Capturing nascent extracellular vesicles by metabolic glycan labeling-assisted microfluidics

Extracellular vesicle (EV) secretion is a dynamic process crucial to cellular communication. Temporally sorting EVs, i.e., separating the newly-produced ones from the pre-existing, can allow not only deep understanding of EV dynamics, but also the discovery of potential EV biomarkers that are related to disease progression or responsible to drug intervention. However, the high similarity between the nascent and pre-existing EVs makes temporal separation extremely challenging. By co-translational introduction of azido groups to act as a timestamp for click chemistry labelling, researchers at Xiamen University develop a microfluidic-based strategy to enable selective isolation of nascent EVs stimulated by an external cue. In two mouse models of anti-PD-L1 immunotherapy, they demonstrate the strategy’s feasibility and reveal the high positive correlation of nascent PD-L1⁺ EV level to tumor volume, suggesting an important role of nascent EVs in response to immunotherapy in cancer treatment.

Microfluidic isolation of EVs with in vitro MGL

a Schematic illustration of biotin-linked MGL EVs captured by a streptavidin modified chip and detected by a fluorescence enzyme immunoassay. b Numerical simulations of velocity streamlines and turbulence flows formed on microgrooves in Z-X and Z-Y vertical cross sections of the herringbone chip channel. c The fluorescence intensity of MGL A375 EVs (in orange) and non-MGL A375 EVs (in gray) captured by Melac-chip. ΔFL = FL – FL₀, where FL₀ and FL are the fluorescence intensity detected by Melac-Chip before and after the addition of EVs. Statistical significance was determined by a two-tailed unpaired t-test. P = 6.8479 × 10^-10, ****P < 0.0001. n = 3 biologically independent experiments. Data shown as mean ± SD. d Representative fluorescence images of MGL A375 EVs and non-MGL A375 EVs captured by Melac-Chip. e Calibration curve for quantifying MGL EVs by Melac-Chip. n = 4 biologically independent experiments. Data shown as mean ± SD. f Scanning electron microscope (SEM) imaging of herringbone chip to show the surface morphology of the captured MGL EVs. g 3D confocal fluorescence microscopy showing the DiI-stained MGL EVs selectively captured by Melac-Chip. h Time curve for quantifying MGL EVs by Melac-Chip (left) and the corresponding representative fluorescence images (right). A375 cells were treated with 50 μM Ac₄ManNAz for different time (0, 4, 6, 36 and 60 h), then labeled with DBCO-PEG₄-biotin (12.5 μM) for 1 h, and finally captured by SA-Chip. n = 6 biologically independent experiments. Data shown as mean ± SD. Statistical significance was determined using Tukey’s Method with One-Way ANOVA. 0 h vs 4 h, P = 6.9914 × 10^-5. 6 h vs 36 h, P = 2.1714 × 10^-5. 36 h vs 60 h, P = 3.1468 × 10^-8. ****P < 0.0001.