	<li>Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend by pipetting up and down several times with 5 mL of complete RPM1 1640 medium.</li>
	<li>Count cells using Trypan blue exclusion on a compatible cell counter.</li>
	<li>Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend in RPMI medium to obtain 5 x 103 cells/mL.</li>
	<li>Coat a 96-well round bottom plate with 100 &#181;L/well of 100 &#181;g/mL of poly-L-lysine (PLL) in PBS during 1 hour at room temperature. Wash twice with PBS and let the plate dry.</li>
	<li>Transfer the cell suspension to a sterile reservoir and dispense 200 &#181;L/well into the PLL-coated&#160;96-well round bottom plates using a multichannel pipette.</li>
	<li>Transfer the plate to the automated imaging apparatus inside an incubator (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Log into the acquisition software, select Schedule To Acquire | Launch Add Vessel | Scan On Schedule | Create Vessel: New.</li>
	<li>Select Scan Type: Spheroid. Select the channels of interest: Phase + Brightfield (to follow spheroids growth), Green (to follow tumor signal, acquisition time 300 ms) and Red (to follow apoptosis, acquisition time 400 ms).
	<ol>
		<li>Select the desired magnification: 10x.</li>
		<li>Pick the plate model and its position in the drawer. Select the position of wells to image. Enter the description of the experiment: name, type of cells, number of cells.</li>
	</ol>
	</li>
	<li>For the analysis setup, select Defer Analysis Until Later. Right click on the timeline and select Set Selected Scan Group Interval option and set Add scans every to 4 h and For a total of to 24 h. Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus).</li>
	<li>Check every 2 days for the growth of spheroids by logging into the imaging software.
	<ol>
		<li>Pick the View Recent Scans option and double-click on the desired experiment. Select Brightfield in the image channels panel and then use the Measure image features tool to measure the diameter of the spheroids. It takes 6 days for a spheroid to reach the desired size: 0.5 mm of diameter. Add 50 &#181;L of complete RPMI medium per well at day 4 to limit medium evaporation effect.</li>
	</ol>
	</li>
</ol>
2. Generation of CD19 CAR T cells
<ol>
	<li>Expansion of CD19 CAR T cells 
	NOTE: Stable expression of CD19 CAR T cells was acquired by bulk retroviral transduction of the healthy donor PBMCs as previously described16. The retroviral construct coding for CD19 CAR is a second-generation CAR and consists of fmc63 scFv chain, CD8 hinge and transmembrane domain, a 4-1BB co-stimulatory domain and finally a CD3&#950; domain.
	<ol>
		<li>To expand, culture the transduced T cells in the presence of anti-CD3/28 magnetic beads with a cell to bead ratio of 1:1 for 10&#8211;11 days. During the expansion, cells are in complete medium (X-VIVO 15, 5% Serum Replacement, and 100 U/mL recombinant human IL-2). 
		NOTE: The ideal density for an efficient expansion is 1 to 2 x 106 cells/mL. Depending on the initial number, cells can be expanded in flasks (25 cm2 flasks to 20 mL, 75 cm2 flasks to 40 mL of total volume) in a cell culture incubator (37 &#176;C, 5% CO2, 95% humidity).</li>
		<li>As a negative control group for the following assays, include non-transduced PBMCs (will be referred as Mock) to the expansion protocol parallel to the CD19 CAR T cells.</li>
		<li>On day 3 and onwards, add fresh medium every day and divide the cells into more culture flasks if necessary.</li>
		<li>On day 10&#8211;11, centrifuge cells at 500 x g for 5 min. Remove supernatant and combine all the cells into one 50 mL tube and resuspend the cells in ~30 mL of fresh complete media.</li>
		<li>Place the 50 mL tube containing the resuspended cells on a magnetic stand to separate the magnetic beads from the culture medium.</li>
		<li>Wait for 2&#8211;3 min for the beads to collect on the side of the tube.</li>
		<li>Remove the culture medium with a pipette and transfer to a new tube without touching the magnetic bead collection zones.</li>
		<li>Repeat steps regarding the bead removal (2.1.5&#8211;2.1.7) once more to limit the number of beads in the final culture medium.</li>
		<li>Resuspend and count the cells, adjust the density to 1 to 2 x 106 cells/mL in complete medium.</li>
		<li>Rest the cells for at least 4 h up to overnight. Then directly freeze them down at -80 &#176;C and transfer the vials to a liquid nitrogen tank on the following day for long-term storage. Alternatively, one can prolong the rest up to overnight for immediate use.</li>
	</ol>
	</li>
	<li>CD19 CAR expression control on primary T cells
	<ol>
		<li>Count the number of expanded T cells as the numbers might vary slightly after overnight culture or moderately after freeze/thaw.</li>
		<li>Transfer 5 x 105 cells from both CD19 CAR and Mock primary T cells to separate flow cytometry tubes.</li>
		<li>Wash the cells with 200 &#956;L of Flow Buffer (2% FBS in PBS) and centrifuge the tubes at 500 x g for 5 min. Repeat the washing steps to get rid of any artifacts caused by the culture medium.</li>
