In vitro evaluation of CAR-T cells in patient-derived glioblastoma models.

Advances in chimeric antigen receptor (CAR) T cell therapies have led to the modality dominating translational cancer research; however, a standardized protocol for evaluating such therapies in vitro is needed. This protocol details the in vitro preclinical evaluation of CAR-T cell therapies for glioblastoma (GBM), including target cell cytotoxicity and T cell proliferation, activation, and cytokine release assays. For complete details on the use and execution of this protocol, please refer to Vora et al. (2020).

Before you begin

GBM tumor samples are dissociated and propagated as described previously Chokshi et al. (2020). Please refer to Tatari et al. (2020) for methods detailing lentiviral production and generation of CAR-T cells.

For all assays, tumor antigen-targeting CAR-T cells and control CAR-T cells are needed. Appropriate control CAR-T cells should not target any antigen expressed on target cells.

Generation of GBM cells expressing firefly luciferase

Timing: 2–3 days

Plate a single-cell suspension of 150,000 GBM cells in 250 μL of Neurocult Complete (NCC) media per well in a 24 well plate. Refer to materials and equipment for details on NCC media.

Carefully add luciferase lentivirus at an MOI of 1 in 50 μL NCC per well. Incubate cells at 37°C, 5% CO2 for 24 h.

Collect cells, centrifuge at 300 × g for 5 min at 20°C–22°C and discard supernatant. Resuspend cells in 2 mL of warm NCC media supplemented with a compatible antibiotic at the minimum inhibitory concentration. Plate cells back into the same wells and incubate at 37°C, 5% CO2 for 48–72 h depending on the antibiotic being used.

To determine the minimum inhibitory concentration for a given antibiotic, perform a dose-response experiment with antibiotic concentrations ranging from 0.5 to 2 μg/mL over 48–72 hours. Duration of antibiotic treatment is specific to the type of antibiotic.

Collect cells, centrifuge at 300 × g for 5 min at 20°C–22°C and discard supernatant. Resuspend cell pellet in 250 μL of TrypLE and incubate at 37°C for 2–5 min until solution contains no visible clumps. If needed, use a pipette to aid in dissociation.

Add 4 mL of PBS and centrifuge cells at 300 × g for 5 min at 20°C–22°C. Discard supernatant.

Resuspend cells in 1 mL of PBS, filter cell suspension through a 35 μm cell strainer cap and determine live cell concentration of each cell solution using Trypan Blue solution.

Key resources table

Materials and equipment

Step-by-step method details

Preparation of CAR-T cells

Timing: 10 min

Warm required amounts of basal XSFM media at 37°C.

Collect CAR-T cells (culture day 12–15) and centrifuge at 450 × g for 5 min at 20°C–22°C.

Aspirate supernatant and resuspend cell pellet in 1 mL of warmed basal XSFM.

Filter the cell suspension into a 12 × 75 mm tube with 35 μm cell strainer cap.

Determine cell number and viability using Trypan Blue solution, and adjust cell count as required for specific assays.

Cytotoxicity assay

Timing: 5–9 h

This assay determines the cytotoxicity of CAR-T cells against target cells using a bioluminescence-based measure of target cell lysis.

This protocol was adapted from Karimi et al. (2014) and performed in Vora et al. (2020).

Preparing target cells (GBM cells)

Prepare luciferase-expressing target cells at a concentration of 3 × 105 live cells/mL of warmed basal XSFM with 75 μg/mL D-firefly luciferin potassium salt.

Add 100 μL of cell mixture per well in a tissue culture-treated, flat-bottom 96-well plate for a total of 3 × 104 live cells per well, plating 6 wells per effector: target (E:T) ratio.

Cells must also be plated for a lysis control and media-only control. Each sample and control should be run in triplicates (Figure 1). The media-only control adjusts luminescence readings for background signal and provides a reference of spontaneous death occurring in the target cells in the absence of effector cells, while the lysis control provides a reference of maximal target cell killing through the addition of a lysis buffer.

