Dynamic architectural interplay between leucocytes and mammary epithelial cells

Abstract

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.Abbreviations 2D, two dimensions; 3D, three dimensions; CD, cluster of differentiation; DAPI, 4 0 ,6-diamidino-2-phenylindole; DCIS, ductal carcinoma in situ; DCs, dendritic cells; DNA, deoxyribonucleic acid; GPI, glycosylphosphatidylinositol; H&E, haematoxylin and eosin; IgA, immunoglobulin A; K5, keratin 5; LIF, leukaemia inhibitory factor; MHCII, major histocompatibility complex class II; MIP, maximum intensity projection; MMPs, matrix metalloproteinases; NF-jB, nuclear factor kappa-light-chain-enhancer of activated B cells; SEMA, semaphorin; SMA, smooth muscle actin isoform a-actin; Stat, signal transducer and activator of transcription; TEBs, terminal end buds; Th, T helper; VEGF, vascular endothelial growth factor.

Multidimensional Imaging of Mammary Gland Development: A Window Into Breast Form and Function

“…The mammary gland is a case in point, as the adipocyte-rich stroma poses significant barriers for high-resolution, deep tissue 3D imaging. Consequently, a number of strategies are used to improve mammary gland wholemount immunostaining and depth of imaging in fixed tissues, including microdissection (Rios et al, 2014(Rios et al, , 2016b, enzymatic digestion (Wuidart et al, 2016(Wuidart et al, , 2018Scheele et al, 2017;Lilja et al, 2018), and optical tissue clearing (Davis et al, 2016;Lloyd-Lewis et al, 2016Elias et al, 2017;Seong et al, 2018;Chen et al, 2019;Hitchcock et al, 2019;Rios et al, 2019;Stewart et al, 2019). Tissue microdissection facilitates high-resolution 3D imaging of large areas of the ductal epithelium within stroma-divested mammary glands (Rios et al, 2014).…”

“…This provides opportunities, therefore, to explore interactions between mammary epithelial cells and their surrounding cellular and non-cellular [e.g., extracellular matrix (ECM)] niche by deep tissue 3D imaging (Inman et al, 2015). In this vein, two recent studies used optical tissue clearing and deep tissue 3D imaging to characterize mammary resident CD45+ leucocyte (Hitchcock et al, 2019), and more specifically macrophage (Stewart et al, 2019), populations at different stages of mammary gland development. Whilst CD45+ cells/macrophages were observed at all developmental stages, their prevalence, morphology, localization and interactions with the mammary epithelial bilayer exhibited stage-specific differences (Hitchcock et al, 2019;Stewart et al, 2019).…”

Deep imaging reveals new insights into mammary gland architecture and breast cancer susceptibility

“…in The FEBS Journal employed a new technique of deep tissue imaging (Fig. ) to reveal striking new insights into the development and function of the mammary gland across the reproductive life course . The technology utilises a urea‐based clearing reagent called CUBIC that removes lipids, reduces light scatter caused by different refractive indices and decolourises blood .…”

“…During lactation, contraction of myoepithelial cells in response to oxytocin causes milk to be squeezed out from the alveoli into ducts connected to the nipple, known as the milk ejection reflex. Deep tissue imaging has revealed the dynamic nature of myoepithelial cell structural organisation during different developmental stages . In the nonpregnant state, myoepithelial cells are aligned parallel to the direction of ductal elongation.…”

“…The method of deep tissue imaging has also enabled investigation into the structure and location of immune cells in the mammary gland . Immune cells, immunostained to detect the pan immune cell marker CD45, dramatically alter in morphology during pregnancy as compared to the nonpregnant state.…”