Integrating single-cell RNA-sequencing and functional assays to decipher mammary cell states and lineage hierarchies

Abstract

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER − ) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER + ) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit + progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

Single-cell evaluation reveals shifts in the tumor-immune niches that shape and maintain aggressive lesions in the breast

“…In particular, our observations agree with previous ndings that luminal-like cells acquire basal-like features following oncogenic or homeostatic disruption (Hein et al, 2016;Lim et al, 2009;Meyer et al, 2011;Molyneux et al, 2010;Van Keymeulen et al, 2015;Wahl and Spike, 2017;Wuidart et al, 2018). Conversely, we cannot rule out the possibility that the LB population arises from basal-like cells, consistent with basalcompartment cells giving rise to luminal cells in proposed hierarches of normal mammary gland development (Fu et al, 2020;Regan and Smalley, 2020), and with studies showing that adult committed basal cells can be reprogrammed to acquire more luminal-like states (Koren et al, 2015;Lilja et al, 2018;Van Keymeulen et al, 2015).…”

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

“…S1B). Further gene expression analysis identified a series of genes, previously described to define specific lineage states of mammary epithelial populations, to be also expressed by cells from non-epithelial clusters [18,21,33,34] (Supplementary Fig. S1C).…”

“…The arrows serve to suggest that cells tending toward a bipotential progenitor fate (mEC9) give rise to myoepithelial progenitors (mEC1) and predicted luminal common progenitors (mEC7). (D) Feature UMAP plots showing expression levels of specific genes in mEC clusters ◂ previously proposed to define a progenitor-like state, such as E74 Like ETS Transcription Factor 5 (Elf5), Monocyte differentiation antigen 14 (Cd14), KIT Proto-Oncogene, Receptor Tyrosine Kinase (Kit) and Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (Ezh2), [8,18,21,49,50]. These results suggest an accumulation of partially differentiated luminal secretory cells in mammary tissue from a never pregnant, post-pubescent female mouse.…”

“…For example, combining flow cytometric isolation with functional cellular markers has improved our understanding of the dynamics of lineage commitment, differentiation processes and mammary tissue development [5,[14][15][16][17]. More recently, single-cell sequencing strategies have enabled the interpretation of contiguous cellular cues and epithelial lineage dynamics in the developing mammary gland [8,15,[18][19][20][21][22][23].…”

Nutritional Regulation of Mammary Gland Development and Milk Synthesis in Animal Models and Dairy Species

“…For example, a recent study suggested that the expression of miRNAs could be specific to different cells [ 181 ]. The access to single cell RNA-seq, already used in breast cancer studies [ 182 , 183 ], will provide a deeper understanding of fine regulatory mechanisms of milk synthesis.…”