Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences , Tohoku University , Sendai 980-8578 , Japan.
Mol Pharm. 2019 Jan 7;16(1):292-304. doi: 10.1021/acs.molpharmaceut.8b00985. Epub 2018 Dec 3.
Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).
脑转移是癌症的常见并发症，至少部分是通过癌细胞衍生的外泌体被内化到脑毛细血管内皮细胞来介导的。阐明这种内化的机制很有意义，因为它可以帮助我们开发阻止脑转移的方法，同时为药物递送到大脑提供一种新的潜在途径。因此，本研究的目的是通过鉴定参与脑转移癌细胞系（SK-Mel-28）衍生的外泌体内化到血脑屏障内皮细胞（hCMEC/D3 细胞）的受体来解决这个问题。基于 sulfo-SBED 的交联和综合蛋白质组学的组合产生了 20 种蛋白质作为 hCMEC/D3 细胞中外泌体受体的候选物。在 37°C 下测量的 PKH67 标记的外泌体在 4°C 下的摄取显著降低了 95.6%，在存在 1mM RGD 肽（整合素配体）时降低了 15.3%。因此，我们专注于鉴定的 RGD 受体，整合素α5 和整合素αV，以及被报道作为腺病毒受体的 CD46，与整合素αV 一起。针对整合素α5 和整合素αV 的中和抗体混合物显著降低了 11.8%的外泌体摄取，而 CD46 siRNA 的应用则降低了 39.0%。免疫组织化学分析证实了 CD46 存在于人脑毛细血管内皮细胞中。这些结果表明 CD46 是 SK-Mel-28 衍生的外泌体被血脑屏障内皮细胞（hCMEC/D3 细胞）摄取的主要受体。
