期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：38
DOI：10.1073/pnas.2106630118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Extracellular vesicles (EVs) are omnipresent in humans and contribute to the intercellular communication between cells. Their content, size, quantity, and surface markers depend on their cytosolic origin and the protein complex involved in membrane trafficking. Hence, vesicles may serve as valuable biomarkers, accessible via liquid biopsies. As of today, however, phenotype-specific EV subpopulations have not been clearly identified, mainly because EVs can be taken up and modified by other cells, hindering the heterogeneous EV production of individual cells. The proposed method enables robust classification of the EVs secreted by single cells, assessing their heterogeneity with respect to surface markers. In the future, this tool can be employed to assess the release and uptake dynamics of EVs in a controlled environment.
Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.
