Establishment and characterization of equine mammary organoids using a method translatable to other non-traditional model species.

Mammary organoid (MaO) models are only available for a few traditional model organisms, limiting our ability to investigate mammary gland development and cancer across mammals. This study established equine mammary organoids (EqMaOs) from cryopreserved mammary tissue, in which mammary tissue fragments were isolated and embedded into a 3D matrix to produce EqMaOs. We evaluated viability, proliferation and budding capacity of EqMaOs at different time points during culture, showing that although the number of proliferative cells decreased over time, viability was maintained and budding increased. We further characterized EqMaOs based on expression of stem cell, myoepithelial and luminal markers, and found that EqMaOs expressed these markers throughout culture and that a bilayered structure as seen in vivo was recapitulated. We used the milk-stimulating hormone prolactin to induce milk production, which was verified by the upregulation of milk proteins, most notably ß-casein. Additionally, we showed that our method is also applicable to additional non-traditional mammalian species, particularly domesticated animals such as cats, pigs and rabbits. Collectively, MaO models across species will be a useful tool for comparative developmental and cancer studies.

DC-SCRIPT affects mammary organoids branching morphogenesis by modulating the FGFR1-pERK signaling axis.

Loss of expression of the transcription regulator DC-SCRIPT (Zfp366) is a prominent prognostic event in estrogen receptor-positive breast cancer patients. Studying the inherent link between breast morphogenesis and tumorigenesis, we recently reported that DC-SCRIPT affects normal mammary branching morphogenesis and mammary epithelium homeostasis. Here we investigated the molecular mechanism involved in DC-SCRIPT mediated regulation of FGF2 induced mammary branching morphogenesis in a 3D organoid culture system. Our data show that the delayed mammary organoid branching observed in DC-SCRIPT-/- organoids cannot be compensated for by increasing FGF2 levels. Interestingly, FGFR1, the dominant FGF2 receptor, was expressed at a significantly lower level in basal epithelial cells of DC-SCRIPT deficient organoids relative to wildtype organoids. A potential link between DC-SCRIPT and FGFR1 was further supported by the predicted locations of the DC-SCRIPT DNA binding motif at the Fgfr1 gene. Moreover, ERK1/2 phosphorylation downstream of the FGFR1 pathway was decreased in basal epithelial cells of DC-SCRIPT deficient organoids. Altogether, this study shows a relationship between DC-SCRIPT and FGFR1 related pERK signaling in modulating the branching morphogenesis of mammary organoids in vitro.

Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures.

Epithelial tissues are highly organized structures that are structured at both the cellular and tissue levels. Individual cells are characterized by an apical membrane facing a central lumen, and a basolateral membrane that contacts adjacent cells and the basement membrane. The maintenance of apical-basal polarity is crucial for maintaining epithelial homeostasis and is considered a barrier to carcinogenesis. Apical-basal cell polarity is compromised in many epithelial cancers, such as breast, lung, and prostate, and has been associated with disease progression. Three-dimensional (3D) organotypic cultures recapitulate the 3D tissue architecture and mechanical properties found in vivo. This chapter describes methods to establish 3D organoids from human cell lines or mouse primary cells with inducible oncogene expression in polarized epithelial structures to investigate mechanisms of tumor initiation, luminal filling, and growth. The method is versatile, and simple modifications can be made to study diverse cell/tissue types and oncogenes.