Heterotypic cell-cell communication regulates glandular stem cell multipotency.

Glandular epithelia, including the mammary and prostate glands, are composed of basal cells (BCs) and luminal cells (LCs)1,2. Many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent stem cells1,3-8. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression3,9-13. This suggests that an active mechanism restricts BSC multipotency under normal physiological conditions, although the nature of this mechanism is unknown. Here we show that the ablation of LCs reactivates the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids. Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs-reminiscent of the genetic program that regulates multipotency during embryonic development7. By predicting ligand-receptor pairs from single-cell data14, we find that TNF-which is secreted by LCs-restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.

Identification of the tumour transition states occurring during EMT.

In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.

Quantitative lineage tracing strategies to resolve multipotency in tissue-specific stem cells.

Lineage tracing has become the method of choice to study the fate and dynamics of stem cells (SCs) during development, homeostasis, and regeneration. However, transgenic and knock-in Cre drivers used to perform lineage tracing experiments are often dynamically, temporally, and heterogeneously expressed, leading to the initial labeling of different cell types and thereby complicating their interpretation. Here, we developed two methods: the first one based on statistical analysis of multicolor lineage tracing, allowing the definition of multipotency potential to be achieved with high confidence, and the second one based on lineage tracing at saturation to assess the fate of all SCs within a given lineage and the "flux" of cells between different lineages. Our analysis clearly shows that, whereas the prostate develops from multipotent SCs, only unipotent SCs mediate mammary gland (MG) development and adult tissue remodeling. These methods offer a rigorous framework to assess the lineage relationship and SC fate in different organs and tissues.

Twelfth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The twelfth annual workshop of the European Network for Breast Development and Cancer focused on methods in mammary gland biology and breast cancer, was scheduled to take place on March 26-28, 2020, in Weggis, Switzerland. Due to the COVID-19 pandemic, the meeting was rescheduled twice and eventually happened as a virtual meeting on April 22 and 23, 2021. The main topics of the meeting were branching and development of the mammary gland, tumor microenvironment, circulating tumor cells, tumor dormancy and breast cancer metastasis. Novel and unpublished findings related to these topics were presented, with a particular focus on the methods used to obtain them. Virtual poster sessions were a success, with many constructive and fruitful interactions between researchers and covered many areas of mammary gland biology and breast cancer.

The mononuclear phagocyte system contributes to fibrosis in post-transplant obliterans bronchiolitis.

Bronchiolitis obliterans syndrome (BOS) is a fibrotic disease that is heavily responsible for the high mortality rates after lung transplantation. Myofibroblasts are primary effectors of this fibrotic process, but their origin is still debated. The purpose of this work was to identify the precursors of mesenchymal cells responsible for post-transplant airway fibro-obliteration.Lineage-tracing tools were used to track or deplete potential sources of myofibroblasts in the heterotopic tracheal transplantation model. Allografts were analysed by histology, confocal microscopy, flow cytometry or single-cell transcriptomic analysis. BOS explants were evaluated by histology and confocal microscopy.Myofibroblasts in the allografts were recipient-derived. When recipient mice were treated with tacrolimus, we observed rare epithelial-to-mesenchymal transition phenomena and an overall increase in donor-derived myofibroblasts (p=0.0467), but the proportion of these cells remained low (7%). Haematopoietic cells, and specifically the mononuclear phagocyte system, gave rise to the majority of myofibroblasts found in occluded airways. Ablation of Cx3cR1+ cells decreased fibro-obliteration (p=0.0151) and myofibroblast accumulation (p=0.0020). Single-cell RNA sequencing revealed similarities between myeloid-derived cells from allografts and both murine and human samples of lung fibrosis. Finally, myofibroblasts expressing the macrophage marker CD68 were increased in BOS explants when compared to controls (14.4% versus 8.5%, p=0.0249).Recipient-derived myeloid progenitors represent a clinically relevant source of mesenchymal cells infiltrating the airways after allogeneic transplantation. Therapies targeting the mononuclear phagocyte system could improve long-term outcomes after lung transplantation.

Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.

Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes. PIK3CA and P53 (also known as TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3ca(H1047R) mutant expression at physiological levels in basal cells using keratin (K)5-CreER(T2) mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReER(T2) mice gave rise to luminal ER(+)PR(+) tumours or basal-like ER(-)PR(-) tumours. Concomitant deletion of p53 and expression of Pik3ca(H1047R) accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca(H1047R) in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3ca(H1047R) expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3ca(H1047R) expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca(H1047R) activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.

Distinct stem cells contribute to mammary gland development and maintenance.

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

The long noncoding RNA Neat1 is required for mammary gland development and lactation.

The lncRNA Neat1 is an essential architectural component of paraspeckle nuclear bodies. Although cell-based studies identified Neat1-paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of paraspeckles nuclear bodies in vivo.

Cell type and stage specific transcriptional, chromatin and cell-cell communication landscapes in the mammary gland.

The mammary gland (MG) is composed of three main epithelial lineages, the basal cells (BC), the estrogen receptor (ER) positive luminal cells (ER+ LC), and the ER negative LC (ER- LC). Defining the cell identity of each lineage and how it is modulated throughout the different stages of life is important to understand how these cells function and communicate throughout life. Here, we used transgenic mice specifically labelling ER+ LC combined to cell surface markers to isolate with high purity the 3 distinct cell lineages of the mammary gland and defined their expression profiles and chromatin landscapes by performing bulk RNAseq and ATACseq of these isolated populations in puberty, adulthood and mid-pregnancy. Our analysis identified conserved genes, ligands and transcription factor (TF) associated with a specific lineage throughout life as well as genes, ligands and TFs specific for a particular stage of the MG. In summary, our study identified genes and TF network associated with the identity, function and cell-cell communication of the different epithelial lineages of the MG at different stages of life.

To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front.

Chemoattractants like f-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent signals that promote the formation of protrusive filamentous actin (F-actin; frontness) and RhoA-dependent actomyosin contraction (backness). Frontness locally inhibits backness and vice versa. In neutrophil-like HL60 cells, blocking phosphatidylinositol-3,4,5-tris-phosphate (PIP3) accumulation with selective inhibitors of PIP3 synthesis completely prevents fMLP from activating a PIP3-dependent kinase and Cdc42 but not from stimulating F-actin accumulation. PIP3-deficient cells show reduced fMLP-dependent Rac activity and unstable pseudopods, which is consistent with the established role of PIP3 as a mediator of positive feedback pathways that augment Rac activation at the front. Surprisingly, such cells also show reduced RhoA activation and RhoA-dependent contraction at the trailing edge, leading to the formation of multiple lateral pseudopods. Cdc42 mediates PIP3's positive effect on RhoA activity. Thus, PIP3 and Cdc42 maintain stable polarity with a single front and a single back not only by strengthening pseudopods but also, at longer range, by promoting RhoA-dependent actomyosin contraction at the trailing edge.

Repression of endogenous retroviruses prevents antiviral immune response and is required for mammary gland development.

The role of heterochromatin in cell fate specification during development is unclear. We demonstrate that loss of the lysine 9 of histone H3 (H3K9) methyltransferase G9a in the mammary epithelium results in de novo chromatin opening, aberrant formation of the mammary ductal tree, impaired stem cell potential, disrupted intraductal polarity, and loss of tissue function. G9a loss derepresses long terminal repeat (LTR) retroviral sequences (predominantly the ERVK family). Transcriptionally activated endogenous retroviruses generate double-stranded DNA (dsDNA) that triggers an antiviral innate immune response, and knockdown of the cytosolic dsDNA sensor Aim2 in G9a knockout (G9acKO) mammary epithelium rescues mammary ductal invasion. Mammary stem cell transplantation into immunocompromised or G9acKO-conditioned hosts shows partial dependence of the G9acKO mammary morphological defects on the inflammatory milieu of the host mammary fat pad. Thus, altering the chromatin accessibility of retroviral elements disrupts mammary gland development and stem cell activity through both cell-autonomous and non-autonomous mechanisms.

