Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with β1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα vesicles are present within human breast tissues, and colocalization with β1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.
Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions among the cells—and the myriad of molecules they synthesize. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, that is, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue as a whole. This biological exploration has required cell purification, deep-RNA sequencing— and a thorough dissection of the genes and pathways defining each cell type, which we present in an adjoining article. Here, we present an exhaustive cellular dissection of the human breast, where we explore its cellular composition and histological organization. Moreover, we introduce a novel fluorescence-activated cell sorting (FACS) antibody panel and rigorous gating strategy capable of isolating each of the twelve major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every one of these breast cell types—some being the first of their kind— and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work and derived resources deliver a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.
Purpose Despite the prevalence and significant morbidity resulting from estrogen receptor positive (ER+) breast adenocarcinomas, there are only a few models of this cancer subtype available for drug development, and arguably none for studying etiology. Those models that do exist have questionable clinical relevance. Methods Given our goal of developing luminal models, we focused on six cell lines derived by minimal mutagenesis from normal human breast cells, and asked if any could generate clinically relevant xenografts, which we then extensively characterized. Results Xenografts of one cell line, 184AA3, consistently formed ER+ adenocarcinomas that had a high proliferative rate and other features consistent with “luminal B” intrinsic subtype. Squamous and spindle cell/mesenchymal differentiation was absent, in stark contrast to other cell lines that we examined or others have reported. We explored intratumoral heterogeneity produced by 184AA3 by immunophenotyping xenograft tumors and cultured cells, and characterized marker expression by immunofluorescence and flow cytometry. A CD44High subpopulation was discovered, yet their tumor forming ability was far less than CD44Low cells. Single cell cloning revealed the phenotypic plasticity of 184AA3, consistent with the intratumoral heterogeneity observed in xenografts. Characterization of ER expression in cultures revealed ER protein and signaling is intact, yet when estrogen was depleted in culture, and in vivo, it did not impact cell or tumor growth, analogous to therapeutically resistant ER+ cancers. Conclusions This model is appropriate for studies of the etiology of ovarian hormone independent adenocarcinomas, for identification of therapeutic targets, predictive testing and drug development.
The evidence for the importance of the microenvironment in regulating cell behavior and maintaining tissue architecture is clear 1-3. Given this fact, it is essential to characterize the elaborate composition of tissues, which allows us to study the elaborate and dynamic interplay among the cellular and molecular constituents. Understanding these processes and determining how they coordinate to form and maintain tissues is a key question in biology. Yet, despite all that has been learned about the breast over the years, so much remains unknown about the orchestration and functional capabilities of these elements and how and why they sometimes lead to tumor formation. Although there is general agreement to the fundamental cellular composition of the breast, newer technologies and reagents permit exploring the cellular heterogeneity with unprecedented detail and clarity. Our goal here was to develop a fluorescence-assisted cell sorting (FACS) antibody panel and comprehensive gating strategy capable of resolving every major cell type in the breast, permitting their isolation to near purity (>99.5%). Using extensive immunostaining of reduction mammoplasty specimens to guide panel design, we developed a method capable of resolving 12 different epithelial and stromal cell populations, including several different epithelial fractions, myoepithelial cells, fibroblasts, adipocytes, leukocytes, pericytes, erythrocytes, adipose-derived mesenchymal stem cells, and both lymphatic and capillary endothelial cells. RNA from each population has been extracted and the transcriptional profiles will be determined by RNA sequencing to confirm each population's identity and lend insight to a number of biological questions. Functional studies are also ongoing, but we have to date successfully cultured each population (excluding blood cells) for several passages, using different media and ECM substrata. Future studies will be aimed at developing heterotypic culture models using these cells, but to also use our new knowledge and experience to similarly explore the heterogeneity of ductal carcinoma in situ (DCIS) and invasive breast cancers. We will present the development of this method and characterization to date of the sorted cell populations. 1. Bissell, M.J. & Hines, W.C. Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat Med 17, 320-329 (2011). 2. Witkiewicz, A.K., Dasgupta, A., Sotgia, F., Mercier, I., Pestell, R.G., Sabel, M., Kleer, C.G., Brody, J.R. & Lisanti, M.P. An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers. The American journal of pathology 174, 2023-2034 (2009). 3. Ghajar, C.M., Meier, R. & Bissell, M.J. Quis custodiet ipsos custodies: who watches the watchmen? The American journal of pathology 174, 1996-1999 (2009). Citation Format: William C. Hines, Kate Thi, Maria Rojec, Gaelen Stanford-Moore, Mina J. Bissell. A cytometric atlas of the human breast: Comprehensive characterization reveals 12 distinct cell populations. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B86. doi:10.1158/1538-7445.CHTME14-B86
A fundamental question in biology, central to our understanding of cancer and other pathologies, is determining how different cell types coordinate to form and maintain tissues. Recognizing the distinct features and capabilities of the cells that compose these tissues is critical. Unfortunately, the complexity of tissues often hinders our ability to distinguish between neighboring cell types and, in turn, scrutinize their transcriptomes and generate reliable and tractable cell models for studying their inherently different biologies. A lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues have impeded progress. Here, we will describe such a method for the breadth of cell types composing the human breast. Furthermore, we have sequenced mRNAs from each purified population and investigated transcriptional patterns that reveal their distinguishing features. These analyses have exposed differentially expressed genes and enriched biological pathways that capture the essence of each cell type, along with transcripts that display intriguing expression patterns. These data, analytic tools, and transcriptional analyses form a rich resource whose exploration provides remarkable insights into the inner workings of the cell types composing the breast, thus furthering our understanding of the rules governing normal cell and tissue function. Citation Format: Katelyn Del Toro, Rosalyn W. Sayaman, Kate Thi, Yamhilette Licon-Munoz, William C. Hines. A cellular and transcriptomic dissection of the human breast for studying mechanisms of cell and tissue function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4652.
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