Fibroblasts are stromal cells found in connective tissue that are critical for organ development, tissue homeostasis and pathology. Single-cell transcriptomic analyses have revealed a high level of inter- and intra-organ heterogeneity of fibroblasts. However, the functional implications and lineage relations of different fibroblast subtypes remained unexplored, especially in the mammary gland. Here, we provide a comprehensive characterization of pubertal mouse mammary fibroblasts, through single-cell RNA sequencing, spatial mapping, functional assays, and in vivo lineage tracing. We unravel a transient niche-forming population of specialized contractile fibroblasts that exclusively localize around the tips of the growing mammary epithelium and are recruited from preadipocytes in the surrounding fat pad stroma. Using organoid-fibroblast co-cultures we reveal that different fibroblast populations can acquire contractile features when in direct contact with the epithelium, promoting organoid branching. The detailed in vivo characterization of these specialized cells and their lineage history provides insights into fibroblast heterogeneity and implicates their importance for creating a signalling niche during mouse mammary gland development.

• MeSH
• analýza jednotlivých buněk MeSH
• buněčný rodokmen MeSH
• epitel metabolismus   růst a vývoj   MeSH
• epitelové buňky cytologie   MeSH
• fibroblasty * cytologie   metabolismus   fyziologie   MeSH
• kokultivační techniky MeSH
• mléčné žlázy zvířat * cytologie   růst a vývoj   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• organoidy cytologie   metabolismus   MeSH
• tukové buňky cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Fibroblasts are an integral cell type of mammary gland stroma, which plays crucial roles in development, homeostasis, and tumorigenesis of mammary epithelium. Fibroblasts produce and remodel extracellular matrix proteins and secrete a plethora of paracrine signals, which instruct both epithelial and other stromal cells of the mammary gland through mechanisms, which have not been fully understood. To enable deciphering of the intricate fibroblast-epithelial interactions, we developed several 3D co-culture methods. In this chapter, we describe methods for establishment of various types of embedded 3D co-cultures of mammary fibroblasts with mammary epithelial organoids, mammary tumor organoids, or breast cancer spheroids to investigate the role of fibroblasts in mammary epithelial development, morphogenesis, and tumorigenesis. The co-culture types include dispersed, aggregated, and transwell cultures.

• Klíčová slova
• 3D culture, Breast cancer, Co-culture, Epithelium, Extracellular matrix, Fibroblast, Mammary gland, Organoid, Spheroid,
• MeSH
• buněčné linie MeSH
• epitel metabolismus   MeSH
• epitelové buňky * MeSH
• fibroblasty metabolismus   MeSH
• karcinogeneze patologie   MeSH
• kokultivační techniky MeSH
• lidé MeSH
• mléčné žlázy zvířat * MeSH
• organoidy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.

• MeSH
• epitelové buňky * metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• kokultivační techniky MeSH
• mléčné žlázy zvířat * metabolismus   MeSH
• morfogeneze fyziologie   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Epithelial-stromal interactions play an essential role in regulation of mammary gland development, homeostasis, and tumorigenesis. Fibroblasts constitute a substantial proportion of mammary gland stromal cells in human breast and have been recognized for their paracrine signaling and extracellular matrix production and remodeling roles during normal breast development as well as in breast cancer. However, our current knowledge on human breast fibroblast functions is incomplete. Here we provide a detailed protocol for an organotypic human breast assay to facilitate research in the roles of human breast fibroblasts in mammary epithelial morphogenesis and early tumorigenesis.

• Klíčová slova
• 3D culture, Breast, Epithelial-stromal interactions, Fibroblast, Mammary gland, Spheroid,
• MeSH
• buňky stromatu MeSH
• epitelové buňky MeSH
• fibroblasty MeSH
• lidé MeSH
• mléčné žlázy lidské * MeSH
• mléčné žlázy zvířat MeSH
• nádory prsu * MeSH
• parakrinní signalizace MeSH
• prsy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH