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// Liting Jin 1, 2, * , Ying Qu 2, * , Liliana J. Gomez 2 , Stacey Chung 2 , Bingchen Han 2 , Bowen Gao 2 , Yong Yue 3 , Yiping Gong 1 , Xuefeng Liu 4 , Farin Amersi 2 , Catherine Dang 2 , Armando E. Giuliano 2 and Xiaojiang Cui 2 1 Department of Breast Surgery, Hubei Cancer Hospital, Wuhan, 430079, China 2 Department of Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 3 Department of Radiation, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 4 Department of Pathology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA * These authors contributed equally to the work Correspondence to: Xiaojiang Cui, email: Xiaojiang.Cui@cshs.org Keywords: conditional reprogramming; estrogen receptor; mammary epithelial cells; luminal cells; myoepithelial cells Received: April 07, 2017 Accepted: October 30, 2017 Published: December 22, 2017 ABSTRACT Purpose: Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. Results: We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. Materials and Methods: We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. Conclusions: The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Human mammary gland development and differentiation are tightly regulated by hormones, growth factors, and microenvironmental cues. Rodent models have been used to help gain knowledge about mammary gland biology, but there are significant structural and hormonal response differences between the human and rodent mammary glands. Moreover, cultured immortalized human mammary cell lines have been widely used for in vitro experiments to study epithelial cell biology, but it has been questioned whether they faithfully recapitulate normal breast cells. Conditional reprogramming has recently emerged as an efficient method to induce rapid and inexhaustible in vitro proliferation of primary epithelial cells from normal and malignant tissues in two-dimensional (2D) culture conditions. However, studies using this method have not shown whether conditionally reprogrammed mammary epithelial cells can form defined structures in three-dimensional (3D) culture conditions. Therefore, our goal is to develop an appropriate in vitro model using conditional reprogramming to study human mammary cell and tissue function under 2D and 3D culture conditions. We cultured primary human mammary cells from normal prophylactic tissues or breast tumors using this method. Cell type heterogeneity, cellular marker expression, and structural arrangement were examined using immunofluorescence staining. We found that normal breast cells grown under this culture condition exhibited morphologic features of luminal cells (CK18, desmoglein 3, and CK19) and myoepithelial cells (vimentin, p63, and CK14), indicating maintenance of in vivo heterogeneity. CD49f and EpCAM double staining is commonly used to separate luminal, basal, and progenitor populations. Immunofluorescence and FACS analysis further revealed subpopulations with varying CD49f and EpCAM expression profiles in the normal primary cultures, as well as detectable expression of ERα in earlier passages. Treatment with estradiol also stimulated cellular proliferation as detected by positive EdU staining. When grown in Matrigel/Collagen I gel, normal primary cells self-organize into two distinct 3D structures that are composed of densely packed cells or a spherical structure containing a lumen, which express either luminal or myoepithelial cell markers, respectively. CK8-positive luminal cells that form the lumen can differentiate into milk-producing cells in the presence of a prolactogenic growth condition. Tumor cells extracted from breast cancer patients showed expression for either basal (CK18 and FOXC1) or luminal (CK14 and ER-positive) markers in 2D cultures. Our ongoing work entails delineating the long-term culture effect on primary mammary cell fate and function and the tumorigenic property of primary breast tumor cells. The current findings uncover an in vitro model that may be a valuable tool to study mammary cell function and can potentially be used to elucidate mechanisms involved in mammary tumorigenesis. Citation Format: Stacey Chung, Liting Jin, Ying Qu, Liliana J. Gomez, Bingchen Han, Bowen Gao, Xuefeng Liu, Farin Amersi, Catherine Dang, Armando E. Giuliano, Xiaojiang Cui. Assessment of conditional reprogramming to generate 2D and 3D primary human mammary cell culture models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A59.