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. 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. G., Mensali, 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=Unpredicted+phenotypes+of+two+mutants+of+the+TcR+DMF5.">Unpredicted phenotypes of two mutants of the TcR DMF5.</a> Journal of Immunological Methods. 425, 37-44 (2015).</li></ol>

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>
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			<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

JoVE Journal

Cancer Research

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

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

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

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

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

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

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

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 ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...