Fibroblasts direct differentiation of human breast epithelial progenitors

Background: Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we established and characterized a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics. Methods: Primary lobular and interlobular fibroblasts were transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the established cell lines along lobular and interlobular pathways was determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties were further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo. The cells' physiological relevance for parenchymal differentiation was examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal or myoepithelial progenitors. The co-cultures were immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth, and luminal epithelial maturation. In extension, myoepithelial progenitors were tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-ß)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts were incubated with the TGF-ß signaling inhibitor, SB431542, prior to heterotypic co-culture with luminal cells. Results: hTERT immortalized fibroblast cell lines retained critical phenotypic traits in culture and linked to primary fibroblasts. Cell culture assays and transplantation to mice showed that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts supported a high level of branching morphogenesis by luminal cells, interlobular fibroblasts supported ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relied on intact TGF-ß signaling. Conclusions: The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines, we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer. Overall design: Duplicate primary lobular and interlobular breast fibroblastic cells. Duplicate immortalized lobular and interlobular breast fibroblastic cells.

研究背景：乳腺癌起源于人类乳腺内被称为终末导管小叶单位（terminal duct lobular units, TDLUs）的特定区域。这类结构属于相对动态的组织，其特征为受性激素驱动的周期性上皮更新。TDLUs由独特的实质成分构成，嵌入于富含成纤维细胞的小叶间质中。本研究建立并鉴定了一种新型人类乳腺小叶成纤维细胞系，并以小叶间成纤维细胞作为对照，旨在探究区域特异性间质信号在调控TDLU动态平衡中的作用。

实验方法：原代小叶成纤维细胞与小叶间成纤维细胞经转导以表达人端粒酶逆转录酶（human telomerase reverse transcriptase, hTERT）。通过免疫细胞化学染色与全基因组RNA测序，鉴定所建立细胞系沿小叶及小叶间通路的分化特征。进一步通过体外培养及体内实验分析间充质干细胞（mesenchymal stem cell, MSC）的分化谱，以表征细胞的功能特性。将细胞与经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化的正常乳腺原代管腔上皮祖细胞或肌上皮祖细胞进行异型共培养，以检验其对实质细胞分化的生理相关性。对共培养体系进行免疫染色，以定量评估上皮分支形态发生、极化、增殖及管腔上皮成熟情况。此外，还在体外培养及小鼠异种移植模型中检测了肌上皮祖细胞的管腔分化能力。为阐明转化生长因子-beta（transforming growth factor-beta, TGF-β）介导的信号串扰在TDLU样形态发生与分化中的意义，在与管腔细胞进行异型共培养前，先用TGF-β信号通路抑制剂SB431542处理成纤维细胞。

实验结果：经hTERT永生化的成纤维细胞系在体外培养中保留了原代成纤维细胞的关键表型特征。细胞培养实验与小鼠移植实验均证实，成纤维细胞的来源决定了上皮祖细胞的TDLU样与导管样分化方向。小叶成纤维细胞可促进管腔上皮细胞发生高水平的分支形态发生，而小叶间成纤维细胞则支持导管样肌上皮特征的形成。TDLU样形态发生至少部分依赖于完整的TGF-β信号通路。

研究结论：正常乳腺间质中最主要的细胞类型——成纤维细胞，在指导上皮分化过程中的重要作用目前仍知之甚少。通过建立小叶成纤维细胞与小叶间成纤维细胞系，本研究证实上皮祖细胞的位点特异性分化受到间质信号的调控。本研究结果印证了间质微环境的细微差异在正常乳腺发育乃至乳腺癌发生过程中具有至关重要的意义。

实验整体设计：设置两组原代乳腺成纤维细胞（小叶型与小叶间型）生物学重复，以及两组永生化乳腺成纤维细胞（小叶型与小叶间型）生物学重复。