Exosomal miR-19a and IBSP cooperate to induce osteolytic bone metastasis of estrogen receptor-positive breast cancer

Bone metastasis is an incurable complication of breast cancer. In advanced stages, patients with estrogen-positive tumors experience a significantly higher incidence of bone metastasis (>87%) compared to estrogen-negative patients (<56%). To understand the mechanism of this bone-tropism of ER+ tumor, and to identify liquid biopsy biomarkers for patients with high risk of bone metastasis, the secreted extracellular vesicles and cytokines from bone-tropic breast cancer cells are examined in this study. Both exosomal miR-19a and Integrin-Binding Sialoprotein (IBSP) are found to be significantly upregulated and secreted from bone-tropic ER+ breast cancer cells, increasing their levels in the circulation of patients. IBSP is found to attract osteoclast cells and create an osteoclast-enriched environment in the bone, assisting the delivery of exosomal miR-19a to osteoclast to induce osteoclastogenesis. Our findings reveal a mechanism by which ER+ breast cancer cells create a microenvironment favorable for colonization in the bone. These two secreted factors can also serve as effective biomarkers for ER+ breast cancer to predict their risks of bone metastasis. Furthermore, our screening of a natural compound library identifies chlorogenic acid as a potent inhibitor for IBSP-receptor binding to suppress bone metastasis of ER+ tumor, suggesting its preventive use for bone recurrence in ER+ patients.

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Software and code
Policy information about availability of computer code Data collection Immunofluorescence, immunohistochemistry and cellular images were acquired using Keyence All-in-one fluorescence microscope (BZ-X700).Laser fluorescence scanning was performed with GenePix® 4000B Microarray Scanner. Time lapse was performed with Olympus IX-70 system. Electron microscopy was performed with FEI Tecnai BioTwin Transmission Electron Microscope (120 keV). Confocal Microscopy was performed with Zeiss LSCM 510 Laser Scanning Confocal Microscope. Bioluminescence was acquired using Xenogen IVIS imaging system (Caliper Life Science). Immunoblotting images were captured by Amersham Imager 600 as well as X-ray film processor. Quantitative PCR was performed with CFX Connect Real Time PCR system (Bio-rad). Absorbance of light was measured by FilterMax F5 (Molecular Devices). Flow cytometry was performed with BD FACS Canto II Analyzer. X-ray images of mice was acquired through Faxitron Multifocus 10x15 Digital Radiography System.

Data analysis
Bioluminescence data was analyzed using Living Image (Caliper Life Science) version 4.7.3 software and Aura software (Spectral Instruments Imaging, LLC) version 3.2. ImageJ (FIJI, 1.52n) was used for image quantification analysis. Statistical analysis was performed by GraphPad Prism (version 8.0). Cell migration was analyzed with Chemotaxis and Migration Tool (Version 2.0, IBIDI).
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

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Sample size
Sample size was calculated based on previous experiment done (Liu Y, 2019; Xing F, 2018). Using power analysis, the anticipated means and standard error of means were included to determine the sample size that is expected to yield a power of approximately 80 percent using a pvalue of 0.05.
Data exclusions No data were excluded in our studies.

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Numbers of the experimental replication or the experiments that were performed independently for each specific result were indicated in the Figure Legends.
Randomization For cell experiments, all cells in each experiment were from the same pool of parental cells. All mice were age-and sex-matched (female mice) and then randomized into different experimental groups. All animals were maintained in the same environment and handled by the same procedure.

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For data collected by objective instruments, such as plate readers, qPCR cyclers, microscopy software, flow cytometers, animal IVIS systems, and western blotting, the investigators were not blinded to group allocation during data collection. However, investigator bias is not considered to contribute to the data because the investigator was blinded at the time of data analysis. Laboratory personnel were blind to animal randomization for drug treatment, which was performed by the PI. However, laboratory personnel could not be blinded during the experiment as they needed to know which groups to treat with which drugs. However, the laboratory personnel was blinded during the data analysis from each individual mice. Data analyses was performed by a biostatistician who was blinded to experimental groups.

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