		<li>Prepare the primary antibody (Biotin Goat Anti-Mouse IgG, F(ab&#39;)&#8322; Fragment Specific) by performing 1:200 dilution in Flow Buffer.</li>
		<li>Resuspend the cells in 100 &#956;L of antibody mix per tube and incubate on ice for 15 min. Repeat the previous washing step twice to remove excess antibody.</li>
		<li>Prepare the secondary antibody (Streptavidin-PE) as 1:400 dilution in Flow Buffer.</li>
		<li>Resuspend the cells in 100 &#956;L of antibody mix per tube and incubate on ice for 15 min. Repeat the previous washing step twice to get rid of excess antibody.</li>
		<li>Resuspend the cells in 200 &#956;L of Flow Buffer per tube and analyze it on a flow cytometer.</li>
		<li>Use Mock T cells to set up the negative and positive gate and analyze CD19 CAR transduced T cells accordingly.</li>
	</ol>
	</li>
</ol>
3. 3D Tumor Spheroid Killing Assay
<ol>
	<li>After 6 days or once spheroids reach the desired size, remove the plate from the incubator. Using a multichannel pipette, gently remove 100 &#181;L/well of complete RMPI 1640 medium from the spheroid plates.
	<ol>
		<li>For this step, angle the tips towards the inside wall of the 96-wells plate, avoiding contact with the bottom of the well in order to minimize disturbance of the spheroids. Remaining volume should be around 100 &#181;L.</li>
	</ol>
	</li>
	<li>Prepare a 1:200 solution of Annexin V red by mixing 50 &#181;L of Annexin V red with 9.95 mL of complete RPMI 1640 medium.</li>
	<li>Add 100 &#181;L/well of the 1:200 Annexin V red solution.</li>
	<li>Transfer the plate to an incubator (37&#176;C, 5% CO2, 95% humidity) for 15 min.</li>
	<li>Harvest the transduced CAR CD19 T cells in a 15 mL tube and centrifuge them at 500 x g for 5 min. Remove the supernatant using a pipette and resuspend by pipetting up and down several times with 2 mL of complete RPM1 1640 medium.</li>
	<li>Count cells using Trypan blue exclusion on a compatible cell counter.</li>
	<li>Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend in RPMI medium to obtain 2 x 105 cells/mL.</li>
	<li>Transfer the cell suspension to a sterile reservoir and dispense 100 &#181;L/well into a 96-well round bottom spheroid plate using a multichannel pipette.</li>
	<li>Transfer the plate back to the automated imaging apparatus inside an incubator (37 &#176;C, 5% CO2, 95% humidity).</li>
	<li>Log into the acquisition software, select Schedule To Acquire.
	<ol>
		<li>Right-click on the Scan timeline and select Edit Timeline. Right-click on the scan group and delete it. Right-click on the timeline and select Set Selected Scan Group Interval option and set Add scans every to 1.5 h and For a total of to 24 h.</li>
		<li>Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus). Select Save schedule scans.</li>
	</ol>
	</li>
</ol>
4. Automated Image Analysis
<ol>
	<li>Log into acquisition, pick the View Recent Scans option and select Launch Analysis option. Select Create New Analysis Definition | Analysis Type: Spheroid. Tick the Image channels to analyze (Phase + Brightfield, Green and Red).</li>
	<li>Select at least 10 representative images: typically, 1 per condition and at least 3 time points (beginning, middle and end of the acquisition).</li>
	<li>Preview the default analyze procedure on the whole image stack.</li>
	<li>Modify the parameters for brightfield mask. Typical parameters are: Sensitivity 10, Hole fill 1,000 &#181;m2, min area 1,000 &#181;m2. Preview on the whole image stack and check that the selected parameters detect the spheroids accurately.</li>
	<li>Modify the parameters for green mask (GFP). Typical parameters are: Top hat segmentation with radius 200 &#181;m and threshold 3 GCU, Edge split off, Hole fill 5,000 &#181;m2, Adjust size -2 pixels, Area min 3,000 &#181;m2. Preview on the whole image stack and check that the selected parameters detect the spheroids accurately.</li>
	<li>Modify the parameters for red mask (Annexin V). Typical parameters are: Top hat segmentation with radius 150 &#181;m and threshold 2 GCU, Edge split off, Hole fill 5,000 &#181;m2, Adjust size 0 pixels, Area min 1,000 &#181;m2. Preview on the whole image stack and check that the selected parameters detect the spheroids accurately.</li>
	<li>Launch the analyzer.
	<ol>
		<li>Once the analysis is done, extract the measurement of interest. Select the analyzed file and then the Graph Metrics option. Select the metrics of interest, the scan and the well. Typically, total red and green intensity within brightfield boundaries give the most accurate measurements by restricting the signal of fluorescence to the spheroids boundaries determined by the brightfield mask (Figure 3). Extract selected metrics in several file format by clicking on &#8220;Export Data&#8221;.</li>
	</ol>
	</li>
	<li>Proceed to the extraction of the images and movies by selecting the analyzed file and then select the Export Images and Movies option. Two options are available, either &#8220;As Displayed&#8221; to retrieve images and movies as seen on the imaging software (usually composite images), either &#8220;As Stored&#8221; to retrieve raw data for external analysis through third-part software.</li>
</ol>

<ol><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chimeric+antigen+receptor-modified+T+cells+in+chronic+lymphoid+leukemia%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.</a> New England Journal of Medicine. 365 (8), 725-733 (2011).</li>
<li>Kochenderfer, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eradication+of+B-lineage+cells+and+regression+of+lymphoma+in+a+patient+treated+with+autologous+T+cells+genetically+engineered+to+recognize+CD19%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.</a> Blood. 116 (20), 4099-4102 (2010).</li>
<li>Brentjens, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD19-targeted+T+cells+rapidly+induce+molecular+remissions+in+adults+with+chemotherapy-refractory+acute+lymphoblastic+leukemia%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.</a> Science Translational Medicine. 5 (177), 177ra138(2013).</li>
<li>Grupp, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.</a> New England Journal of Medicine. 368 (16), 1509-1518 (2013).</li>
<li>D'Aloia, M. M., Zizzari, I. G., Sacchetti, B., Pierelli, L., Alimandi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR-T+cells%3A+the+long+and+winding+road+to+solid+tumors%5BTitle%5D)+AND+%22Cell+Death+%26+Disease%22%5BJournal%5D)">CAR-T cells: the long and winding road to solid tumors.</a> Cell Death & Disease. 9 (3), 282(2018).</li>
<li>Amann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+innovative+3D+cell+culture+system+to+study+tumour--stroma+interactions+in+non-small+cell+lung+cancer+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells.</a> PLoS One. 9 (3), e92511(2014).</li>
<li>Enzerink, A., Salmenpera, P., Kankuri, E., Vaheri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clustering+of+fibroblasts+induces+proinflammatory+chemokine+secretion+promoting+leukocyte+migration%5BTitle%5D)+AND+%22Molecular+Immunology%22%5BJournal%5D)">Clustering of fibroblasts induces proinflammatory chemokine secretion promoting leukocyte migration.</a> Molecular Immunology. 46 (8-9), 1787-1795 (2009).</li>
<li>Birgersdotter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+culturing+of+the+Hodgkin+lymphoma+cell-line+L1236+induces+a+HL+tissue-like+gene+expression+pattern%5BTitle%5D)+AND+%22Leukemia+%26+Lymphoma%22%5BJournal%5D)">Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern.</a> Leukemia & Lymphoma. 48 (10), 2042-2053 (2007).</li>
<li>Pickl, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+3D+and+2D+tumor+models+reveals+enhanced+HER2+activation+in+3D+associated+with+an+increased+response+to+trastuzumab%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab.</a> Oncogene. 28 (3), 461-468 (2009).</li>
<li>Galateanu, B., et al. <a target="_blank" href="https://www.spandidos-publications.com/10.3892/ijo.2016.3467?text=abstract">Impact of multicellular tumor spheroids as an in vivo-like tumor model on anticancer drug response.</a> International Journal of Oncology. 48 (6), 2295-2302 (2016).</li>
<li>Shaheen, S., Ahmed, M., Lorenzi, F., Nateri, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spheroid-Formation+%28Colonosphere%29+Assay+for+in+Vitro+Assessment+and+Expansion+of+Stem+Cells+in+Colon+Cancer%5BTitle%5D)+AND+%22Stem+Cell+Reviews+and+Reports%22%5BJournal%5D)">Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer.</a> Stem Cell Reviews and Reports. 12 (4), 492-499 (2016).</li>
<li>Izraely, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Metastatic+Microenvironment%3A+Melanoma-Microglia+Cross-Talk+Promotes+the+Malignant+Phenotype+of+Melanoma+Cells%5BTitle%5D)+AND+%22International+Journal+of+Cancer%22%5BJournal%5D)">The Metastatic Microenvironment: Melanoma-Microglia Cross-Talk Promotes the Malignant Phenotype of Melanoma Cells.</a> International Journal of Cancer. , (2018).</li>
<li>Khawar, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+Dimensional+Mixed-Cell+Spheroids+Mimic+Stroma-Mediated+Chemoresistance+and+Invasive+Migration+in+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Neoplasia%22%5BJournal%5D)">Three Dimensional Mixed-Cell Spheroids Mimic Stroma-Mediated Chemoresistance and Invasive Migration in hepatocellular carcinoma.</a> Neoplasia. 20 (8), 800-812 (2018).</li>
<li>Merker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+and+characterization+of+ErbB2-CAR-engineered+cytokine-induced+killer+cells+for+the+treatment+of+high-risk+soft+tissue+sarcoma+in+children%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children.</a> Oncotarget. 8 (39), 66137-66153 (2017).</li>
<li>Mittler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Content+Monitoring+of+Drug+Effects+in+a+3D+Spheroid+Model%5BTitle%5D)+AND+%22Frontiers+in+Oncolology%22%5BJournal%5D)">High-Content Monitoring of Drug Effects in a 3D Spheroid Model.</a> Frontiers in Oncolology. 7, 293(2017).</li>
<li>X Tadesse, F. G., Mensali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unpredicted+phenotypes+of+two+mutants+of+the+TcR+DMF5%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Unpredicted phenotypes of two mutants of the TcR DMF5.</a> Journal of Immunological Methods. 425, 37-44 (2015).</li>
</ol>