Figure 1

Layout for cytotoxicity assay with E:T ratios from 1:1 to 4:1

Red and green represent wells in which CAR-T cells and control CAR-T cells are added, respectively. Yellow represents wells in which target cells are added without effector cells.

For all E:T ratios, target cell count remains constant while CAR-T cell count is adjusted. E:T ratios typically range from 1:1 to 10:1.

Read the plate with a luminometer (measured as relative luminescence units or RLU) to ensure equal distribution of target cells. Luminometer (FLUOstar Omega, BMG Labtech) settings should be as follows: bottom optic read, gain: 3600, time: 10 s.

Luminescence values should be within a small range of variability (<10%).

Preparing CAR-T cells

Adjust the CAR-T cell concentrations to 3×105 CAR-T cells/mL basal XSFM with 75 μg/mL D-firefly luciferin potassium salt. Plate 100 μL of cell mixture per well for a 1:1 E:T ratio, or adjust to a higher cell concentration for other E:T ratios to obtain a final volume of 200 μL per well.

CAR-T cells consist of live, CAR-transduced T cells. This can be determined by measuring transduction efficiency, calculated when producing CAR-T cells using flow cytometry (Tatari ). For instance, if the CAR-T cell transduction efficiency is 80%, a concentration of 3.75×105 live cells/mL will yield 3×105 CAR-T cells/mL.

For lysis controls, add 100 μL of XSFM with 75 μg/mL D-firefly luciferin potassium salt and 1% nonidet P-40 lysis buffer.

Add 100 μL of basal XSFM with 75 μg/mL D-firefly luciferin potassium salt to media-only wells.

Plate readoutTest: RLU of individual well for which percent specific lysis is being determined.

Incubate plate at 37°C, 5% CO2 for 4–8 h. After incubation, measure bioluminescence with luminometer using same settings previously described (step 8).

You may take multiple readings over 24 hours, however, increased cytotoxicity in control wells may occur as a result of non-specific effects. See troubleshooting problem 4 for further details.

To analyze data, use the following equation:

Spontaneous lysis: average RLU of media-only wells.

Lysis control: average RLU of lysis control wells.

CAR-T cell proliferation assay

Timing: 4.5 days

This assay quantifies the proliferation of CAR-T cells upon co-culturing with tumor cells.

Co-culture

Prepare target cells at a concentration of 4×106 live cells/mL of basal XSFM and plate 250 μL per well in a tissue culture-treated, flat-bottom 24-well plate. One well is needed for each CAR-T cell being used.

In the same plate, add 250 μL per well of basal XSFM without target cells as media-only controls.

Adjust the CAR-T cell concentration to 4×106 CAR-T cells/mL of basal XSFM and plate 250 μL per well.

CAR-T cells consist of live, CAR-transduced T cells. This can be determined by measuring transduction efficiency, calculated when producing CAR-T cells using flow cytometry (Tatari ).

This results in a 1:1 E:T ratio in co-culture wells.

Incubate at 37°C, 5% CO2 for 24–48 h.

Prepare cells for flow cytometry

Collect cell solution from each well.

Centrifuge at 300 × g for 5 min at 20°C–22°C and resuspend pellet gently in 1 mL of PBS.

Filter cell suspension into a 12 × 75 mm tube through 35μm cell strainer cap.

Determine live cell concentration of each cell solution using Trypan Blue solution and aliquot between 500,000 and 1,000,000 live cells per well into 12 × 75 mm tubes, collecting three tubes per well.

Three samples are needed per experimental condition: CD3 antibody, isotype control antibody or unstained control, and a viability stain control. The number of cells used per sample can vary depending on cell availability but must be consistent throughout all samples.

Cell staining

Centrifuge tubes at 300 × g for 5 min at 20°C–22°C. Remove supernatant and resuspend cells being stained in 100 μL of PBS with 2 mM EDTA (PBS-EDTA). Stain cells with the respective antibody as per manufacturer’s recommendations (i.e., antibody concentration, duration, temperature). Skip to step 24 for viability stain control.

