Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood–brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell–cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destruction of BBB through the abnormal localization of actin via the downregulation of its target gene,PDPK1. PDPK1 degradation by miR-181c leads to the downregulation of phosphorylated cofilin and the resultant activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate that systemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis of breast cancer cell lines and are preferentially incorporated into the brainin vivo. Taken together, these results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB.

Breast cancer patients often develop metastatic disease years after resection of the primary tumor. The patients are asymptomatic because the disseminated cells appear to become dormant and are undetectable. Because the proliferation of these cells is slowed, dormant cells are often unresponsive to traditional chemotherapies that exploit the rapid cell cycling of most cancer cells. We generated a bone marrow–metastatic human breast cancer cell line (BM2) by tracking and isolating fluorescent-labeled MDA-MB-231 cells that disseminated to the bone marrow in mice. Coculturing BM2 cells with bone marrow mesenchymal stem cells (BM-MSCs) isolated from human donors revealed that BM-MSCs suppressed the proliferation of BM2 cells, decreased the abundance of stem cell–like surface markers, inhibited their invasion through Matrigel Transwells, and decreased their sensitivity to docetaxel, a common chemotherapy agent. Acquisition of these dormant phenotypes in BM2 cells was also observed by culturing the cells in BM-MSC–conditioned medium or with exosomes isolated from BM-MSC cultures, which were taken up by BM2 cells. Among various microRNAs (miRNAs) increased in BM-MSC–derived exosomes compared with those from adult fibroblasts, overexpression of miR-23b in BM2 cells induced dormant phenotypes through the suppression of a target gene, MARCKS, which encodes a protein that promotes cell cycling and motility. Metastatic breast cancer cells in patient bone marrow had increased miR-23b and decreased MARCKS expression. Together, these findings suggest that exosomal transfer of miRNAs from the bone marrow may promote breast cancer cell dormancy in a metastatic niche.

Metastasis is the main cause of cancer mortality for many types of cancer; however, difficulties remain in effectively preventing metastasis. It has been recently and widely reported that cancer-derived extracellular vesicles (EVs) contribute to cancer metastasis. Thus, therapeutic strategies targeting cancer-derived EVs hold great promise because of the possibility of EVs driving the cancer microenvironment toward metastasis. Here, we provide a novel strategy for therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model, establishing a rationale for further clinical investigation. Treatment with human-specific anti-CD9 or anti-CD63 antibodies significantly decreased metastasis to the lungs, lymph nodes, and thoracic cavity, although no obvious effects on primary xenograft tumor growths were observed. In in vitro and in vivo experiments, the EVs incubated with the targeted antibodies were preferentially internalized by macrophages, suggesting that antibody-tagged cancer-derived EVs would be eliminated by macrophages. Our results suggested that therapeutic antibody administration effectively suppresses EV-triggered metastasis in cancer and that the removal of EVs could be a novel strategy for cancer therapy.

Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs), were isolated from multiple human body fluids, and these “circulating miRNAs” have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs), which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs, however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

The tetraspanin CD63 is a highly N-glycosylated protein that is known to regulate cancer malignancy. However, the contribution of glycosylation of CD63 to cancer malignancy remains unclear. Previously, we reported that ribophorin II (RPN2), which is part of an N-oligosaccharyle transferase complex, is responsible for drug resistance in breast cancer cells. In this study, we demonstrate that cancer malignancy associated with the glycosylation of CD63 is regulated by RPN2. Inhibition of RPN2 expression led to a reduction in CD63 glycosylation. In addition, the localization of CD63 was deregulated by knockdown of RPN2. Interestingly, multidrug resistance protein 1 (MDR1) localization was displaced from the cell surface in CD63-silenced cells. CD63 silencing reduced the chemoresistance and invasion ability of malignant breast cancer cells. Furthermore, the enrichment of CD63/MDR1-double positive cells was associated with lymph node metastasis. Taken together, these results indicated that high glycosylation of CD63 by RPN2 is implicated in clinical outcomes in breast cancer patients. These findings describe a novel and important function of RPN2-mediated CD63 glycosylation, which regulates MDR1 localization and cancer malignancy, including drug resistance and invasion.

Extracellular vesicles (EVs) are nanometer-sized membranous vesicles and are involved in cell-to-cell communication. EVs contain several types of functional molecules, such as proteins, mRNAs, and microRNAs (miRNAs). Over the past several years, EVs have emerged as potential tools for a drug delivery system (DDS) that can target organs or cells. EVs have a function of organ tropism and are naturally occurring from cells. Therefore, EVs have expected as naturally DDSs, which have the organ tropism and a low side effect. Actually, some reports showed that EVs delivered drugs to specific organ. However, despite observed the organ tropism, the mechanisms of organ tropism of EVs are still unclear. Moreover, preservation and efficient collection of EVs are desired to be investigated. Here, we provide an overview of the methods for using EVs as DDSs.