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. G., Sacchetti, B., Pierelli, L., Alimandi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR-T+cells:+the+long+and+winding+road+to+solid+tumors.">CAR-T cells: the long and winding road to solid tumors.</a> Cell Death &amp; Disease. 9 (3), 282 (2018).</li><li>Amann, 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=Development+of+an+innovative+3D+cell+culture+system+to+study+tumour--stroma+interactions+in+non-small+cell+lung+cancer+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+of+fibroblasts+induces+proinflammatory+chemokine+secretion+promoting+leukocyte+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culturing+of+the+Hodgkin+lymphoma+cell-line+L1236+induces+a+HL+tissue-like+gene+expression+pattern.">Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern.</a> Leukemia &amp; Lymphoma. 48 (10), 2042-2053 (2007).</li><li>Pickl, M., Ries, 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=Comparison+of+3D+and+2D+tumor+models+reveals+enhanced+HER2+activation+in+3D+associated+with+an+increased+response+to+trastuzumab.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+multicellular+tumor+spheroids+as+an+in+vivo-like+tumor+model+on+anticancer+drug+response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-Formation+(Colonosphere)+Assay+for+in+Vitro+Assessment+and+Expansion+of+Stem+Cells+in+Colon+Cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Metastatic+Microenvironment:+Melanoma-Microglia+Cross-Talk+Promotes+the+Malignant+Phenotype+of+Melanoma+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+Dimensional+Mixed-Cell+Spheroids+Mimic+Stroma-Mediated+Chemoresistance+and+Invasive+Migration+in+hepatocellular+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+ErbB2-CAR-engineered+cytokine-induced+killer+cells+for+the+treatment+of+high-risk+soft+tissue+sarcoma+in+children.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Content+Monitoring+of+Drug+Effects+in+a+3D+Spheroid+Model.">High-Content Monitoring of Drug Effects in a 3D Spheroid Model.</a> Frontiers in Oncolology. 7, 293 (2017).</li><li>X Tadesse, F. 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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

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 ...

This method can help to answer key question in the immunotherapy field about the assessment of immune cell cytotoxicity against 3D structure tumor cells. The main advantages of this method are that it is straightforward and it combines advanced imaging technology and a sensitive immunological assay. Begin by washing the colorectal cancer cell line of interest with five milliliters of PBS and removing the cells from the flask bottom with 0.5 milliliters of trypsin for five minutes at 37 degrees Celsius.After confirming detachment under a microscope, neutralize the cell dissociation enzyme with 10 milliliters of complete medium and collect the cells by centrifugation. Resuspend the pellet in five milliliters of fresh complete medium for counting and resuspend the cells at a 1.5 times 10 to the third cells per milliliter concentration. Then seed 200 microliters of cells into each well of 96-well round bottom plate and place the plate in an automated imaging apparatus inside a 37 degree Celsius incubator with 5%CO2 and 95%humidity.Log in to the acquisition software of the apparatus and select Schedule to Acquire, Launch Add Vessel, Scan on Schedule, and Create Vessel New. Next, select scan type Spheroid and select the appropriate bright-field and fluorescence channels of interest. Set the magnification to 10 times and select the plate model and its position within the imaging apparatus drawer.Select the position of the wells to be imaged and enter the description of the experiment including the name, type of cells, and number of cells. For the analysis setup, select Defer Analysis Until Later, right click on the timeline, and select Set Selected Scan Group Interval and set Add Scans Every to four hours and For a Total of to 24 hours. Then set the starting time to at least one hour after the incubation in the automated imaging apparatus.To check the spheroid growth progress, every two days, log in to the imaging software and select View Recent Scans. Double click on the experiment of interest and select Brightfield in the Image Channels panel. Then use the Measure Image Features tool to measure the diameter of the spheroids, adding 50 microliters of complete medium per well at day four to limit any medium evaporation effects.When the spheroids reach the appropriate experimental size, carefully angle the plate and use a multi-channel pipette to gently remove 150 microliters of complete medium from each well without disturbing the spheroids. Next, replace the discarded medium with 50 microliters of a one to 200 solution of Annexin V Red and place the plate in a cell culture incubator for 15 minutes. Centrifuge transduced CAR CD19 T cells in a 15 milliliter conical tube and resuspend the pellet in two milliliters of complete medium.After counting, dilute the cells to a two times 10 to the fifth cells per milliliter of complete medium concentration. Then add 100 microliters of CAR CD19 T cells to each spheroid-containing well and return the plate to the automated imaging apparatus. In the acquisition software, select Schedule to Acquire and right click on the scan timeline.Select Edit Timeline and right click on the scan group to delete it. Right click on the timeline again and select Set Selected Scan Group Interval and set Add Scans Every to 1 1/2 hours and For a Total of to 24 hours. Then set the imaging start time to at least one hour after the start of the incubation in the automated imaging apparatus and select Save Schedule Scans.For automated image analysis, in the scan software, select View Recent Scans and Launch Analysis. Select Create New Analysis Definition and analysis type Spheroid and set the image channels to be analyzed. Select at least 10 representative images and preview the default analyze procedure on the entire image stack.Modify the parameters for the bright-field mask and preview the image stack, confirming that the selected parameters detect the spheroids accurately. Modify the parameters for green mask and preview the image stack to confirm that the selected parameters detect the spheroids accurately. Modify the parameters for red mask and preview the image stack to confirm that the selected parameters detect the spheroids accurately.Take care to achieve the best signal over noise and sensitivity over specificity ratios, keeping in mind that you won't be able to find a set of parameters that will permit to capture every event at every time point. Then launch the analyzer. When the analysis is complete, extract the measurement of interest and select the analyzed file and the graph metrics option.Select the metrics of interest, the scan, and the well. Click Export Data to extract the selected metrics in several file formats. Then, to extract the images, select the analyzed file and select Export Images and Movies.CD19 CAR expression on transduced T cells and CD19 expression on transduced colorectal cancer cells can be confirmed by flow cytometry. Here, the outcome of a typical spheroid experiment can be observed. Unlike mock T cells, CD19 CAR T cells are able to specifically kill the colorectal cancer cell line-derived spheroids, greatly diminishing the number of live tumor cells.Tracking of the evolution of the total green and red signals within the spheroid boundaries over time reveals that shortly after CD19 CAR T cell injection, the size of the spheroids shrinks quickly as the apoptosis signal increases quickly. Following this procedure, other methods like toxicity assays, cell migration assays, or spheroid structure measurements can be performed to answer additional questions about drug screening, T cell motility, or spheroid formation, respectively.

Research

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Cancer Research

15.0K Views.  Oslo University Hospital-Radiumhospitalet. This method can help to answer key question in the immunotherapy field about the assessment of immune cell cytotoxicity against 3D structure tumor cells. The main advantages of this method are that it is straightforward and it combines advanced imaging technology and a sensitive immunological assay. Begin by washing the colorectal cancer cell line of interest with five milliliters of PBS and removing the cells from the flask bottom with 0.5 milliliters of trypsin for five minutes at 37 degree...