Viability stain samples should be resuspended promptly after centrifuging, as leaving cells pelleted will reduce cell viability.

Add 1mL of PBS-EDTA to the tubes and mix gently to wash cells. Centrifuge at 300 × g for 5 min at 20°C–22°C.

Resuspend all pellets in 150 μL of 1× 7-AAD viability stain (100-fold dilution of 7-AAD from stock in PBS-EDTA).

Isolate T cells

Prepare a 96 well plate with 200 μL basal XSFM per well (n = 3 replicates per sample). Sort CD3+ cells into the plate at a cell density of 1000 cells/well.

Cells should be gated by selecting live cells (7-AAD-negative population), then from this population the CD3-positive cells.

Add 200 μL basal XSFM as a blank in the same plate (n = 3).

Incubate at 37°C, 5% CO2 for 3 days.

Plate readout

Add 1× presto blue reagent into all wells (20 μL into 200 μL well; 10-fold dilution). Incubate plate at 37°C, 5% CO2 for 4 h.

Read plate for relative fluorescence intensity (RFI) at an Excitation and Emission wavelength of 544nm and 590nm respectively.

The presto blue assay is a measure of viable cells, but reflects differences in proliferation when same number of cells are seeded and culture conditions are constant as is the case with this assay.

Proliferation is calculated for each well by subtracting the average RFI of all blank wells from the RFI of that well. Mean RFI can be plotted for each CAR-T cell being tested as a side-by-side comparison of proliferation in the presence and absence of target cells.

CAR-T cell activation assay

Timing: 30 h

This assay detects markers of activation on the CAR-T cell surface to assess activation upon co-culturing with target cells.

Co-culture

Prepare target cells at a concentration of 4×106 live cells/mL of basal XSFM and plate 250 μL per well in a tissue culture-treated, flat-bottom 24-well plate. Three wells are needed for each CAR-T cell being used.

Add 250 μL of basal XSFM per well in the same plate as media-only controls.

Adjust the CAR-T cell concentration to 4×106 cells/mL of basal XSFM and plate 250 μL of the cell solution per well.

This results in a 1:1 E:T ratio in co-culture wells.

Incubate at 37°C, 5% CO2 for 24 h.

Prepare cells for flow cytometry

Refer to steps 18–24 for preparing and staining cells for flow cytometry analysis.

Three samples are needed per well: CD25/CD69/CD3 co-stained, isotype co-stained or unstained control, and a viability stain control. Subsequent co-staining can be done by repeating steps 22 and 23 for each antibody. Fluorescence minus one controls can also be used to improve gating accuracy, whereby cells are stained with all fluorophores except one. Doing this for each fluorophore reveals the spread of all other fluorophores into the missing channel and gates can be set accordingly.

Data analysis

Analyze the CD3+ population for CD25 and CD69 markers of activation.

Flow gating strategy: Forward scatter (FSC)-height vs. side scatter (SSC)-height is used as the initial gate to exclude debris. Doublet cells are excluded using an FSC-height vs FSC-width gate. Viability gate is set using 7-AAD to exclude non-viable cells. Isotype control or an unstained control is used to set the gate for expression of CD3, CD25, and CD69 where the gate is drawn to exclude baseline expression of fluorophores. Expression of CD25/CD69 is analyzed on gated CD3 population.

The percentage of T cells positive for each marker, in addition to MFI (median fluorescence intensity) per cell reflects the degree of activation occurring in the cells, which can be compared between CAR-T and control CAR-T cells to determine specificity of the CAR and any off-target effects that may be taking place.

Cytokine release assay

Timing: 30 h

This assay measures cytokine levels secreted by CAR-T cells upon co-culturing with cancer cells.

Co-culture target cells and CAR-T cells or CAR-T cells only at a 1:1 ratio in basal XSFM (repeat activation assay co-culture, steps 31–34).

For each well, transfer cells to sterile tube and centrifuge at 300 × g for 5 min at 20°C–22°C.