Early lineage segregation of multipotent embryonic mammary gland progenitors.

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.

Publisher Correction: Early lineage segregation of multipotent embryonic mammary gland progenitors.

In the version of this Article originally published, ref. 52 was incorrectly only attributed to its corresponding author, Fre, S., and an older title was used. The correct citation should have been: Lilja, A. M. et al. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nat. Cell Biol. https://doi.org/10.1038/s41556-018-0108-1 (2018)'. This has now been amended in all online versions of the Article.

Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation.

Adipocytes undergo pronounced changes in size and behavior to support diverse tissue functions, but the mechanisms that control these changes are not well understood. Mammary gland-associated white adipose tissue (mgWAT) regresses in support of milk fat production during lactation and expands during the subsequent involution of milk-producing epithelial cells, providing one of the most marked physiological examples of adipose growth. We examined cellular mechanisms and functional implications of adipocyte and lipid dynamics in the mouse mammary gland (MG). Using in vivo analysis of adipocyte precursors and genetic tracing of mature adipocytes, we find mature adipocyte hypertrophy to be a primary mechanism of mgWAT expansion during involution. Lipid tracking and lipidomics demonstrate that adipocytes fill with epithelial-derived milk lipid. Furthermore, ablation of mgWAT during involution reveals an essential role for adipocytes in milk trafficking from, and proper restructuring of, the mammary epithelium. This work advances our understanding of MG remodeling and tissue-specific roles for adipocytes.

Lineage-Restricted Mammary Stem Cells Sustain the Development, Homeostasis, and Regeneration of the Estrogen Receptor Positive Lineage.

The mammary gland (MG) is composed of different cell lineages, including the basal and the luminal cells (LCs) that are maintained by distinct stem cell (SC) populations. LCs can be subdivided into estrogen receptor (ER)+ and ER- cells. LCs act as the cancer cell of origin in different types of mammary tumors. It remains unclear whether the heterogeneity found in luminal-derived mammary tumors arises from a pre-existing heterogeneity within LCs. To investigate LC heterogeneity, we used lineage tracing to assess whether the ER+ lineage is maintained by multipotent SCs or by lineage-restricted SCs. To this end, we generated doxycycline-inducible ER-rtTA mice that allowed us to perform genetic lineage tracing of ER+ LCs and study their fate and long-term maintenance. Our results show that ER+ cells are maintained by lineage-restricted SCs that exclusively contribute to the expansion of the ER+ lineage during puberty and their maintenance during adult life.

Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium.

Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.

Tracing epithelial stem cells during development, homeostasis, and repair.

Epithelia ensure many critical functions of the body, including protection against the external environment, nutrition, respiration, and reproduction. Stem cells (SCs) located in the various epithelia ensure the homeostasis and repair of these tissues throughout the lifetime of the animal. Genetic lineage tracing in mice has allowed the labeling of SCs and their progeny. This technique has been instrumental in characterizing the origin and heterogeneity of epithelial SCs, their tissue location, and their differentiation potential under physiological conditions and during tissue regeneration.

Multipotent and unipotent progenitors contribute to prostate postnatal development.

The prostate is a glandular epithelium composed of basal, luminal and neuroendocrine cells that originate from the urogenital sinus during embryonic development. After birth, the prostate keeps developing until the end of puberty. Here, we used inducible genetic lineage tracing experiments in mice to investigate the cellular hierarchy that governs prostate postnatal development. We found that prostate postnatal development is mediated by basal multipotent stem cells that differentiate into basal, luminal and neuroendocrine cells, as well as by unipotent basal and luminal progenitors. Clonal analysis of basal cells revealed the existence of bipotent and unipotent basal progenitors as well as basal cells already committed to the luminal lineage with intermediate cells co-expressing basal and luminal markers associated with this commitment step. The existence of multipotent basal progenitors during prostate postnatal development contrasts with the distinct pools of unipotent basal and luminal stem cells that mediate adult prostate regeneration. Our results uncover the cellular hierarchy acting during prostate development and will be instrumental in defining the cellular origin and the mechanisms underlying prostate cancer initiation.