Optimized media supports growth of hormone receptor–positive patient-derived organoid cultures.A, schematic illustration of processing of PDOs and CAFs from tumor. B, take-rate and confirmed ER/PR positivity of established PDO lines. C and D, representative brightfield images (C) and quantification of diameter (D) in patient #27 PDOs grown in breast organoid media and modified media #2 at passage 2. E and F, representative brightfield images (E) and quantification of diameter (F) in patient #26 PDOs grown in modified media #2 and bone metastasis media at passage 2. G and H, representative confocal images (G) and quantifications (H) of 5-ethynyl-2′-deoxyuridine (EdU) positive cells (red) in PDOs grown in modified media #2 and bone metastasis media at passage 2. The scale bars represent 200 μm (C and E), 40 μm (G). Proliferation was assessed by pulsing PDOs with EdU for 4 h and quantifying the ratio of EdU+ cells per total (DAPI+) number of cells in 20 to 30 PDOs from one experiment. Each data point represents the percentage of EdU+ cells in one PDO, red line indicates the mean ratio of EdU+ cells per treatment group. The diameter of PDOs was measured using CellProfiler software and normalized to breast organoid media (D) or modified media #2 (F). Student’s t test was used to assess significance. CAFs, cancer-associated fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; ER, estrogen receptor; PDO, patient-derived organoids; PR, progesterone receptor.