Akagi Takanori, Kato Kei, Kobayashi Masashi, Kosaka Nobuyoshi, Ochiya Takahiro, Ichiki Takanori
Department of Bioengineering, School of Engineering, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, Japan.
Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo Japan.
PLoS One. 2015 Apr 30;10(4):e0123603. doi: 10.1371/journal.pone.0123603. eCollection 2015.
Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.
包括外泌体和微囊泡在内的细胞外囊泡（EVs）在细胞生物学和医学领域引起了广泛关注。为了更好地理解EVs并进一步探索其应用，需要开发用于生物纳米囊泡的分析方法。特别是，考虑到EVs的异质性，需要能够测量单个囊泡的方法。在此，我们报告芯片上免疫电泳可为单个EVs的差异蛋白表达谱分析提供一种有用的方法。使用包含微毛细管电泳芯片和激光暗场显微成像系统的测量平台，对从MDA-MB-231人乳腺癌细胞培养上清液中收集的EVs进行了电泳实验。与正常免疫球蛋白G（IgG）同型对照相比，与抗人CD63（外泌体和微囊泡标志物）抗体反应的EVs的zeta电位分布显示出明显的正向偏移。因此，芯片上免疫电泳可以灵敏地检测EVs上CD63糖蛋白的过表达。此外，为了探索芯片上免疫电泳在癌症诊断中的适用性，用该方法分析了从小鼠肿瘤模型血液中收集的EVs。通过比较EVs与正常IgG、抗人CD63和抗人CD44（癌症干细胞标志物）抗体进行免疫化学反应后的zeta电位分布，在实际样品中差异检测到了血液中循环的肿瘤来源的EVs。结果表明，本方法可能适用于液体活检，这是一种有前景的低侵入性癌症诊断方法。
