Tumor cells and CAFs with a concentration of 2 × 105 cells per milliliter were suspended into a mixture of pre-cold breast cancer culture medium (40) and Matrigel (Corning, 354234) with a ratio of 1:1. The cells were split into each well of a Costar 6-well culture plate with an ultralow attachment surface (Corning) and incubated in a humidified 37°C, 5% CO2 incubator. The medium was replaced by fresh 10% Matrigel breast cancer culture medium every 2 days, and the organoids were collected by using cell strainers with 40 μm nylon mesh (Thermo Fisher Scientific). At day 7, the organoids with diameter between 70 and 100 μm were collected by the corresponding cell strainers (Thermo Fisher Scientific) and gently resuspended in Matrigel-free DMEM/F-12 medium containing 10% FBS, 1% penicillin/streptomycin, and 2 ng/mL IL2. Organoid cell numbers were counted by digesting the organoids with trypLe Express (Thermo Fisher Scientific) at 37°C with a shaking velocity of 500 rpm for 15 minutes. The organoids were mixed with OT-I–specific CD8+ T cells with a ratio of 1:5 (organoid cell vs. T cell) in the Costar 6-well culture plate. After coculturing for 24–30 hours, the killing efficiency was evaluated by optical imaging and flow cytometry.

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