Abstract A114: Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium

Abstract High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of epithelial ovarian cancer. Despite its name, HGSOC originates in the fallopian tube (FT), but metastasizes quickly in the adnexal region and is the most lethal gynecological cancer worldwide. Organoid culture is emerging as a powerful model for studying normal FT cell biology and ovarian cancer. To standardize primary human FT and HGSOC organoid culture, we have developed GyneCult Fallopian Tube Organoid Medium (FTOM), an optimized serum-free medium and a workflow that supports robust and representative FT organoid culture from freshly isolated or cryopreserved primary human FT cells. FT cultures were initiated by seeding 4000 fresh or cryopreserved single cells (8 donors) directly into 20 µL Corning® Matrigel® domes and overlaying them with FT Organoid Medium. Cultures were maintained with full-medium changes every 3 - 4 days and were passaged every 8 - 14 days into new 20 µL Matrigel® droplets, either at a passage ratio of 1: 3 or by seeding at 4000 cells per droplet. Cultures were analyzed by immunocytochemistry (ICC) to detect secretory markers keratin 7 (KRT7), oviductal glycoprotein 1 (OVGP1), and PAX8, as well as ciliated cell markers acetylated alpha tubulin (TUBA1A) and FOXJ1. Across all donors, 16 ± 8% (mean ± SD) of dissociated EpCAM+ FT cells formed 50 - 300 µm diameter cystic organoids within 14 days of seeding (n = 8). Cultures can be maintained for at least 5 passages with 14 - 20 cumulative population doublings, at 3 - 4 doublings per passage. ICC analysis confirmed that organoids contained both polarized secretory cells (i. e. , KRT7+, OVGP1+, PAX8+) and ciliated cells (i. e. acetylated TUBA1A+, FOXJ1+), indicating multilineage capacity (n = 5). We also tested the compatibility of GyneCult FTOM with culturing HGSOC samples and observed that one of three tumor samples tested generated organoids. These tumour organoids had a solid and irregular shaped morphology and were composed of PAX8+ cells. These cells underwent ≥ 5 cumulative population doublings over 5 passages, with 0. 5 - 2 doublings per passage; this performance is consistent with recently published HGSOC organoid culture methods. These results demonstrate that GyneCult Fallopian Tube Organoid Medium is a robust medium for initiating and culturing FT epithelium as organoids, can support HGSOC organoid culture, and is a valuable tool for studying FT biology. Citation Format: Victor Ho, Maggie Chan, Manreet Chehal, Alice Liang, Allen C Eaves, Sharon A Louis, John Stingl. Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium abstract. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84 (5 Suppl₂): Abstract nr A114.