3D immune mediated Tumour Killing Models: Tools for screening immune or tumour targeted therapeutics

Figure 1: SKOV-3 NLR tumour cells were seeded into 96 well plates, PBMC were added once spheroids were established in the presence of Pembrolizumab or IgG4 control or vehicle (untreated) and imaged every 4 hours for 96 hours using the CellCyte X. Relative spheroid area (%) was measured (A). AUC statistics were calculated using GraphPad Prism v9.5.0

ELISpot is a highly sensitive method to test immune modulators, novel vaccine candidates or de-risk immunogenicity testing in an antigen-specific CD4 and/or CD8 T cell assay.

Condition	Aim
No Stimulation	Negative control
PMA	Positive control
CERI (CMV, EBV, RSV, Influenza)	MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
CPI (CMV, Parainfluenza, Influenza)	Positive protein antigens to evaluate modulation of CD4+ T-cell memory response
CEF (CMV, EBV, Influenza)	MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
Cyclosporin A (CsA)	Inhibition of immune response

Figure 1: The ELISpot visual image results showing four donors under four conditions: Unstimulated (negative control), stimulated with PMA (positive control), stimulated with CERI, and stimulated with CPI antigens. Each purple spot represents one IFNγ producing cell.

Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors

Figure 2: T cell responses from three donors using stimuli, CERI (I), CPI (II) and CEF (III). Expressed as spot forming units per 100,000 donor PBMC. Depletion of CD4 or CD8 T cells demonstrates specificity of response to peptide pools. Each value represents the mean of triplicate wells ±SEM.

I. T cell response to Tetanus Toxoid, Influenza and PPD antigens

II. Dose response to Influenza antigen

Antigen specific T cells response to a recall antigens. (I) Healthy donors PBMC were stimulated with PHA-M or triple antigen cocktail (Tetanus Toxoid, Influenza and PPD). Cyclosporin was used as a reference treatment. (II) Dose response to Influenza antigen. CD4 and CD8 T cells proliferation was measured by flow cytometry using CTV dilutions.

Antibody dependent cell cytotoxicity (ADCC) in a 3D tumour spheroid model

Figure 1: SK-OV-3 NLR cells expressing HER2 were plated were seeded into 96 well plates and once spheroids were established purified NK cells were added to cultures in the presence of Trastuzumab or IgG1 Isotype or IL-12/IL-15 or vehicle (untreated) and imaged every 4 hours for 96 hours using the CellCyte X. Relative spheroid area (%) was measured (A) and the AUC (B) calculated using GraphPad Prism v9.5.0. Line graphs show the mean of triplicate wells and the bar graph shows mean +/- SEM of 9 donors. (C) Representative images from the CellCyte X at 96 hours post NK cell addition to tumour spheroids.

Assessing antibody dependent cell cytotoxicity (ADCC) driving the depletion of pathogenic immune cell subsets

ADCC PBMC assay to test specificity of the immune cell depleting antibody Alemtuzumab. Anti-CD52 antibody (Alemtuzumab) mediates killing via ADCC of CD52 expressing immune cells including T and B cells. The viability of CD4, CD8 and B cells was measured by flow cytometry after 4 and 18h of culture in the presence of an anti-CD52 IgG1 (Alemtuzumab) antibody or isotype at two concentrations. Graphs show mean +/- SEM, n=3 donors.

Monocyte derived macrophages (moMΦ) are differentiated from monocytes isolated from healthy blood donors followed by polarisation into a range of phenotypes M1/ M2a-d and TAM phenotypes using the stimuli described below. Readouts include flow cytometric analysis of surface markers associated with different subsets, production of cytokines and chemokines and ability to phagocytose. Therapeutics can be added at the monocyte to macrophage differentiation phase or polarisation phase to prevent or drive polarisation into a distinct macrophage phenotype.

We present a medium-throughput human macrophage assay to screen novel therapeutics that modulate pro-inflammatory M1 (IFNγ/LPS) and anti-inflammatory M2 (IL-4/IL-10) macrophage subsets. Dexamethasone and tofacitinib are used as reference drugs for benchmarking.

