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Introduction

Protocol

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Acknowledgements

Materials

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Cancer Research

A Spheroid Killing Assay by CAR T Cells

Published: December 12th, 2018

DOI:

10.3791/58785

1Department of Cellular Therapy, Department of Oncology, Oslo University Hospital-Radiumhospitalet

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 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.

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 variab....

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1. Generation of Spheroids from Colorectal Cancer Cell Line

  1. 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 °C for 5 min.
  2. Check cell detachment under a microscope and neutralize cell dissociation en.......

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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.......

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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.......

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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.

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NameCompanyCatalog NumberComments
Dulbecco’s Phosphate Buffered SalineSIGMA-ALDRICHD8537-500MLLot Number: RNBG7037
75 cm² growth area flasksVWR430639Lot Number: 2218002
75 cm² growth area flasksVWR734-2705Lot Number: 3718006
Trypsin-EDTASIGM-ALDRICHT3924-100mlLot Number: SLBTO777
RPMI 1640 med L-glutamin, 10 x 500 mlLife Technology (Gibco)21875-091Lot Number: 1926384
Fetal Bovine SerumGibco10500064Lot Number: 08Q3066K
GentamicinThermo Fischer15750060Lot Number: 1904924A
Trypan Blue Solution, 0.4%Thermo Fischer15250061Lot Number: 1886513
96 well plate, round bottomVWR734-1797Lot Number: 33117036
Dynabeads Human T-Activator CD3/CD28Thermo Fischer11132D-
X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 LBioNordikaBE02-060QLot Number: 8MB036
CTS Immune Cell Serum ReplacementThermo FischerA2596102Lot Number: 1939319
IL-2 ProleukinNovartisLot Number: 505938M
IncuCyte Annexin V Red ReagentEssen Bioscience4641Lot Number: 17A1025-122117
Reagent ReservoirVWR89094-672Lot Number: 89094-672
15 ml tubesVWR734-1867Lot Number: 19317044
anti-human CD19-PEBD Biosciences555413Lot Number: 4016990
RRID: AB_395813
Biotin-SP (long spacer) AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgGJackson ImmunoResearch115-066-072Lot Number: 129474:
RRID: AB_2338583
Streptavidin-PEBD Biosciences554061Lot Number: 5191579:
RRID: AB_10053328
HCT 116 Colorectal Carcinoma LineATCCCCL-247-
Incucyte S3Essen Bioscience

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