Multiplexed profiling of single-cell extracellular vesicles secretion

Abstract

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

Microfluidic Single‐Cell Omics Analysis

“…Its merits, including simple microfabrication, well‐shaped structure, convenient operation without pumps, make it a widespread platform for high‐throughput single‐cell culture and in situ analysis . Additionally, the roofless microwell structure permits rapid and direct sample retrieval, and flexible capping manipulation endows the capability for secretion capture and detection . Tang et al adopted 200 000 addressable microwells to detect single metabolically active tumor cells.…”

Application of Microfluidic Chips in Separation and Analysis of Extracellular Vesicles in Liquid Biopsy for Cancer

“…The microchamber array contained 6343 identical units for single cell culture, and the antibody barcode array consisted of 9 parallel microchannels coated with different antibodies for EV capture and detection. Based on this high-throughput multiplexed platform, we revealed the single-cell heterogeneity of EV secretion ability [61].…”

“…In this section, we only discuss on-chip EV analysis. To confirm the size and morphology of separated EVs directly on chips, scanning electron microscopy and atomic force microscopy are generally performed [60,61,62,64,68,70,71,72,73,78,80,85]. However, both methods are not suitable for clinical application due to the complicated sample preparation steps and expensive machines.…”

“…Fluorescent imaging is the most common way to detect and quantify EVs on microfluidic chips. The captured EVs can be stained with fluorescence dyes that specifically stain EV membranes [60,73], or with fluorecein-labeled antibodies [61,65,66,67,71,72,73,81,82,83]. On-chip ELISA is another common method to quantitatively detect the expression of EV biomarkers [62,69,76,80].…”

“…Ji et al presented a single-cell EV capture and detection microfluidic platform based on immunoaffinity approach. Based on this high-throughput multiplexed platform, they revealed that the decrement of certain EV phenotypes (e.g., CD63+EV) was associated with the invasive feature of both oral squamous cell carcinoma (OSCC) cell lines and primary OSCC cells [61]. Lewis et al used an ACE microarray chip to separate EVs from plasma of colon cancer patients.…”