High-throughput surface marker screen on primary human breast tissues reveals further cellular heterogeneity.

BACKGROUND: Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. METHODS: To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. RESULTS: We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. CONCLUSIONS: Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity

Stem and progenitor cell division kinetics during postnatal mouse mammary gland development.

The cycling properties of mammary stem and progenitor cells is not well understood. To determine the division properties of these cells, we administered synthetic nucleosides for varying periods of time to mice at different stages of postnatal development and monitored the rate of uptake of these nucleosides in the different mammary cell compartments. Here we show that most cell division in the adult virgin gland is restricted to the oestrogen receptor-expressing luminal cell lineage. Our data also demonstrate that the oestrogen receptor-expressing, milk and basal cell subpopulations have telomere lengths and cell division kinetics that are not compatible with these cells being hierarchically organized; instead, our data indicate that in the adult homeostatic gland, each cell type is largely maintained by its own restricted progenitors. We also observe that transplantable stem cells are largely quiescent during oestrus, but are cycling during dioestrus when progesterone levels are high.We thank the members of Stingl lab, Doug Winton, Jason Carroll, Robert Clarke, Phil Jones and Hamid Raza Ali for scientific discussions. We thank the core facilities at the Cancer Research UK-CI for enabling experiments. In particular, Loic Tauzin, Nina Lane and Mateuz Strzelecki for assistance with cell sorting; the Biological Resources Unit for animal husbandry; and Histopathology staff, in particular Leigh-Anne McDuffus and Cara Walters. J. Stingl’s laboratory acknowledges the support of The University of Cambridge, Cancer Research UK (core grant number C14303/A17197) and Hutchison Whampoa Limited. M.A. Blasco’s laboratory is funded by the Spanish Ministry of Economy and Competitiveness Project SAF2013-45111RETOS, the European Union FP7 Project EUROBATS, the European Research Council (ERC) Project TEL STEM CELL (GA#232854), the Regional Government of Madrid project 2+2 ReCaRe, the AXA Research Fund and the Fundación Botín.This is the final version of the article. It was first available from Nature via http://dx.doi.org/10.1038/ncomms948

Proteomic characterization of microdissected breast tissue environment provides a protein-level overview of malignant transformation

Both healthy and cancerous breast tissue is heterogeneous, which is a bottleneck for proteomics‐based biomarker analysis, as it obscures the cellular origin of a measured protein. We therefore aimed at obtaining a protein‐level interpretation of malignant transformation through global proteome analysis of a variety of laser capture microdissected cells originating from benign and malignant breast tissues. We compared proteomic differences between these tissues, both from cells of epithelial origin and the stromal environment, and performed string analysis. Differences in protein abundances corresponded with several hallmarks of cancer, including loss of cell adhesion, transformation to a migratory phenotype, and enhanced energy metabolism. Furthermore, despite enriching for (tumor) epithelial cells, many changes to the extracellular matrix were detected in microdissected cells of epithelial origin. The stromal compartment was heterogeneous and richer in the number of fibroblast and immune cells in malignant sections, compared to benign tissue sections. Furthermore, stroma could be clearly divided into reactive and nonreactive based on extracellular matrix disassembly proteins. We conclude that proteomics analysis of both microdissected epithelium and stroma gives an additional layer of information and more detailed insight into malignant transformation

Characterisation of microRNA expression in post-natal mouse mammary gland development.

BACKGROUND: The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the mammary epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development.We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained. RESULTS: One third (n = 102) of all murine miRNAs analysed were detected during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show changes, we observed inverse patterns of miRNA and target expression. The data sets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research. CONCLUSION: MicroRNAs were expressed in likely co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation provide new avenues for research into mammary gland biology and generate candidates for functional validation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Confinement and the analytic structure of the one body propagator in Scalar QED

We investigate the behavior of the one body propagator in SQED. The self energy is calculated using three different methods: i) the simple bubble summation, ii) the Dyson-Schwinger equation, and iii) the Feynman-Schwinger represantation. The Feynman-Schwinger representation allows an {\em exact} analytical result. It is shown that, while the exact result produces a real mass pole for all couplings, the bubble sum and the Dyson-Schwinger approach in rainbow approximation leads to complex mass poles beyond a certain critical coupling. The model exhibits confinement, yet the exact solution still has one body propagators with {\it real} mass poles.Comment: 5 pages 2 figures, accepted for publication in Phys. Rev.