Collect supernatant and store at −80°C for cytokine analysis or proceed directly to step 40

Perform flow cytometry quantification of TNF-α and IFN-γ cytokines using Cytometric Bead Array Kit (BD Biosciences). For a detailed protocol, please refer to the BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit Instruction Manual (BD Biosciences, 2019). Briefly, supernatant or provided cytokine standard solutions are serially diluted and cytokine bead suspensions are added. Beads are then washed and used in flow cytometry to quantify presence of TNF-α and IFN-γ cytokines.

Expected outcomes

The cytotoxicity assay should show increasing target cell lysis with increasing E:T ratios, while control CAR-T cells should continuously show low lysis.

The proliferation assay should show CAR-T cell proliferation increasing significantly upon co-culture with target cells compared to control CAR-T cells and CAR-T cells without target cell co-culture.

The activation assay should show a large fraction of CAR-T cells expressing CD25 and CD69 activation markers as shown in figure 2, whilst this expression should be limited for control CAR-T cells.

Figure 2

Representative flow cytometry plot of activation assay

Live, CD3+ cells were analyzed for expression of both CD25 and CD69 activation markers.

Similarly, the cytokine release assay should show high levels of both TNF-α and IFN-γ cytokines in CAR-T cells, whilst showing limited amounts in control CAR-T cells.

Limitations

In vitro co-culture-based assays provide valuable insights into the efficacy of CAR-T cell therapies but are inherently limited by several factors. Culturing of cell lines can allow for selection and cultural artifacts, and depending on the cell line, variation may occur. Additionally, the complex tumor immune microenvironment can have varying and unpredictable effects on immunotherapies not seen in vitro.

The in vitro cytotoxicity assays are done in small volumes where effector molecules are in close proximity to each other. CAR-T cell cytotoxicity can be enhanced by this clustering and intracellular signaling can be amplified, potentially exaggerating cytotoxicity values, known as the bystander effect. However, this assay has been effective at demonstrating relative cell killing and continues to be a reliable proxy of CAR-T cell efficacy.

CAR-T cell proliferation is measured via the presto blue assay which measures cell viability and thus cannot distinguish between an actively dividing cell and one in a quiescent state. Proliferation is thus calculated as an arbitrary fluorescence reading correlated to the number of live cells. Regardless, changes in viable cell count directly reflect and can be used to accurately assess CAR-T cell proliferation when the same number of cells are initially plated. Further, changes in viable cell count upon co-culture of CAR-T cells with target cells can be used to assess target cell-induced proliferation.

Troubleshooting

Problem 1

Low CAR transduction efficiency.

Potential solution

Due to the “pooled” approach of assessing the entire T cell population, low transduction efficiency can make differences between CAR-T and control CAR-T cell results indistinguishable, even for effective therapies. Enrichment for CAR-transduced T cells can be done prior to assays using a CAR-expressed marker to resolve this. See (Vora et al., 2020) for conceptual details on this.

Problem 2

Transfection of target cells with luciferase plasmid is not feasible or has low efficiency.

For cells in which stable transfection with luciferase plasmid (before you begin, steps 1–6) is not feasible, other measures of cytotoxicity are available which don’t require a reporter gene (Kiesgen et al., 2021). Low transfection can be overcome by electroporating target cells with luciferase mRNA (Omokoko et al., 2016).

Problem 3

Low CAR-T cell viability.

T cells are very sensitive to handling, and as such, resuspending vigorously can result in high amounts of cell death. T cells should be handled gently at all points in the protocol and handling should be kept to a minimum.

Problem 4

High cytotoxicity or activation in non-targeting control CAR-T cells.

Prior to final reading during the cytotoxicity (step 12) or activation (step 34) assays, prolonged co-culturing may lead to non-specific effects and increase killing or activation for both CAR-T and control CAR-T cells. If controls show high cytotoxicity or activation, the readout time may need to be optimized and include shorter incubation time.

Non-targeted control CAR may express activation markers due to culturing and expansion. This is acceptable so long as the targeting CAR shows much greater activation.

Problem 5

Optimum cytotoxicity occurs prior to readout timepoint.

