SUMMARY Aging is closely associated with increased susceptibility to breast cancer, yet there have been limited systematic studies of aging-induced alterations in the mammary gland. Here, we leverage high-throughput single-cell RNA sequencing to generate a detailed transcriptomic atlas of young and aged murine mammary tissues. By analyzing epithelial, stromal, and immune cells, we identify age-dependent alterations in cell proportions and gene expression, providing evidence that suggests alveolar maturation and physiological decline. The analysis also uncovers potential pro-tumorigenic mechanisms coupled to the age-associated loss of tumor suppressor function and change in microenvironment. In addition, we identify a rare, age-dependent luminal population co-expressing hormone-sensing and secretory-alveolar lineage markers, as well as two macrophage populations expressing distinct gene signatures, underscoring the complex heterogeneity of the mammary epithelia and stroma. Collectively, this rich single-cell atlas reveals the effects of aging on mammary physiology and can serve as a useful resource for understanding aging-associated cancer risk.

Aging of the mammary gland is closely associated with increased susceptibility to diseases such as cancer, but there have been limited systematic studies of aging-induced alterations within this organ. We performed high-throughput single-cell RNA-sequencing (scRNA-seq) profiling of mammary tissues from young and old nulliparous mice, including both epithelial and stromal cell types. Our analysis identified altered proportions and distinct gene expression patterns in numerous cell populations as a consequence of the aging process, independent of parity and lactation. In addition, we detected a subset of luminal cells that express both hormone-sensing and alveolar markers and decrease in relative abundance with age. These data provide a high-resolution landscape of aging mammary tissues, with potential implications for normal tissue functions and cancer predisposition.

Epithelial cell hierarchy is a highly disputed topic in mammary gland biology. Studying the differentiation potential among cell types within the mammary gland is crucial for understanding normal development and would inform studies in tumorigenesis on a molecular and cellular level. Our recent study uncovers alterations occurring in the aging mouse mammary gland by utilizing single‐cell RNA‐sequencing to study the transcriptomic changes that occur between young and aged mice. From this data, we identified a cluster of luminal cells that expressed canonical lineage markers for two cell types, hormone‐sensing (HS) and alveolar (AV) cells, and that these HS‐AV cells decrease in abundance with age. The co‐expression of these markers contests the presently accepted epithelial hierarchy, as the HS and AV cells are currently thought to be maintained by separate pools of unipotent progenitor cells. Here, we characterized these murine HS‐AV cells by their gene expression pattern consisting of canonical HS and AV markers and confirmed their presence using immunofluorescence staining. In these stainings, we used two sets of canonical HS and AV markers to show co‐localization of both types in single cells, thus uncovering their histological localization within the luminal layer. We further quantified the abundance of these cells and confirmed the population’s decrease with age. Additionally, we have examined human tissues for the presence of HS‐AV cells and have confirmed their existence in the human mammary gland. Future multi‐variate analysis on a larger set of human samples is needed to determine the correlation of HS‐AV cell abundance with age in human mammary glands, as confounding factors such as history of pregnancy, family history, or environmental factors makes it challenging to exclusively analyze the age‐dependent effect on the cell type’s population size. Finally, the lineage relationship of HS‐AV cells to HS and AV cells remains unclear. We are performing functional experiments on isolated HS‐AV cells in order to study their differentiation lineages in vitro as organoid cultures. Unmasking the function of these cells within the normal mammary gland could provide significant insight into the study of the mammary epithelial differentiation hierarchy. A better understanding of this previously less well understood cell type may improve our fundamental knowledge of mammary cell states during normal physiology. Given that the mammary gland is a major organ for cancer development, a more detailed understanding of its cell states may also inform mechanisms of disease development. Support or Funding Information Susan G. Komen Postdoctoral Fellowship, Croucher Postdoctoral Fellowship (CML), NIH F31 Fellowship (GKG), Hope Funds Postdoctoral Fellowship, Marsha Rivkin Center for Ovarian Cancer Research (YO), Chan Zuckerberg Initiative Donor‐Advised Fund, Silicon Valley Community Foundation, NHGRI Career Development Award (LP), Susan G. Komen Breast Cancer Foundation, and Breast Cancer Research Foundation (JSB).