ODP575 Single-cell analysis reveals heterogeneity of mouse mammary gland fibroblasts with discrete functions, estrogen responses, differentiation, and communications with epithelium.
Authors: Yoshitake Ryohei , Chang Gregory , Saeki Kohei , Ha Desiree , Wu Xiwei , Wang Jinhui , Chen Shiuan 
Published: November 2022

Abstract
Mammary gland is an essential organ for milk production. It continues to develop after birth under the influence of ovarian hormones, especially estrogen. Among its cellular components, fibroblasts are recognized as an important player for mammary gland organization. Although human mammary gland fibroblasts have been classified into two subtypes based on histological localizations and different marker expressions (i. e., interlobular and intralobular fibroblasts), their detailed characteristics and functions are still limited. Moreover, despite the importance of experimental mouse models for studying mammary gland biology, heterogeneity of mouse mammary gland fibroblasts has not been fully defined. Here, we performed single-cell RNA sequencing (scRNA-seq) to determine cellular populations and their potential contributions to mammary gland organization. Based on the comprehensive gene expression profiles at a single cell level, we identified two major clusters of the mouse mammary gland fibroblasts. One of the major clusters showed the highly specific expression of Dpp4 gene, which is a known marker for human interlobular fibroblasts, and the upregulation of the inflammatory response gene signature. The other major cluster was consisted of three subclusters without Dpp4 expression, which suggested the distinct contributions to adipogenesis and/or ECM modeling in mammary gland stroma. Through the histological validation, we defined these two major subsets as "DPP4+" and "DPP4-" fibroblasts. Since our scRNA-seq was performed on two different ovarian hormone-depleted mouse models followed with hormone treatments, we evaluated the effect of the hormones, especially estrogen. The results demonstrated that the estrogen treatment altered the gene expression profiles of the DPP4+ and a subcluster of the DPP4- fibroblasts in a population-specific manner. Estrogen upregulated interferon-regulated genes and ECM genes in these fibroblast populations, respectively, but not typical estrogen-regulated genes, even in the ERα+ DPP4+ fibroblasts. To define these mammary gland fibroblasts better, we integrated our datasets with a recently established "steady-state mouse fibroblasts atlas" (1) as well as four other scRNA-seq datasets containing normal mouse mammary gland fibroblasts. Using this integrated dataset, we revealed the directional differentiation among the mammary gland fibroblasts, going from the fibroblasts and branching toward the DPP4- fibroblasts with more specialized functions. Moreover, the cell-cell interaction analysis combined with a mammary epithelium dataset from our previous publication (2) indicated possible cell-type-specific communications between mammary gland fibroblasts and epithelial cells. In conclusion, this study provided a comprehensive view of mouse mammary gland fibroblast biology at the single-cell level. Our findings would provide fundamental insights for further investigations on the roles of different fibroblast populations in mammary gland organization and pathological conditions, such as breast cancer. Reference: (1) Buechler et al., Nature. 2021 May;593(7860): 575–579. (2) Saeki et al., Commun Biol. 2021 Jun;4(1): 1–16.
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