The authors have nothing to disclose.

The use of spheroids as an innovative tool to validate future cancer treatment has become a field of growing interest in the past years. Spheroids represent an intermediate step between classical 2D in vitro analysis and in vivo assessment. The method further holds a lot of promise regarding their potency in terms of tumor micro-environment mimicking as well as gene profiling7. The protocol presented in this publication was adapted from Saheen et al.11 to the Incuc...

Adoptive cell transfer (ACT) represents the next generation cancer treatment. It relies on the injection of effector cells (T- or NK-cells) into a patient. These cells can be genetically modified with a receptor that will guide them to their target, the tumor, and destroy it. Recently this approach was shown to be feasible when a Chimeric Antigen Receptor (CAR) directed against the B-cell marker CD19 was introduced into the patient T cells to kill his/her cancer1. In the case of CAR, which is an artificial receptor, the design consists of specific antibody fragments, the antigen binding domain reduced to an entity designated single chain variable fragment (scFv), linked to the T-cell signaling domains. Although there are several designs, the most commonly used versions referred to as second-generation CAR designs, consist of CD3z for TCR signaling and one co-stimulatory domain (CD28, 4-1BB, OX40, etc.)1,2. The immunotherapy field directed most of its attention to this new form of ACT when CD19 CAR-T cells efficaciously treated numerous patients with B cell malignancies3,4. Following this success, researchers tried to exploit the similar designs by targeting other epitopes for solid tumors with limited success. Unfortunately, the scarcity of tumor specific antigens and the harsher tumor microenvironments rendered CAR T cells less effective towards solid tumors5.
Currently, the most commonly used in vitro validation strategies rely on two-dimensional (2D) systems that only address a fragment of the already mentioned solid tumor challenges. Classically, 2D in vitro systems involve a mixture of CAR T cells and target cancer cell lines as monolayers to assess the functionality and specificity of these effector cells. Although these strategies are important and vital parts of the studies, they do not take into consideration the complex morphology and three-dimensional (3D) structure of the cancer cells6. Cancer cells cultured in 3D systems, referred to as spheroids, acquire new phenotypic traits through changes in gene expression profile7, which might influence the recognition by redirected effector cells. Birgersdotter and colleagues demonstrated that a Hodgkin lymphoma (HL) cell line when only grown in a 3D culture model acquires a gene expression profile that is similar to primary tumor samples8. Therefore, spheroids or similar 3D culture methodologies offer more relevant in vitro models as opposed to standard 2D systems. Such systems are also similar to in vivo studies which are seen as the final step in the validation process of a given CAR. Considering that 2D systems fail to mimic the morphology of cancer clusters, spheroids offer similar formations to assess the functionality of CAR T cells prior to in vivo models. In one study, Pickl et al. identified that a spheroid model of human epidermal growth factor receptor (HER2) overexpressing cancer cells demonstrated similar signaling profiles to in vivo models9. This further supports that spheroids offer more relevant and close-to-in vivo assessment of the CAR T cells. Additionally, CAR T-cell validation against spheroids might help assess their efficacy more critically and prevent some of the designs from moving to in vivo studies prematurely10; thus, contributing to ethically concerned research by sacrificing fewer animals. Moreover, protocols using spheroids are not more expensive than classical 2D systems and much faster as compared to classical in vivo studies. Taken together, one can predict that the inclusion of spheroid studies will soon become standard practice to link in vitro and in vivo studies.