The human squamous oesophagus has widespread capacity for clonal expansion from cells at diverse stages of differentiation.

OBJECTIVE: Knowledge of the cellular mechanisms involved in homeostasis of human squamous oesophagus in the steady state and following chronic injury is limited. We aimed to better understand these mechanisms by using a functional 3D approach. DESIGN: Proliferation, mitosis and the expression of progenitor lineage markers were assessed in normal squamous oesophagus from 10 patients by immunofluorescence on 3D epithelial whole mounts. Cells expressing differential levels of epithelial and progenitor markers were isolated using flow cytometry sorting and characterised by qPCR and IF. Their self-renewing potential was investigated by colony forming cells assays and in vitro organotypic culture models. RESULTS: Proliferation and mitotic activity was highest in the interpapillary basal layer and decreased linearly towards the tip of the papilla (p<0.0001). The orientation of mitosis was random throughout the basal layer, and asymmetric divisions were not restricted to specific cell compartments. Cells sorted into distinct populations based on the expression of epithelial and progenitor cell markers (CD34 and EpCAM) showed no difference in self-renewal in 2D culture, either as whole populations or as single cells. In 3D organotypic cultures, all cell subtypes were able to recapitulate the architecture of the tissue of origin and the main factor determining the success of the 3D culture was the number of cells plated, rather than the cell type. CONCLUSIONS: Oesophageal epithelial cells demonstrate remarkable plasticity for self-renewal. This situation could be viewed as an ex vivo wounding response and is compatible with recent findings in murine models

Proteomic characterization of microdissected breast tissue environment provides a protein-level overview of malignant transformation

Both healthy and cancerous breast tissue is heterogeneous, which is a bottleneck for proteomics-based biomarker analysis, as it obscures the cellular origin of a measured protein. We therefore aimed at obtaining a protein-level interpretation of malignant transformation through global proteome analysis of a variety of laser capture microdissected cells originating from benign and malignant breast tissues. We compared proteomic differences between these tissues, both from cells of epithelial origin and the stromal environment, and performed string analysis. Differences in protein abundances corresponded with several hallmarks of cancer, including loss of cell adhesion, transformation to a migratory phenotype, and enhanced energy metabolism. Furthermore, despite enriching for (tumor) epithelial cells, many changes to the extracellular matrix were detected in microdissected cells of epithelial origin. The stromal compartment was heterogeneous and richer in the number of fibroblast and immune cells in malignant sections, compared to benign tissue sections. Furthermore, stroma could be clearly divided into reactive and nonreactive based on extracellular matrix disassembly proteins. We conclude that proteomics analysis of both microdissected epithelium and stroma gives an additional layer of information and more detailed insight into malignant transformation

Proteomics pipeline for biomarker discovery of laser capture microdissected breast cancer tissue

Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while inappropriate statistical methods will lead to false positive hits. All these issues have hampered the identification of reliable protein markers. A workflow, which integrates reproducible and robust sample preparation and data handling methods, is highly desirable in clinical proteomics investigations. Here we describe a label-free tissue proteomics pipeline, which encompasses laser capture microdissection (LCM) followed by nanoscale liquid chromatography and high resolution MS. This pipeline routinely identifies on average ̃10,000 peptides corresponding to ̃1,800 proteins from sub-microgram amounts of protein extracted from ̃4,000 LCM breast cancer epithelial cells. Highly reproducible abundance data were generated from different technical and biological replicates. As a proof-of-principle, comparative proteome analysis was performed on estrogen receptor a positive or negative (ER+/-) samples, and commonly known differentially expressed proteins related to ER expression in breast cancer were identified. Therefore, we show that our tissue proteomics pipeline is robust and applicable for the identification of breast cancer specific protein markers