The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. ...Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D ...imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.
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•A single-step, non-toxic clearing agent for 3D imaging of whole organs and tumors•Derivation of an integrative platform to interrogate intratumoral heterogeneity•Profound clonal restriction occurs during neoplastic progression•The epithelial-mesenchymal transition occurs clonally as a frequent event
Rios et al. develop a rapid, large-scale single-cell resolution 3D imaging protocol and use the protocol together with RNA sequencing to explore the cellular dynamics of mammary tumorigenesis and show that a molecular epithelial-to-mesenchymal transition is a prominent feature of tumor clones.
Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations ...that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2
tissue, including expansion of aberrant ERBB3
luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3
progenitors in BRCA2
breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3
progenitors could generate both ER
and ER
cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.
The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf ...deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.
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•The transcription factor EHF is essential for mammary development in pregnancy•Ehf drives differentiation of progenitor cells into milk-producing alveolar cells•Ehf deletion increased tumor development in the mouse mammary gland
Nightingale and Reehorst et al. identify a key role for the transcription factor EHF in mouse mammary gland development and in controlling the differentiation of mammary epithelial cells into milk-producing cells during pregnancy. They also show that Ehf is an important suppressor of tumor development in this tissue.
Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is ...known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.
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•Foxp1 is an essential transcription factor for mammary gland development•Foxp1 controls the activation of quiescent MaSCs marked by Tspan8•Foxp1 directly represses the transcription of Tspan8 in basal, but not luminal, cells•Tspan8 deletion rescues the Foxp1-deficient mammary phenotype
Fu et al. show that the transcriptional repressor Foxp1 is crucial for governing the exit of mammary stem cells from quiescence. Moreover, Tspan8 was identified as a direct Foxp1 target that plays a functional role in regulating the stem cell state, and its deletion reversed the effects of Foxp1 loss.
White-spotting patterns in mammals can be caused by mutations in the gene KIT, whose protein is necessary for the normal migration and survival of melanocytes from the neural crest. The alpaca ...(Vicugna pacos) blue-eyed white (BEW) phenotype is characterized by 2 blue eyes and a solid white coat over the whole body. Breeders hypothesize that the BEW phenotype in alpacas is caused by the combination of the gene causing gray fleece and a white-spotting gene. We performed an association study using KIT flanking and intragenic markers with 40 unrelated alpacas, of which 17 were BEW. Two microsatellite alleles at KIT-related markers were significantly associated (P < 0.0001) with the BEW phenotype (bew1 and bew2). In a larger cohort of 171 related individuals, we identify an abundance of an allele (bew1) in gray animals and the occurrence of bew2 homozygotes that are solid white with pigmented eyes. Association tests accounting for population structure and familial relatedness are consistent with a proposed model where these alleles are in linkage disequilibrium with a mutation or mutations that contribute to the BEW phenotype and to individual differences in fleece color.
Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5
basal ...epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5
Tspan8
) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5
Tspan8
cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.
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