Fluorescent biosensors offer a powerful tool for tracking and quantifying protein activity in living systems with high temporospatial resolution. However, the expression of genetically encoded fluorescent proteins can interfere with endogenous signaling pathways, potentially leading to developmental and physiological abnormalities. The EKAREV-NLS mouse model, which carries a FRET-based biosensor for monitoring extracellular signal-regulated kinase (ERK) activity, has been widely utilized both in vivo and in vitro across various cell types and organs. In this study, we report a significant defect in mammary epithelial development in EKAREV-NLS C57BL/6J female mice. Our findings reveal that these mice exhibit severely impaired mammary epithelial outgrowth, linked to systemic defects including disrupted estrous cycling, impaired ovarian follicle maturation, anovulation, and reduced reproductive fitness. Notably, estrogen supplementation was sufficient to enhance mammary epithelial growth in the EKAREV-NLS C57BL/6J females. Furthermore, outcrossing to the ICR genetic background fully restored normal mammary epithelial outgrowth, indicating that the observed phenotype is dependent on genetic background. We also confirmed the functional performance of the biosensor in hormone-supplemented and outcrossed tissues through time-lapse imaging of primary mammary epithelial cells. Our results underscore the critical need for thorough characterization of biosensor-carrying models before their application in specific research contexts. Additionally, this work highlights the influence of hormonal and genetic factors on mammary gland development and emphasizes the importance of careful consideration when selecting biosensor strains for mammary studies.

• Keywords
• Biosensor, EKAREV–NLS mouse, ERK signaling, Estradiol supplementation, Genetic background, Hormonal imbalance, Mammary epithelium,
• MeSH
• Biosensing Techniques methods   MeSH
• Epithelial Cells * drug effects   metabolism   pathology   MeSH
• Estrogens * pharmacology   administration & dosage   MeSH
• Genetic Background MeSH
• Mammary Glands, Animal * drug effects   pathology   growth & development   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Estrogens * 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
• Epithelial Cells * metabolism   MeSH
• Fibroblasts metabolism   MeSH
• Coculture Techniques MeSH
• Mammary Glands, Animal * metabolism   MeSH
• Morphogenesis physiology   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article 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.

• Keywords
• 3D culture, Breast, Epithelial-stromal interactions, Fibroblast, Mammary gland, Spheroid,
• MeSH
• Stromal Cells MeSH
• Epithelial Cells MeSH
• Fibroblasts MeSH
• Humans MeSH
• Mammary Glands, Human * MeSH
• Mammary Glands, Animal MeSH
• Breast Neoplasms * MeSH
• Paracrine Communication MeSH
• Breast MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

3D cell culture methods have been an integral part of and an essential tool for mammary gland and breast cancer research for half a century. In fact, mammary gland researchers, who discovered and deciphered the instructive role of extracellular matrix (ECM) in mammary epithelial cell functional differentiation and morphogenesis, were the pioneers of the 3D cell culture techniques, including organoid cultures. The last decade has brought a tremendous increase in the 3D cell culture techniques, including modifications and innovations of the existing techniques, novel biomaterials and matrices, new technological approaches, and increase in 3D culture complexity, accompanied by several redefinitions of the terms "3D cell culture" and "organoid". In this review, we provide an overview of the 3D cell culture and organoid techniques used in mammary gland biology and breast cancer research. We discuss their advantages, shortcomings and current challenges, highlight the recent progress in reconstructing the complex mammary gland microenvironment in vitro and ex vivo, and identify the missing 3D cell cultures, urgently needed to aid our understanding of mammary gland development, function, physiology, and disease, including breast cancer.

• Keywords
• 3D cell culture, Breast, Co-culture, Extracellular matrix, Imaging, Microenvironment, Organoid, Screening, Stromal cells,
• MeSH
• Cell Differentiation MeSH
• Cell Culture Techniques instrumentation   MeSH
• Spheroids, Cellular pathology   MeSH
• Epithelial Cells pathology   MeSH
• Extracellular Matrix pathology   MeSH
• Coculture Techniques methods   MeSH
• Humans MeSH
• Mammary Glands, Human cytology   pathology   MeSH
• Mammary Glands, Animal cytology   pathology   MeSH
• Models, Animal MeSH
• Mice MeSH
• Breast Neoplasms pathology   MeSH
• Organoids MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH