Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland

Abstract

The developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1

“…Several sub-clusters of basal cells were identified in both analyses, and MaSC proceeded to give rise to more mature basal populations and to luminal lineages on separate branches. Interestingly, another analysis recently identified a cluster expressing markers of both the luminal and basal lineages 25 , consistent with a “luminobasal” cell state, previously described in both normal breast 27 and in breast tumors 28 . In our analysis, the genes enriched in this cluster are most consistent with cluster 4 (mature luminal), and it will be interesting to further investigate what regulates a luminobasal cell state, and what are its potential cell fates.…”

“…Other studies have described a profiling of human breast epithelial cells using clustering based on scRNA-seq 25 , 26 . Nguyen et al 26 profiled breast epithelial cells isolated from donated tissue.…”

Characterization of transcriptome diversity and in vitro behavior of primary human high-risk breast cells

“…Protein expression validation was also not able to be included, however, in previous studies we have validated concordance between quantitative and specific RNA and qualitative protein measurements 19 , 20 . The pregnancy gene expression profile was developed from RNAseq of whole mammary gland tissue, which would also contain populations of stromal and other cells that could influence gene expression levels shown 53 . To help address this issue, the pregnancy-related gene profile was limited to genes known to be expressed in mammary adenocarcinoma cell lines.…”

Single-cell transcription mapping of murine and human mammary organoids responses to female hormones

“…From these, 10,508 cells were from untreated samples, 9,695 cells were from samples treated with a low dose of estrogen (33.3 ng/mL), and 11,599 cells were from samples treated with a high dose of estrogen (66.6 ng/mL). Each of the cell cluster identities were determined once more using previously described lineage commitment markers in intact mammary tissue (Henry et al, 2021).…”