Multiplexed Profiling of Single-cell Extracellular Vesicles Secretion

Abstract

Extracellular vesicles (EVs) are important intercellular mediators regulating health and disease. Conventional EVs surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EVs secretion. Herein, by using spatially patterned antibodies barcode, we realized multiplexed profiling of single-cell EVs secretion from more than 1000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to deep understanding of previously undifferentiated single cell heterogeneity underlying EVs secretion. Notably, we observed the decrement of certain EV phenotypes (e.g. CD63+ EVs) were associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EVs secretion and cytokines secretion simultaneously from the same single cells to investigate multidimensional spectrum of intercellular communications, from which we resolved three functional subgroups with distinct secretion profiles by visualized clustering. In particular, we found EVs secretion and cytokines secretion were generally dominated by different cell subgroups. The technology introduced here enables comprehensive evaluation of EVs secretion heterogeneity at single cell level, which may become an indispensable tool to complement current single cell analysis and EV research. Significance Extracellular vesicles (EVs) are cell derived nano-sized particles medicating cell-cell communication and transferring biology information molecules like nucleic acids to regulate human health and disease. Conventional methods for EV surface markers profiling can't tell the differences in the quantity and phenotypes of EVs secretion between cells. To address this need, we developed a platform for profiling an array of surface markers on EVs from large numbers of single cells, enabling more comprehensive monitoring of cellular communications. Single cell EVs secretion assay led to previously unobserved cell heterogeneity underlying EVs secretion, which might open up new avenues for studying cell communication and cell microenvironment in both basic and clinical research.

Cargo and cell‐specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis

“…An advantage of the EV-MAC approach described here is the relative ease by which EVs can be spinnoculated onto coverslips and subsequently analysed using conventional immunofluorescent imaging techniques [29,31]. Notably, other studies have previously demonstrated that immunofluorescent interrogation of EV populations is possible [35][36][37][38][39][40][41][42][43]. However, the workflow we describe allows the heterogeneity of EV populations to be rapidly assessed by investigators without super-resolution or singlemolecule approaches described in prior studies.…”

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

“…In a single-cell in vitro analysis, some cells secreted little to no EVs, while other cells exhibited “super-secretor” phenotypes and produced ten-times more than an average cell. Furthermore, EV secretion increases proportionally with the number of neighboring cells indicating paracrine signaling effects regulate EV secretion (Ji et al, 2019 ). In vitro live-tracking of transgenic CD63 fused with a pH-sensitive optical (green fluorescent protein) reporter suggests that a single cell can have between 1 and 15 multivesicular endosome-plasma membrane fusion events (intralumenal vesicle release) per minute (~10 3 to 2 × 10 4 release events per cell, per day) considering variance within and between cell lines among the three human cell lines tested (Bebelman et al, 2020 ).…”

“…For more detail on Dynabeads and bead-based EV capture (see Ugelstad et al, 1987 ; Jorgedal et al, 2007 ; Oksvold et al, 2015 ). Due to the extensive EV heterogeneity we are beginning to uncover with single-EV analytics (Lee et al, 2018 ; Ji et al, 2019 ), both the strengths and weaknesses of biochemical specificity are becoming apparent. We suspect that IAC and biochemical capturing techniques as a whole are limited with regard to biological insights, insofar as their outcomes may not be generalizable between biological systems.…”

“…They then applied three fluorescently-labeled antibodies per imaging cycle, and up to 11 protein markers in total per EV (Lee et al, 2018 ). For single-cell derived EV analysis, a microwell platform with fluorescent, multiplexed antibody barcoding was recently described (Ji et al, 2019 ). Furthermore, a custom, high-sensitivity flow cytometer was capable of detecting single phycoerythrin-conjugated antibodies, and two-color fluorescence flow cytometry was implemented to analyze EV surface protein expression in plasma-derived EVs from colorectal cancer patients (Tian et al, 2018 ).…”

Localized cell-surface sampling of a secreted factor using cell-targeting beads

“…12 However, these methods often rely on sampling processes wherein important effector molecules may be degraded, sequestered, or converted on time scales faster than those required for sample preparation and analysis, resulting in diminished signal; further, these readouts are typically used as end-point analyses that lack the temporal resolution provided by in situ methods and analyses. 13 More targeted approaches that integrate sample collection and readout, such as compartmentalized microfluidic cell culture platforms for in situ bead-based assays 14,15 , integrated microchip single-cell culture and analysis devices 16 , small-volume cell-encapsulation and -sensor systems [17][18][19][20][21] and enzyme-linked immunosorbent spot (ELISpot) assays 22,23 , address the limitations posed by traditional techniques and enable precise in situ analysis of culture systems at flexible timepoints throughout the experiment. These integrated culture and analysis platforms allow users to probe specific phenomena using systems with excellent spatial and temporal detection resolution, as well as single-cell resolution for secretome analysis.…”