Multidimensional Fluorescence Imaging of Embryonic and Postnatal Mammary Gland Development

Multidimensional fluorescence imaging represents a powerful approach for studying the dynamic cellular processes underpinning the development, function, and maintenance of the mammary gland. Here, we describe key multidimensional imaging strategies that enable visualization of mammary branching morphogenesis and epithelial cell fate dynamics during postnatal and embryonic mammary gland development. These include 4-dimensional intravital microscopy and ex vivo imaging of embryonic mammary cultures, in addition to methods that facilitate 3-dimensional imaging of the ductal epithelium at single-cell resolution within its native stroma. Collectively, these approaches provide a window into mammary developmental dynamics, and the perturbations underlying tissue dysfunction and disease

High-throughput screening for novel regulators of Beta-catenin in Wnt signalling

Beta-catenin is a crucial component of the Wnt signalling pathway, which is imperative in many developmental processes and aberrantly regulated in several different cancers. The standard model of Wnt/Beta-catenin signalling states that, upon stimulation by Wnt ligand, Beta-catenin accumulates and subsequently translocates to the nucleus to activate TCF-dependent transcription of a variety of target genes, including oncogenes. However, the mechanisms regulating the nuclear localisation of Beta-catenin and its correlation with TCF-dependent transcription are poorly understood. In order to identify novel regulators of Beta-catenin levels and localisation in Wnt signalling imaging-based high-throughput knockout screens were developed in a Wnt inducible cell line, in addition to a cancer cell line in the presence of normal and downregulated APC. Results from the screens show that, in addition to known Wnt signalling components, genes not previously ascribed to the pathway appeared to modulate Beta-catenin. The study has provided sources of possible mechanistic insights into a number of areas of biology that may be involved in β-catenin regulation. Furthermore, it reveals an unprecedented degree of cross talk between Wnt and many other major signalling pathways. Moreover, the data indicated a degree of cell-type specificity in the regulators identified and, significantly, a lack of correlation between β-catenin levels and transcriptional activity

The immune environment of the mammary gland fluctuates during post-lactational regression and correlates with tumour growth rate

Post-lactational mammary gland regression encompasses extensive programmed cell death and removal of milk-producing epithelial cells, breakdown of extracellular matrix components and redifferentiation of stromal adipocytes. This highly regulated involution process is associated with a transient increased risk of breast cancer in women. Using a syngeneic tumour model, we show that tumour growth is significantly altered depending on the stage of involution at which tumour cells are implanted. Tumour cells injected at day 3 involution grew faster than those in nulliparous mice, whereas tumours initiated at day 6 involution grew significantly slower. These differences in tumour progression correlate with distinct changes in innate immune cells, in particular among F4/80-expressing macrophages and among TCRδ(+) unconventional T cells. Breast cancer post-pregnancy risk is exacerbated in older first-time mothers and, in our model, initial tumour growth is moderately faster in aged mice compared with young mice. Our results have implications for breast cancer risk and the use of anti-inflammatory therapeutics for postpartum breast cancers

Stat3-mediated alterations in lysosomal membrane protein composition.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome

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

$\textbf{Background}$: 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. $\textbf{Methods}$: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3DISCO, SeeDB, CUBIC and PACT. Using confocal, twophoton and light sheet microscopy, their compatibility with wholemount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. $\textbf{Results}$: 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 wholemount 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. $\textbf{Conclusions}$: 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 $\textit{in situ}$, and will significantly enhance our understanding of both normal and pathological mammary gland development.This work was supported by a grant from the Medical Research Council (MRC) program grant no. MR/J001023/1 (B.L-L. and C.J.W.). F.M.D. was funded by a National Health and Medical Research Council CJ Martin Biomedical Fellowship (GNT1071074). O.B.H. was funded by a Wellcome Trust PhD Studentship (105377/Z/14/Z). J.R.H was funded by an MRC research grant no. MR/K011014/1. F.C.L. was funded by Cancer Research UK and M.P. was funded by the MRC-LMB (MC_U105178788).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by BioMed Central