Dynamic architectural interplay between leucocytes and mammary epithelial cells.

The adult mammary gland undergoes dynamic changes during puberty and the postnatal developmental cycle. The mammary epithelium is composed of a bilayer of outer basal, or myoepithelial, cells and inner luminal cells, the latter lineage giving rise to the milk-producing alveolar cells during pregnancy. These luminal alveolar cells undergo Stat3-mediated programmed cell death following the cessation of lactation. It is established that immune cells in the microenvironment of the gland have a role to play both in the ductal outgrowth during puberty and in the removal of dead cells and remodelling of the stroma during the process of postlactational regression. However, most studies have focussed on the role of the stromal immune cell compartment or have quantified immune cell populations in tissue extracts. Our recent development of protocols for deep imaging of the mammary gland in three dimensions (3D) has enabled the architectural relationship between immune cells and the epithelium to be examined in detail, and we have discovered a surprisingly dynamic relationship between the basal epithelium and leucocytes. Furthermore, we have observed morphological changes in the myoepithelial cells, as involution progresses, which were not revealed by previous work in 2D tissue sections and whole tissue. This dynamic architecture suggests a role for myoepithelial cells in the orderly progression of involution. We conclude that deep imaging of mammary gland and other tissues is essential for analysing complex interactions between cellular compartments.

Neutral lineage tracing of proliferative embryonic and adult mammary stem/progenitor cells.

Mammary gland development occurs over multiple phases, beginning in the mammalian embryo and continuing throughout reproductive life. The remarkable morphogenetic capacity of the mammary gland at each stage of development is attributed to the activities of distinct populations of mammary stem cells (MaSCs) and progenitor cells. However, the relationship between embryonic and adult MaSCs, and their fate during different waves of mammary gland morphogenesis, remains unclear. By employing a neutral, low-density genetic labelling strategy, we characterised the contribution of proliferative stem/progenitor cells to embryonic, pubertal and reproductive mammary gland development. Our findings further support a model of lineage restriction of MaSCs in the postnatal mammary gland, and highlight extensive redundancy and heterogeneity within the adult stem/progenitor cell pool. Furthermore, our data suggest extensive multiplicity in their foetal precursors that give rise to the primordial mammary epithelium before birth. In addition, using a single-cell labelling approach, we revealed the extraordinary capacity of a single embryonic MaSC to contribute to postnatal ductal development. Together, these findings provide tantalising new insights into the disparate and stage-specific contribution of distinct stem/progenitor cells to mammary gland development.

Mammary Stem Cells: Premise, Properties, and Perspectives.

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape.

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.

BACKGROUND: High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated. METHODS: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. RESULTS: Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue. CONCLUSIONS: The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.

Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny.

The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium.

BMP2/BMPR1A is linked to tumour progression in dedifferentiated liposarcomas.

Bone Morphogenic Protein 2 (BMP2) is a multipurpose cytokine, important in the development of bone and cartilage, and with a role in tumour initiation and progression. BMP2 signal transduction is dependent on two distinct classes of serine/threonine kinase known as the type I and type II receptors. Although the type I receptors (BMPR1A and BMPR1B) are largely thought to have overlapping functions, we find tissue and cellular compartment specific patterns of expression, suggesting potential for distinct BMP2 signalling outcomes dependent on tissue type. Herein, we utilise large publicly available datasets from The Cancer Genome Atlas (TCGA) and Protein Atlas to define a novel role for BMP2 in the progression of dedifferentiated liposarcomas. Using disease free survival as our primary endpoint, we find that BMP2 confers poor prognosis only within the context of high BMPR1A expression. Through further annotation of the TCGA sarcoma dataset, we localise this effect to dedifferentiated liposarcomas but find overall BMP2/BMP receptor expression is equal across subsets. Finally, through gene set enrichment analysis we link the BMP2/BMPR1A axis to increased transcriptional activity of the matrisome and general extracellular matrix remodelling. Our study highlights the importance of continued research into the tumorigenic properties of BMP2 and the potential disadvantages of recombinant human BMP2 (rhBMP2) use in orthopaedic surgery. For the first time, we identify high BMP2 expression within the context of high BMPR1A expression as a biomarker of disease relapse in dedifferentiated liposarcomas.