Dysregulated macrophage polarisation exacerbates various diseases. Therapeutics that target macrophage re-polarisation could modify the disease state by shifting macrophages to a beneficial phenotype. For example, promoting an inflammatory M1 phenotype could benefit the tumor microenvironment, while fostering an anti-inflammatory phenotype could help in autoimmune and inflammatory diseases.

Macrophages were differentiated from monocytes and polarised to M1 or M2a phenotypes following the schema to the left. Polarisation was assessed through CD80 staining and TNFα secretion for M1, and CD206 staining and CCL18 secretion for M2a; staining quantified via HCS analysis. Inhibition of M1 and M2a polarisation was demonstrated using dexamethasone and tofacitinib as reference compounds. Novel therapeutics can be benchmarked against these references.

A THP-1 macrophage cytokine release assay (CRA) can be used to explore modulators of an inflammatory response or “de-risk” any potential unwanted effects of novel therapeutic delivery systems.

THP-1 cells were differentiated with PMA and then stimulated with LPS to induce cytokine release in the presence of different concentrations of the corticosteroid dexamethasone. Cytokine release was measured at 4h and 24h post LPS stimulation.

Inflammasome complexes form in response to infection, tissue damage or metabolic imbalances. Once formed the inflammasomes activate Caspase 1 which in turn activated the pro-inflammatory cytokines IL-1β and IL-18. Targeting the inflammasome and the resulting signaling pathways is a useful tool in modulating the immune system.

Effect of MCC950 on Inflammasome activation – IL-1β and IL-18. PBMC from three healthy donors were pretreated with MCC950, vehicle (DMSO) or media for 1 hours prior to stimulation with LPS for 4 hours. Nigericin was then added for a further 45 minutes prior to harvest of the cell culture supernatant for quantification of (A) IL-1β and (B) IL-18 by TR-FRET and ELISA respectively. Data points show mean +/- SEM of technical replicates. One-way ANOVA with Dunnett’s multiple comparisons test comparing stimulated (media) to unstimulated, LPS alone, vehicle and MCC950 to vehicle; *<0.05, **<0.01, ***<0.001.

Other formats: PBMC, macrophage subsets, microglia

THP-1 macrophage phagocytosis of E.Coli bioparticles and efferocytosis of Heat Shock treated A-375 melanoma tumour cells. THP-1 cells were cultured with E.Coli bioparticles 0-12.5ug/ml; at 1h phagocytosis was evaluated by pHrodo+ cells using flow cytometry. THP-1 cells were cultured with three E:T ratios of A-375 or heat shock treated A-375; at 1h efferocytosis was evaluated by pHrodo+ cells using flow cytometry.

Therapies targeting the modification of tumor cells to increase neoantigen expression and improve immune cell recognition are currently under development. Detecting these subtle changes typically involves several cycles of dendritic cell restimulation of T cells, followed by the sensitive measurement of rare neoantigen-specific T cell responses using IFNγ ELISPOT assays. The schematic below shows the assay design with multiple round of dendritic cell (DC) restimulation to expand rare neoantigen T cell responses.

IFNγ release from activated tumour neoantigen-specific CD4 T cells

IFNγ release from activated tumour neoantigen-specific naive CD8 T cells

Figures: CD4 or naïve CD8 T cells were co-cultured with unloaded dendritic cells (DC), or with DC loaded with tumour cells that had been previously treated with media or vehicle. Three rounds of stimulation of T cells with DC were performed (* single round of DC stimulation). PMA and ionomycin were added as positive controls during the final round of stimulation. T cell stimulation was assessed by measuring the number of spot forming units (SFU) by IFNγ ELISpot. Media and T cell only wells were included as background controls. Number of SFU per 100,000 plated T cells, with mean of two (experimental controls) or three (all others) replicates ± SEM shown. Representative images of CD4 and naïve CD8 T cell IFNγELISpot. Each blue dot represents one IFNγ-producing T cell. ULOD = Upper Limit of Detection

ELISpot is a highly sensitive method to test immune modulators, novel vaccine candidates or de-risk immunogenicity testing in an antigen-specific CD4 and/or CD8 T cell assay.

