Multiplexed analysis of cell-specific single extracellular vesicle

Idiopathic pulmonary fibrosis (IPF) is an irreversible, fatal disease leading to organ failure. Even though accurate and early diagnosis is critical for patient treatment, there is no method for evaluating IPF progression precisely. Extracellular vesicles (EVs) in bronchoalveolar lavage fluid (BALF) could play important roles as cellular surrogates for molecular IPF diagnosis. However, the current bulk EV analysis prevents the accurate evaluation of EV biomarkers derived from various cell types. Here, we developed a multiplexed, single EV analysis platform for IPF markers on EVs from different cellular subtypes.
To select IPF markers that are clinically significant for each cell type, we reanalyzed the single-cell sequencing data of 5 IPF datasets. After making in vitro IPF models using epithelial cells, macrophages, and fibroblasts that expressed IPF markers, EVs were isolated from conditioned media using size-exclusion chromatography. The isolated EVs were labeled with TFP-fluorophores and immunolabeled with antibodies. We used a nanoplasmonic gold chip for a sensitive single EV analysis.
As we hypothesized that cell-specific markers were expressed exclusively in each type of cells. Using multichannel imaging, we quantified all EVs, cell-specific EVs, and IPF marker-positive EVs. The results show that dual-positive (marker and cell-specific) EVs significantly increased or decreased in IPF models (EpCAM+/MMP7+ for epithelial cell, CD44+/Col1A1+ for fibroblast, CD45+/THBS1+ for M1 macrophage, CD45+/SPP1+ for M2 macrophage), which was similar with cells. Even though we mixed the EVs, we could find the cell-specific EVs and evaluate the IPF markers in these EVs.
This study shows that multicolor imaging enabled molecularly profiling of IPF markers in cell-specific EVs by comparing control and IPF in vitro models. We will apply the technology to analyze EVs from human BALF samples and evaluate their diagnostic power for molecular diagnosis of IPF. This opens up a new possibility for detecting the disease by liquid biopsy assays.