1. Research Center for Translational Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200120, China.
2. Department of Bioinformatics, School of Life Sciences and Technology, Tongji University, Shanghai, China.
3. Department of Gastroenterology, Shanghai East Hospital, Tongji University, Shanghai 200120, China.
4. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China.
5. Biomedical Engineering Department, Peking University, Beijing, China.
6. The Third Hospital of BaoGang Group, Baotou, China.
7. Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong, China.
*Equal contribution to this work.
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.
Keywords: bone metastasis, triple-negative breast cancer, cancer stem cell, malignancy