Condition	Aim
No Stimulation	Negative control
PMA	Positive control
CERI (CMV, EBV, RSV, Influenza)	MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
CPI (CMV, Parainfluenza, Influenza)	Positive protein antigens to evaluate modulation of CD4+ T-cell memory response
CEF (CMV, EBV, Influenza)	MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
Cyclosporin A (CsA)	Inhibition of immune response

Figure 1: The ELISpot visual image results showing four donors under four conditions: Unstimulated (negative control), stimulated with PMA (positive control), stimulated with CERI, and stimulated with CPI antigens. Each purple spot represents one IFNγ producing cell.

Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors

Figure 2: T cell responses from three donors using stimuli, CERI (I), CPI (II) and CEF (III). Expressed as spot forming units per 100,000 donor PBMC. Depletion of CD4 or CD8 T cells demonstrates specificity of response to peptide pools. Each value represents the mean of triplicate wells ±SEM.

T helper 17 (Th17) cells, are a subset of CD4 T cells distinguished by their production of IL-17. While crucial for protecting the body against extracellular pathogens, Th17 cells have also been implicated in autoimmune disease. Discovering therapies that target either the differentiation or effector function of Th17 cells could therefore prove beneficial for the treatment of some autoimmune diseases.

Evaluation of therapeutic modulation of Th17 differentiation

Figure 1: Polarisation of Th17 cells. Naïve CD4 cells were cultured under Th17 polarising conditions for 12 days in the presence or absence of Ursolic Acid. CD4 T cells were assessed for proliferation by CTV dilution; intracellular cytokine staining (ICS) of IL-17 and IL-10 by flow cytometry. On Day 12, Supernatants were collected and evaluated for IL-17 and IL-10 levels by MSD. RoRγT inhibitor Ursolic Acid showed selective inhibition of IL-17 production by intracellular cytokine staining (ICS) and MSD.

Evaluation of therapeutic modulation of Th17 effector function

Figure 2: Inhibition of Th17 cell effector function. Magnetically sorted memory Th17 cells were polyclonally stimulated under Th17 conditions in the presence or absence of ursolic acid or FK506 for 5 days. A percentage of Th17 cells are polyfunctional (IL- 17A+IFNγ+) and refractory to inhibition by ursolic acid (an inhibitor of Th17 differentiation) but not FK506.

For many the humble “workhorse” mixed lymphocyte reaction (MLR) is a key screening assay for immuno-therapeutics. 1-way (DC and T cells) and 2-way MLRs (PBMC) provide expression of key ligands on human immune cells for therapeutic modulation.

Schematic showing key therapeutic targets that are blocked (antagonise) or engaged (agonise) to control immune responses in Autoimmunity or boost immune responses for Immuno-Oncology.

1-way or 2-way MLR. One way MLR of CTV labelled T cells in response to allogenic DC, matured with LPS. T cell proliferation and IFNγ production is inhibited by Rapamycin, FK506 and Cyclosporin A, in contrast, PD-1 blockade (Pembrolizumab) increases T cell IFNγ production, but does not enhance T cell proliferation.

This assay provides a relevant human system to test novel therapeutics that block or engage key co-stimulatory or inhibitory ligands expressed on immune cells.

Natural Tregs (nTregs) are a thymus-derived subset of CD4+ T cells found in peripheral blood, capable of suppressing immune responses to maintain tolerance and homeostasis. Suppression assays model Treg function by measuring their ability to inhibit effector T cell proliferation, activation, and cytokine production. Altering Treg suppressive function can have promising therapeutic potential, both in the context of autoimmunity and immuno-oncology.

Schematic showing mechanisms by which regulatory T cells suppress effector T cell responses.

nTreg cells suppress the proliferation of Tresp cells in a ratio-dependent manner
nTregs were co-cultured with stimulated CD3 T cells at three Tresp:Treg ratios. Graphs show increased suppression of Tresponder proliferation in the presence of increasing number of Tregs. Data shown from three healthy donors.

