On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells

Abstract

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.

Measurement of Individual Nanobioparticles on Microfluidic Chips by Laser Dark-field Imaging

“…2(a) [18,19]. Briefly, the platform comprises a microcapillary electrophoresis ( CE) chip, a pair of platinum electrodes, a DC power supply [Matsusada Precision HVL-1.1P(A)], a 404 nm laser source (Coherent Obis laser system, 50 mW), a microscope (Nikon, Ti-U), and a COMS camera (Andor Neo sCOMS).…”

“…Zeta potential of individual exosomes was evaluated by a procedure described previously [17][18][19]. Briefly, after of a sample chip, the chip was placed on the stage of an inverted microscope, and then electrodes were dipped into the reservoirs at both ends of the microchannel.…”

“…Recently, we have developed an on-chip microcapillary electrophoresis ( CE) platform equipped with a laser-dark field microscope that enables the zeta potential of individual nanobioparticles to be obtained [17][18][19]. Additionally, an analytical method for profiling the surface proteins of individual exosomes that employs the principle of on-chip particle immunoelectrophoresis has been proposed [19].…”

“…Additionally, an analytical method for profiling the surface proteins of individual exosomes that employs the principle of on-chip particle immunoelectrophoresis has been proposed [19]. Using the system, surface proteins can be analyzed at the single-particle level.…”

Advances in the Treatment of Breast Cancer-Emphasis on fertility preservation-A Case Report

“…With the recent advances in breast cancer treatment the 5 year survival rate has increased to 89.7% in the United States [2]. With further improvements in the treatment of breast cancer and more sophisticated models one expects there to be an increased number of young women who may require fertility treatments [3][4][5][6]. "With newer methods regarding breast cancer treatment, five year survival rates are anticipated to improve as well as 20 year survival rates.…”