Abstract B86: A cytometric atlas of the human breast: Comprehensive characterization reveals 12 distinct cell populations

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 population9s 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 don9t 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