Construction of an atlas of the mouse and human mammary glands using mass cytometry

Abstract

The mammary gland is a remarkably dynamic organ which develops largely postnatally and whose terminal differentiation is reversible. Three epithelial lineages are known to comprise the postnatal mammary gland: basal/myoepithelial (BA), alveolar luminal (AV), and hormone-sensing luminal (HS). These three lineages change dramatically in form and function during the complex linear development of puberty; the cyclic differentiation of gestation, lactation, and involution; the post-reproductive periods of aging; and tumor initiation. However, the extent of intralineage heterogeneity in these contexts is unclear, thus stymieing functional interrogations of the role of different subpopulations in development and malignant transformation. In this thesis, utilizing primarily mass cytometry, mammary epithelial diversity is investigated in a variety of contexts. In Chapter 2, the adult murine nulliparous glands of both sexes, as well as the pubescent gland, were evaluated, and multiple unique populations were identified in each lineage, including putatively more differentiated subsets which proliferated strongly in response to hormonal fluctuations of the estrous cycle. The pubescent gland possessed four unique luminal populations, including two types of quiescent AV cells and two types of highly proliferative HS cells. Loss of estradiol and progesterone via ovariectomy caused the losses of putative mature populations and of AV lineage specification. Exogenous hormone administration suppressed the misexpression of HS markers in AV cells, thus restoring lineage fidelity among luminal cells. In Chapter 3, epithelial cells were evaluated across nineteen time points in nulliparity, gestation, lactation, involution, and post-involution in order to determine how each epithelial lineage changes across the differentiation cycle. Lineage trajectories of each cell type revealed multiphasic, temporally synchronized differentiation cycles in both luminal lineages. However, BA cells exhibited continuous differentiation leading up to lactation, did not revert to a nulliparous phenotype, and instead developed into two unique subpopulations of post-involution cells distinguished by their quiescent stem-like properties. This contrast between luminal and basal cells demonstrates that functional dedifferentiation of an organ need not require total dedifferentiation of all its constituent cells. In Chapter 4, the human mammary gland was evaluated utilizing a multi-omics approach to explore alterations of the breast across numerous variables, including parity, age, and genetic predisposition to breast cancer. A unique population of basal-like AV cells accumulated in ducts with age and was responsive to EGF and TGF signaling. In addition, tissues containing germline BRCA2 mutations were enriched for PR-negative HS cells. Collectively, the data presented in this thesis provide an exceptionally detailed atlas of the mammary gland in its postnatal states in both the mouse and human and provide a high-resolution portrait of postnatal development of the gland in multiple phases. These studies may inform future functional interrogations of the various novel populations described as well as of the phenotypic changes which precede breast tumorigenesis.