Fig. 1 |. A tumour-organoid-based approach for screening immunotherapy drugs.

The drug screen approach is composed of breast tumour organoids and tumour-specific CD8⁺ T cells. A lentiviral vector expressing OVA was transduced to the Luc⁺ EO771 cells. The resulting OVA+ cells were orthotopically transplanted to C57BL/6 mice to generate syngeneic mammary tumours. The tumours were harvested and dissociated to single cells for 2D culture and only the adherent cells were collected to generate tumour organoids. 2 ml of tumour organoid culture medium (2 × 10⁵ cells/ml) was seeded into 6-well culture plate with ultra-low attachment surface. After 7-day culture, tumour organoids were filtered by cell strainers with nylon mesh between 70 and 150 μm. The tumour organoids with diameter between 70 and 150 μm were treated with drugs for 48 h in the matrigel-free medium. The treated tumour organoids were then co-cultured with the pre-activated and OVA-specific CD8⁺ T cells from OT-I mice in the breast organoid culture medium containing 10 ng ml⁻¹ IL2 without matrigel for 24 h. The luciferase released from the EO771 cells was measured using a Dual-luciferase report assay system (Promega) on a BioTek Cytation5 imaging reader, to assess the T cell-mediated cytotoxicity effect.