Abstract LB-033: A scaffold-free 3D organoid model to study neoplastic progression in breast cancer

Human breast cancers are morphologically heterogeneous and are known to fall mainly under two clinicopathological subgroups, ductal and lobular carcinomas. Toward the goal of modeling oncogenesis with a 3D platform, it is important to generate 3D biological structures in vitro that mimic the phenotypes present in the terminal duct lobular unit (TDLU), where human breast cancers are thought to be derived. Current 3D culture systems have enabled recapitulation of tissue architecture and differentiation resembling the in vivo situation, but rely on the use of laminin-rich gels such as Matrigel or collagen to allow growth of breast acinar structures with hollow lumen. Here, we used a gel-free hanging drop culture system that allows growth of self-organized mammary gland (MG) organoids long-term. To accomplish this, we established a reliable 3D breast cancer model from MCF10A epithelial cells, the most widely used cell line for normal human breast function, using a high-throughput 3D culture technique with a 384-well hanging drop platform at optimized media conditions. We successfully achieved significant organoid expansion with 3D structures up to 1.2 mm in diameter at 16 days that matched a similar phenotype observed in the TDLU. The MCF10A organoid model not only recapitulates acinar morphogenesis in vitro, as previously done, but also exhibits multiple lineage phenotypes, and is capable of undergoing dynamic remodeling upon treatment. We achieved >95% spheroid formation efficiency after 3 days, where cells were seeded with complete media, 0.24% (w/v) MethoCel, and 1.5% (v/v) Matrigel at cell density of 3,000 cells per droplet into a 384-well array plate. Droplets were washed every 2 days with fresh media for a total of 16 days. Organoids were collected from the array plate at a desired time and fixed in 4% PFA solution, embedded in frozen OCT blocks, and cut to 5 μm sections. We performed immunohistochemistry and immunofluorescence studies on the organoids (N=10), in triplicate using anti-cytokeratin 18 (CK 18, luminal marker), anti-CK 5/6 (basal cell marker), anti-E-cadherin (cell adhesion and luminal marker), and DNA staining dye (DAPI). MCF10A organoids were positive for basal marker CK 5/6 on the basolateral side, with luminal markers CK 18 and E-cadherin on the apical side, and also a mixed phenotype of CK 5/6+ and CK18+ cells, known to reflect progenitor status. In addition, we conducted 3D epithelial-to-mesenchymal induction assays using either TGF-β1 or hypoxia mimetic, CoCl2, and discovered a fibrotic-like response with increased collagen I deposition, loss of cell-cell contact, increased vimentin expression, and filled organoid lumen. The changes in normal MCF10A organoids vs. malignant organoids were measured by the morphological parameters: average organoid diameter, sphericity, and hollowness, using standard image segmentation techniques in ImageJ software. Average diameter was determined by the equivalent diameter, sphericity was measured as a ratio from 0 to 1, 1 being a perfect circle, and hollowness was found by the ratio of cell/matrix areas to total organoid bounding area. In conclusion, we propose 3D scaffold-free organoid technology provides a more physiologically relevant 3D model to study neoplastic progression in breast cancer and enables organoid expansion necessary for more robust screening of drug therapies. Citation Format: Sabra Djomehri, Shuichi Takayama, Celina Kleer. A scaffold-free 3D organoid model to study neoplastic progression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-033.