Here, we present the preparation of spheroids from the colon cancer cell line HCT 116. This cell line was modified to express the human CD19 molecule to render it sensitive to CD19 CAR T cells and to provide a clear assessment of the killing using a clinically validated CAR construct.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>SIGMA-ALDRICH</td><td>D8537-500ML</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; RNBG7037</td></tr><tr><td>75 cm&amp;sup2; growth area flasks</td><td>VWR</td><td>430639</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 2218002</td></tr><tr><td>75 cm&amp;sup2; growth area flasks</td><td>VWR</td><td>734-2705</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 3718006</td></tr><tr><td>Trypsin-EDTA</td><td>SIGM-ALDRICH</td><td>T3924-100ml</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; SLBTO777</td></tr><tr><td>RPMI 1640 med L-glutamin, 10 x 500 mL</td><td>Life Technology (Gibco)</td><td>21875-091</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 1926384</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10500064</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 08Q3066K</td></tr><tr><td>Gentamicin</td><td>Thermo Fischer</td><td>15750060</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 1904924A</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fischer</td><td>15250061</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 1886513</td></tr><tr><td>96 well plate, round bottom</td><td>VWR</td><td>734-1797</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 33117036</td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Thermo Fischer</td><td>11132D</td><td>-</td></tr><tr><td>X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L</td><td>BioNordika</td><td>BE02-060Q</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 8MB036</td></tr><tr><td>CTS Immune Cell Serum Replacement</td><td>Thermo Fischer</td><td>A2596102</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 1939319</td></tr><tr><td>IL-2 Proleukin</td><td>Novartis</td><td></td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 505938M</td></tr><tr><td>IncuCyte Annexin V Red Reagent</td><td>Essen Bioscience</td><td>4641</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 17A1025-122117</td></tr><tr><td>Reagent Reservoir</td><td>VWR</td><td>89094-672</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 89094-672</td></tr><tr><td>15 mL tubes</td><td>VWR</td><td>734-1867</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 19317044</td></tr><tr><td>anti-human CD19-PE</td><td>BD Biosciences</td><td>555413</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 4016990&lt;br/&gt; &lt;strong&gt;RRID:&lt;/strong&gt; AB_395813</td></tr><tr><td>Biotin-SP (long spacer) AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115-066-072</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 129474:&lt;br/&gt; &lt;strong&gt;RRID:&lt;/strong&gt; AB_2338583</td></tr><tr><td>Streptavidin-PE</td><td>BD Biosciences</td><td>554061</td><td>&lt;strong&gt;Lot Number:&lt;/strong&gt; 5191579:&lt;br/&gt; &lt;strong&gt;RRID:&lt;/strong&gt; AB_10053328</td></tr><tr><td>HCT 116 Colorectal Carcinoma Line</td><td>ATCC</td><td>CCL-247</td><td>-</td></tr><tr><td>Incucyte S3</td><td>Essen Bioscience</td><td></td><td></td></tr></tbody></table>

