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96-Well live-cell assays for immune cell killing of 3D tumour spheroids

• Blended phase and fluorescent images of A549NucLight Red™ spheroids in the presence andabsence of immune cells.

• A549 cells (2.5K/well) seeded with PBMCsactivated with anti-CD3 (10 ng/ml) and IL-2(10 ng/ml). Cytotoxicity was quantified basedon the red fluorescent intensity.

• Data demonstrates an E:T ratio-dependentdestruction of tumour spheroids by theactivated T-cell population.

• Note that E:T ratio optimisation is required asnon targeted cell death was observed at E:Tratios >5:1.

Summary & Impact

www.essenbioscience.com

96-Well 3D Immune Cell Killing Assay Workflow

Activator-Dependent Tumour Cytotoxicity

Fluorescence as a Measure of Spheroid Cytotoxicity

Effector-to-Target Ratio Dependent Cytotoxicity

Herceptin Induced ADCC in HER2-Positive SKOV-3 cells

Activated T-Cells Non-Activated T-Cells

HER2-Positive SKOV-3 spheroids

HER2-negative A549 spheroids

2D vs. 3D Herceptin Response

Continuous Live-Cell Analysis: Methodology

• A549 NucLight Red™ cells weretreated with isolated PBMCsactivated with Anti-CD3/IL-2 (10ng ml-1) or IL-12/IL2 (10 ng ml-1)targeting T-cell or NK-cellpopulations, respectively.

• Activating different populationsof PBMCs, resulted indifferential effects on tumourspheroid destruction.

• In contrast to activated NK-cells,T-cell activated populationsexhibit increased proliferation.

• T-cell mediated spheroiddestruction occurred morerapidly compared to NK-cellmediated death.

• Data illustrates that T-cellmediated spheroid destructionis significantly driven by thepresence of anti-CD3 antibody.

• The frequencies of cell typeswithin PBMC populations varyfrom donor to donor. TypicallyCD3+ T-cells account for 45-70%of PBMCs, while only 5-20% ofare NK cells. This variability inoccurrence could explain thedifferential effects observed intumour spheroid cytotoxicity.

• A similar assay was conducted in a 2D culture model. SKOV-3cells (1.6K/well) were seeded overnight prior to the additionof PBMCs (8K/well) and subsequent treatment with Herceptin.

• SKOV-3 tumour spheroids appear to exhibit ~300-fold lowerHerceptin sensitivity in comparison to 2D.

• Note the apparent 34% inhibition of the 3D spheroid at thelowest test concentration (0.08 µg ml-1). This may suggest thata biphasic concentration response curve may exist, where theoutermost cells behave as in the 2D model, whereas thespheroid centre has lower sensitivity.

• Further experimentation is required to further understand thedifferential effects of Herceptin in 2D vs 3D models.

• Blended phase and fluorescent images, withcorresponding masks, of A549 human lungepithelial carcinoma cells stably expressing nuclearrestricted RFP (A549 NucLight Red™, EssenBioScience).

• Note the increase in size and fluorescence intensityof the spheroid alone and the shrinkage and

• decline of fluorescence in the presence of immunecells.

• Spheroid proliferation and immune cell-mediatedcytotoxicity can be quantified kinetically using theIncuCyte® size metrics (fluorescence intensity andfluorescence area) which require masking of thefluorescent spheroid.

Spheroid death Time-course Profile of Activators

M. Oliver1, K. Patel1, N. Holtz2, E. Endsley2, T. Dale1 & D. Trezise1

1Essen BioScience, Welwyn Garden City, AL7 3AX UK or 2Ann Arbor, MI, 48108, USA

Natural Killer cell vs T-cell mediated Tumour cytotoxicity

• HER2-positive SKOV-3 or HER2-negative A549 NucLight Red™spheroids (2.5K/well) were seededwith PBMCs (6.25K/well) and treatedwith Herceptin (mAb targeting HER2receptors).

• Herceptin >10 µg ml-1 inducedconcentration-dependent inhibitionof SKOV-3 spheroid growth.

• Herceptin-induced cytotoxicity wasmeasured in SKOV-3 but not A549spheroids.

3D IC50

2.4 µg ml-1

2D IC50

7.3 ng ml-1

Data normalised to vehicle and maximal inhibition controls

• Immunotherapies such as checkpoint inhibitors, CAR-Ts and immune-targeting Abs have great promise forcancer treatment. Translational cell-based assays arerequired to optimise these approaches.

• Here we describe image-based, immune cell-killingassays of 3D tumour spheroids, geared for assessingthe efficacy of novel immune-modulators.

• Human tumour cell lines expressing RFP were used toform spheroids in 96-well ULA plates. Immune cellswere then added and activated to kill. Spheroidviability was assessed over time (up to 10 days) bymeasuring the loss of RFP fluorescence usingIncuCyte live-cell analysis.

• This method is exemplified with a range of immunecell types ((PBMCs, T-cells, NK-cells) and activators,including anti-CD3 & IL-2

• In an ADCC format, Herceptin induced aconcentration-dependent specific killing of Her-2expressing tumours. Higher concentrations ofHerceptin were required in 3D vs 2D ADCC assays.

• These data demonstrate how immune-cell killing andADCC assays can be extended from traditional 2Dmono-cultures to 3D spheroid assays, providing thepotential for greater translational relevance. Theseassays will be highly valuable in the search for novelimmune-modulators.

Phase & fluorescence blended image

Fluorescence mask overlay

A549 cells alone

+ PBMCs+ antiCD3/IL2

E:T 10:1

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A549 NucLight Red™ cells (2.5K/well). Optimised E:T ratios of 2.5:1