The majority of the deaths from breast cancer is due to metastasis. Bone is the most common organ to which breast cancer cells metastasize. The mechanism regulating the bone-metastatic preference remains unclear; there is a lack of a gene signature to distinguish bone-metastatic breast cancer cells. Herein, florescence-labeled MDA-MB-231 cells were transplanted into the fat pads of of the mammary gland in nude mice to generate breast tumors. Tumor cells invaded into the circulation were tracked by in vivo flow cytometry system. Metastatic tumor cells in the bone were isolated using fluorescent-activated cell sorting technique, followed by assays of cell colony formation, migration and invasion, mammosphere formation in vitro, mammary gland tumorigenesis in vivo, and Next-Generation Sequencing analysis as well. Through tumor regeneration and cell sorting, two bone-metastatic cell sublines were derived from MDA-MB-231 cells; which showed higher abilities to proliferate, migrate, invade and epithelial-to-mesenchymal transit in vitro, and stronger ability to regenerate tumors and metastasize to the bone in vivo. Both cell sublines exhibited cancer stem cell-like characteristics including higher expression levels of stem cell markers and stronger ability for mommaspheres formation. Furthermore, a Normal Distribution-like pattern of the bone-metastatic cells invading into circulation was firstly identified. Deep-sequencing analysis indicated upregulation of multiple signaling pathways in regulating EMT, cell membrane budding and morphologic change, lipid metabolism, and protein translation, which are required to provide adequate metabolic enzymes, structural proteins, and energy for the cells undergoing metastasis. In conclusion, we established two bone-metastatic breast cancer cell sublines, carrying higher degree of stemness and malignancy. The gene signature distinguishing the bone-metastatic breast cancer cells holds therapeutic potentials in prevention of breast cancer metastasis to the bone.

中文翻译：

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骨转移性乳腺癌细胞中的癌症干细胞特性和基因特征。

大多数乳腺癌死亡是由于转移所致。骨是乳腺癌细胞最常见的转移器官。调节骨转移偏好的机制尚不清楚。缺乏区分骨转移性乳腺癌细胞的基因特征。在本文中，将荧光标记的MDA-MB-231细胞移植到裸鼠乳腺的脂肪垫中以产生乳腺肿瘤。通过体内流式细胞术系统追踪侵入循环的肿瘤细胞。使用荧光激活细胞分选技术分离骨中的转移性肿瘤细胞，然后分析细胞集落形成、迁移和侵袭、体外乳腺球形成、体内乳腺肿瘤发生。，以及下一代测序分析。通过肿瘤再生和细胞分选，从MDA-MB-231细胞中衍生出两种骨转移细胞亚系；其显示出更高的能力增殖，迁移，侵入和上皮-间充质转运体外，和再生能力更强的肿瘤和转移至骨的体内. 两种细胞亚系都表现出类似癌症干细胞的特征，包括更高的干细胞标志物表达水平和更强的母球形成能力。此外，首先确定了侵入循环的骨转移细胞的正态分布样模式。深度测序分析表明，在调节 EMT、细胞膜出芽和形态变化、脂质代谢和蛋白质翻译方面的多个信号通路上调，这些都是为发生转移的细胞提供足够的代谢酶、结构蛋白和能量所必需的。总之，我们建立了两个骨转移性乳腺癌细胞亚系，具有更高的干性和恶性程度。