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.

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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