a spheroid killing assay by car t cells

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.

As can be seen in Figure 1, it is crucial to check by flow cytometry the level of expression of CD19 CAR on T cells (Figure 1A) and the level of CD19 on HCT116 tumor cell lines (Figure 1B). Figure 2 exemplifies the outcome of a typical spheroid experiment. The automated imaging apparatus takes pictures in four different channels: bright field, phase, green and red fluore...

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 ...

Research

JoVE Journal

Cancer Research

Spheroids are a type of 3D cell culture model used to more accurately represent a cell’s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids’ growth.

Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

In this video, we demonstrate the repetitive tumor challenge assay to evaluate the CAR T cells’ repetitive tumor killing potential during high tumor cell loads. Further, the CAR T cells can be analyzed using flow cytometry to determine the different subpopulations of CAR T cells in the sample.

Repetitive Tumor Challenge Assay: An In Vitro Culture Assay to Evaluate Chimeric Antigen Receptor (CAR) T Cells for Repetitive Tumor Killing Potential

This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic capabilities.

A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

This video demonstrates an in vitro assay evaluating the cytotoxic effects of CAR T cells on 3D tumor spheroids expressing fluorescent proteins. This assessment utilizes the Annexin V red fluorescent probe, which binds to exposed phosphatidylserine residues on the outer leaflet of apoptotic cancer cells after interactions with CAR T cells. The red fluorescence emitted by the probe aids in distinguishing these cells from healthy ones emitting green fluorescence.

An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids

This video demonstrates an in vitro assay to validate chimeric antigen receptor (CAR) T cell activity. B-cell lymphoma cells expressing CD19 antigen and cytoplasmic luciferase are co-cultured with CD19-specific CAR T cells. CAR binding to CD19 on a tumor induces the CAR T cells to release cytokines and toxins that cause tumor cell cytotoxicity. The CAR T cell activity is evaluated by quantifying the released cytokine and decreased luciferase-mediated bioluminescence in the tumor cells.

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

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.

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

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

Learn a spheroid killing assay to evaluate CAR T-cell cytotoxicity against 3D tumor models using real-time imaging. This method combines advanced imaging with sensitive immunological assays for immunotherapy research.

A Spheroid Killing Assay by CAR T Cells

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