Characterization of Mammary Cells Co‐expressing Separate Lineage Markers

Abstract

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).