E. G. Thomas

14-3-3σ (sigma) regulates proliferation and differentiation of multipotent p63-positive cells isolated from human breastmilk

The mammary gland is a dynamic organ that only undergoes complete differentiation during pregnancy. Differentiation is fuelled by asymmetric division of stem cells that reside in normally quiescent niches in the resting gland in response to pregnancy-associated hormones. Loss of regulation of stem cells is believed to underlie some breast cancers. This process is poorly understood in humans since it is difficult to extract stem cells from the lactating gland. We have identified a p63-positive population in breastmilk that proliferates and differentiates into at least two separate mammary lineages in culture. Nuclear translocation of p63 coincides with expression of the cell-cycle arrest protein 14-3-3σ (Sigma) and precedes differentiation. Transient down-regulation of Sigma promotes maintenance of the p63-positive population without affecting normal differentiation. We propose that p63-postive cells from breastmilk represent a novel source of cells to model regulation of mammary gland development and tumorigenesis.

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.

Receptor activator of NF-κB ligand promotes proliferation of a putative mammary stem cell unique to the lactating epithelium

In mice, CD49fhi mammary stem cells (MaSCs) asymmetrically divide to generate CD49f+ committed progenitor cells that differentiate into CD49f− phenotypes of the milk‐secreting tissue at the onset of pregnancy. We show CD49f+ primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy‐associated hormones (PAH) compared with CD49f+ cells from nonlactating tissue. Differentiation of CD49f+ PMEC in extracellular matrix produces CD49f− luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f− luminal cells, while receptor activator of nuclear factor (NF)‐κB ligand (RANKL) specifically stimulates division of basal CD49f+ cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self‐renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH. STEM CELLS2012;30:1255–1264

A new selective fluorescent probe based on tamoxifen

Developing targeted validation probes that can interrogate biology is of interest for both chemists and biologists. The synthesis of suitable compounds provides a means for avoiding the costly labeling of cells with specific antibodies and the bias associated with the interpretation of biological validation experiments. The chemotherapeutic agent, tamoxifen has been routinely used in the treatment of breast cancer for decades. Once metabolized, the active form of tamoxifen (4-hydroxytamoxifen) competes with the binding of estrogens to the estrogen receptors (ER). Its selectivity in ER modulation makes it an ideal candidate for the development of materials to be used as chemical probes. Here we report the synthesis of a fluorescent BODIPY®FL conjugate of tamoxifen linked through an ethylene glycol moiety, and present proof-of-principle results in ER positive and ER negative cell lines. Optical microscopy indicates that the fluorescent probe binds selectively to tamoxifen sensitive breast cancer cell lines. The compound showed no affinity for the tamoxifen resistant breast cancer lines. The specificity of the new compound make it a valuable addition to the chemical probe tool kit for estrogen receptors.

Investigation of tumour-Infiltrating T cells and RANK/RANKL signalling in breast cancer

Aims: First, to investigate the prognostic significance of T cell subsets and RANK/RANKL signalling in the primary tumour of locally advanced breast cancer (LABC) and metastatic breast cancer patients. Second, to investigate the predictive significance of a relationship between clinical response to the anti-RANKL monoclonal antibody denosumab and immunopathological features of the tumour cells. Methods: Multiplex immunohistochemistry (IHC; Biocare) is used to assess the marker pairs CD4/CD8, RANK/PR and RANKL/FOXP3 in 84 patients in full-face serial sections and 60 patients in tissue microarrays with LABC or metastatic breast cancer. Aperio ImageScope software and ImageJ digital cell-counting algorithms are used to quantify total marker-positive cells as a percentage of total cells. Results: Statistical analysis of data collected so far shows an independent positive trend between the presence of FOXP+ T cells or CD4+ cells, but not CD8+ T cells, and shorter disease free survival (DFS) and overall survival (OS) (p = 0.089). Discussion: Previous research on T cell infiltration and breast cancer outcomes has produced conflicting data. Our data demonstrate evidence on a large, targeted cohort on the relevance of the presence of different T cell subsets and outcome. This may represent a novel diagnostic tool.