Name: a spheroid killing assay by car t cells
Uploaded: 2018-12-12T13:00:00
Description: Watch this Scientific Journal Video about A Spheroid Killing Assay by CAR T Cells at JoVE.com

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 en...

<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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+in+chronic+lymphoid+leukemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+B-lineage+cells+and+regression+of+lymphoma+in+a+patient+treated+with+autologous+T+cells+genetically+engineered+to+recognize+CD19.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+T+cells+rapidly+induce+molecular+remissions+in+adults+with+chemotherapy-refractory+acute+lymphoblastic+leukemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia.">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. 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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&#8217;s Phosphate Buffered Saline</td>
			<td>SIGMA-ALDRICH</td>
			<td>D8537-500ML</td>
			<td>Lot Number: RNBG7037</td>
		</tr>
		<tr>
			<td>75 cm&#178; growth area flasks</td>
			<td>VWR</td>
			<td>430639</td>
			<td>Lot Number: 2218002</td>
		</tr>
		<tr>
			<td>75 cm&#178; growth area flasks</td>
			<td>VWR</td>
			<td>734-2705</td>
			<td>Lot Number: 3718006</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>SIGM-ALDRICH</td>
			<td>T3924-100ml</td>
			<td>Lot Number: 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>Lot Number: 1926384</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td>Lot Number: 08Q3066K</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fischer</td>
			<td>15750060</td>
			<td>Lot Number: 1904924A</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Fischer</td>
			<td>15250061</td>
			<td>Lot Number: 1886513</td>
		</tr>
		<tr>
			<td>96 well plate, round bottom</td>
			<td>VWR</td>
			<td>734-1797</td>
			<td>Lot Number: 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>Lot Number: 8MB036</td>
		</tr>
		<tr>
			<td>CTS Immune Cell Serum Replacement</td>
			<td>Thermo Fischer</td>
			<td>A2596102</td>
			<td>Lot Number: 1939319</td>
		</tr>
		<tr>
			<td>IL-2 Proleukin</td>
			<td>Novartis</td>
			<td></td>
			<td>Lot Number: 505938M</td>
		</tr>
		<tr>
			<td>IncuCyte Annexin V Red Reagent</td>
			<td>Essen Bioscience</td>
			<td>4641</td>
			<td>Lot Number: 17A1025-122117</td>
		</tr>
		<tr>
			<td>Reagent Reservoir</td>
			<td>VWR</td>
			<td>89094-672</td>
			<td>Lot Number: 89094-672</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>VWR</td>
			<td>734-1867</td>
			<td>Lot Number: 19317044</td>
		</tr>
		<tr>
			<td>anti-human CD19-PE</td>
			<td>BD Biosciences</td>
			<td>555413</td>
			<td>Lot Number: 4016990 
			RRID: AB_395813</td>
		</tr>
		<tr>
			<td>Biotin-SP (long spacer) AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-066-072</td>
			<td>Lot Number: 129474: 
			RRID: AB_2338583</td>
		</tr>
		<tr>
			<td>Streptavidin-PE</td>
			<td>BD Biosciences</td>
			<td>554061</td>
			<td>Lot Number: 5191579: 
			RRID: 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

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...

Watch this Scientific Journal Video about A Spheroid Killing Assay by CAR T Cells at JoVE.com

A Spheroid Killing Assay by CAR T Cells (Video) | JoVE

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

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 ...

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