While readout of the proliferation assay usually occurs after a 24–48 h co-culture of CAR-T cells with target cells (step 17), this period can be shortened or the assay can be read out several times within the period to gain more resolution of cytotoxicity at earlier time points.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Sheila Singh (ssingh@mcmaster.ca).

Materials availability

Requests for cell lines and information of all other pertinent information can be directed toward Dr. Sheila Singh.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD25-FITC	Miltenyi Biotec	Cat. No. 130-113-283, RRID:AB_2734062
CD3-PECy7	BD Biosciences	Cat. No. 557851, Research Resource Identifier:AB_396896
CD69-APC	BD Biosciences	Cat. No. 555533, Research Resource Identifier:AB_398602
IgG Compbeads	BD Biosciences	Cat. No. 552843
Biological samples
HEK293 T Lenti-X cells	n/a	n/a
Chemicals, peptides, and recombinant proteins
Antibiotic/antimycotic solution	Wisent Bio Products	Product No. 450-115-EL
Basic fibroblast growth factor (bFGF), human, recombinant	STEMCELL Technologies	Cat. No. 78003.2
D-firefly luciferin potassium salt	Perkin Elmer	Part No. 122799
DMEM, high glucose with pyruvate	Thermofisher Scientific	Cat. No. 11995073
DNase vial (D2)	Worthington Biochemical Corporation	Cat. No. NC9216647
EDTA	Millipore Sigma	Product No. 20158
Epidermal growth factor (EGF), human, recombinant	STEMCELL Technologies	Cat. No. 78006
Fetal bovine serum (FBS)	Wisent Bioproducts	Product No. 098-150
Heparin solution	STEMCELL Technologies	Cat. No. 07980
Liberase research grade	Millipore Sigma	Product No. LIBTM-RO
MEM-non-essential amino acids	Thermofisher Scientific	Cat. No. 11140050
MycoZap prophylactic	Lonza	Cat. No. VZA-2031
NeuroCult NS-A proliferation kit (Human)	STEMCELL Technologies	Cat. No. 05751
Nonidet 40 (NP40)	Thermofisher Scientific	Cat. No. 98379
PBS pH 7.4, with calcium and magnesium	Wisent Bio Products	Product No. 311-011-CL
Polybrene infection/transfection reagent	Millipore Sigma	Product No. TR-1003
PrestoBlue reagent	Thermofisher Scientific	Cat. No. A13262
Prime-XV T cell expansion media XSFM (XSFM)	Irvine Scientific	Cat. No. 91141
Trypan blue	Thermofisher Scientific	Cat. No. 15250-061
TrypLE™ express enzyme (1×), phenol red	Thermofisher Scientific	Cat. No. 12605028
7-AAD viability dye	Beckman Coulter	Product No. A07704
Critical commercial assays
Cytometric bead array (CBA) Kit	BD Biosciences	Cat. No. 550749
Experimental models: Cell lines
CAR-T cells	Please refer to Vora et al. (2020)	n/a
Patient-derived GBM stem cell lines	Dr. Sheila Singh’s Laboratory	n/a
Software and algorithms
Kaluza Analysis Software	Beckman Coulter	Web Link: https://www.mybeckman.ca/flow-cytometry/software/kaluza
FLUOstar omega fluorescence 556 microplateReader	BMG Labtech	n/a
Other
Countess chamber slides	Invitrogen	Cat. No. C10228
5 mL round-bottom polystyrene tubes with 35 μm strainer lid	Corning	Cat. No. CA21008-948

NCC media

Reagent	Final concentration	Amount
NeuroCult basal medium	n/a	443.3 mL
NeuroCult supplement	n/a	50 mL
Antibiotic/Antimycotic solution (100×)	1×	5 mL
MycoZap prophylactic (500×)	1×	1 mL
Heparin solution	0.0002% (w/v)	500 μL
EGF	20 ng/mL	100 μL
bFGF	10 ng/mL	100 μL
Total	n/a	500 mL

Storage: Store at 4°C for up to 1 week. Store all reagents as per manufacturer’s instructions.