I. T cell response to Tetanus Toxoid, Influenza and PPD antigens

II. Dose response to Influenza antigen

Antigen specific T cells response to a recall antigens. (I) Healthy donors PBMC were stimulated with PHA-M or triple antigen cocktail (Tetanus Toxoid, Influenza and PPD). Cyclosporin was used as a reference treatment. (II) Dose response to Influenza antigen. CD4 and CD8 T cells proliferation was measured by flow cytometry using CTV dilutions.

In this 3D tumour spheroid model we test antibody-drug conjugates (ADC’s), checkpoint inhibitor (CPI) targeting antibodies and ADCC modalities. The 3D spheroid model provides a robust model for lead candidate selection for in vivo immuno-oncology studies and can be performed with a bank of fluorescently labelled, validated tumour cell types and immune subsets.

Figure 1: SKOV-3 NLR tumour cells were seeded into 96 well plates, PBMC were added once spheroids were established in the presence of Pembrolizumab or IgG4 control or vehicle (untreated) and imaged every 4 hours for 96 hours using the CellCyte X. Relative spheroid area (%) was measured (A). AUC statistics were calculated using GraphPad Prism v9.5.0. Samples size calculations for testing novel CPI n=9 donors; for novel ADCC n=3 donors.

Tumour cell line spheroids co-cultured with immune cells allow the therapeutic assessment of novel targeted monoclonal antibodies (ADCC) in a system which more closely recapitulates solid in vivo tumours by including 3D structure.

.

Figure 1: SK-OV-3 NLR cells expressing HER2 were plated were seeded into 96 well plates and once spheroids were established purified NK cells were added to cultures in the presence of Trastuzumab or IgG1 Isotype or IL-12/IL-15 or vehicle (untreated) and imaged every 4 hours for 96 hours using the CellCyte X. Relative spheroid area (%) was measured (A) and the AUC (B) calculated using GraphPad Prism v9.5.0. Line graphs show the mean of triplicate wells and the bar graph shows mean +/- SEM of 9 donors. (C) Representative images from the CellCyte X at 96 hours post NK cell addition to tumour spheroids.

Fluorescently labelled tumour cells are seeded in 96 well plates and tumour spheroids generated. Antibody-Drug-Conjugate (ADC) mediated killing of tumour cells is measured by reduction of tumour spheroid area via live cell imaging on the CellCyte X. Next generation ADC’s with immune modulatory arms can be assessed in the presence of immune cells.

Figure 1: SK-OV-3, SK-BR-3 and BT-20 NLR cells were seeded into 96 well plates; Trastuzumab-Deruxtecan or Isotype control was added once spheroids were established and imaged every 4 hours for 96 hours using the CellCyte X. Data shows tumour cytotoxicity normalised to isotype control, and example killing kinetics.

Tumour cells initiate cancer and drive disease progression, however many other cell types surrounding the tumour, including stromal and immune cells promote carcinogenesis. Cancer associated fibroblasts (CAFs) play a critical role in supporting tumour growth, shaping the cell microenvironment and promoting therapeutic resistance. Co-culture of tumour cells with fibroblasts has been shown to create a more organotypic tumour-like environment, enabling more physiologically relevant responses for therapeutic testing.

Formation of fibroblast-tumour spheroids

SK-OV-3/Fibroblast Spheroid Formation at 48-hours. SK-OV-3 NLR and CellTrace™ Blue-labelled NHDF cells were seeded into ultra low attachment 96-well plates and allowed to form spheroids over 72-hours. Images were acquired on the Opera Phenix High Content Screening System, 10x magnification. Images displayed above are from representative wells with indicated SKOV-3 and NHDF cell compositions.  

SK-OV-3/Fibroblast Spheroid killing following Cisplatin treatment

Time course of SK-OV-3/Fibroblast Spheroid killing following Cisplatin treatment. Spheroids were generated over 96-hours with a total of 5,000 cells per spheroid. Spheroids were treated with Cisplatin (100µM), vehicle or media control with varying percentages of SKOV-3 and NHDF cells (50-100% SKOV-3 cells) making up the spheroid. TKA was performed with a 1:1 ratio of PBMC: Spheroid. Images were acquired every 4-hours for 72-hours. Images were acquired on the Opera Phenix High Content Screening System, 10x magnification.