Abstract 5201: Modeling human mammary epithelial lineages using patient-derived organoid cultures

Abstract Organoids have emerged as a powerful model for studying normal and tumor cell biology; however, the application of organoid technology for culturing human breast epithelial cells has been challenging due to the difficulty in maintaining estrogen receptor (ER)-expressing cells in culture. To address this problem, we have developed MammoCult™ Organoid Kit (Human), a modular serum- and phenol red-free cell culture kit that promotes the growth of multilineage organoids, as well as organoids composed solely of ER+ and ER- luminal or basal cells. Primary human breast cells from reduction mammoplasty tissues from both pre- and postmenopausal donors were dissociated and seeded in 3D extracellular matrix domes, as either single cells or tissue fragments. Organoids were typically dissociated and passaged every 10 - 20 days. Cultures were analyzed by flow cytometry and immunocytochemistry for the expression of the pan epithelial marker EpCAM, the luminal markers Keratin 8 (K8), KIT, ER, MUC1, progesterone receptor (PR), and the basal markers CD49f, smooth muscle actin (SMA), and K14. Organoid cultures initiated from single cell suspensions generated distinct organoid morphologies reflective of their cell of origin. Luminal cells generated small hollow cystic organoids composed predominantly of cells that expressed K8, MUC1, and variable levels of expression of ER, PR, and KIT (n = 6 - 12). Notably, ER expression was maintained in luminal organoids for a minimum of 5 passages when split at a 1:3 to 1:5 split ratio (n = 3). Basal cells seeded in Corning® Matrigel® supplemented with collagen I (2 mg/mL) formed terminal ductal lobular unit (TDLU)-like branching structures that were composed solely of K14+SMA+ basal cells and could be passaged for a minimum of 2 passages when co-seeded with human mammary fibroblasts (n = 3). Multilineage organoids composed of both luminal and basal cells could be generated when cell aggregates or tissue fragments were initially seeded (n = 5). These results demonstrate that the MammoCult™ Organoid Kit (Human) offers a robust, in vitro model system for studying human mammary gland biology. Citation Format: David A. Rowbotham, Samantha Rothwell, Sharon A. Louis, Allen C. Eaves, John Stingl. Modeling human mammary epithelial lineages using patient-derived organoid cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5201.