Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5–40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring

Wnt/β-catenin signaling pathway inhibits the proliferation and apoptosis of U87 glioma cells via different mechanisms

<div><p>The Wnt signaling pathway is necessary for the development of the central nervous system and is associated with tumorigenesis in various cancers. However, the mechanism of the Wnt signaling pathway in glioma cells has yet to be elucidated. Small-molecule Wnt modulators such as ICG-001 and AZD2858 were used to inhibit and stimulate the Wnt/β-catenin signaling pathway. Techniques including cell proliferation assay, colony formation assay, Matrigel cell invasion assay, cell cycle assay and Genechip microarray were used. Gene Ontology Enrichment Analysis and Gene Set Enrichment Analysis have enriched many biological processes and signaling pathways. Both the inhibiting and stimulating Wnt/β-catenin signaling pathways could influence the cell cycle, moreover, reduce the proliferation and survival of U87 glioma cells. However, Affymetrix expression microarray indicated that biological processes and networks of signaling pathways between stimulating and inhibiting the Wnt/β-catenin signaling pathway largely differ. We propose that Wnt/β-catenin signaling pathway might prove to be a valuable therapeutic target for glioma.</p></div

Differential expression genes analysis base on KEGG in three groups.

<p>Differential expression genes analysis base on KEGG in three groups.</p

KEGG pathways of differential expression genes involved in cell growth, death and signaling transduction.

<p>KEGG pathways of differential expression genes involved in cell growth, death and signaling transduction.</p

Stimulating and inhibiting Wnt/β-catenin in U87 glioma cells.

<p>(A-C) β-catenin localized to cytoplasm and nucleus under the treatment of IWR-1-endo (IWR), ICG-001 (ICG) and AZD2858 (AZD). (D) Stimulating Wnt/β-catenin signaling pathway promoted expression of Wnt/beta-catenin signaling pathway target genes, compared with inhibiting Wnt/β-catenin signaling pathway. *** means <i>p</i> < 0.001.</p

Clustering and characterization of the differential expression of genes.

<p>(A) The differential expression genes in Wnt stimulating group, were selected for cluster analysis. (B) The differential expression genes in Wnt inhibiting group, were slected for cluster analysis. (C) Categories of 10 top enriched biological process GO term in Wnt stimulating group. (D) Categories of 10 top enriched biological process GO term in Wnt inhibiting group. FDR-adjusted p-value<0.05 and fold-change >2.0.</p

Venn diagrams of differentially expressed genes between different groups.

<p>(A) Up-regulated genes between groups. (B) down-regulated genes between groups. The diagram showing the intersection of genes differentially expressed between Wnt stimulating groups and control (red), between Wnt inhibiting group and control (blue), between Wnt stimulating group and Wnt inhibiting group (green). FDR-adjusted p-value<0.05 and fold-change >2.0.</p

Immunofluorescence images of cultured U87 glioma cells.

<p>neural marker Tuj1 (A), neural progenitor cells marker NESTIN (B, green), glial marker GFAP (C, green), stem cell marker OCT4 (D, green), NANOG (G, green), SOX2 (H, green). The glioma stem cell marker CD133 is showed in (F, green). β-catenin (D, green) can be monitored in U87 glioma cells. Nuclei indicated by PI (red) in all images. Bars: A-E, 100um; F-H, 200um.</p

Effects of ICG-001 and AZD2858 on cell proliferation, colony formation and cell cycle of U87 glioma cells.

<p>(A) Cell viability for each media condition. (B) Percentage of single colony forming under different media condition. (C) Single colony forming of ICG-001 treated cells, AZD2858 treated cells and control cells under the same media condition. (D) Ki67 expression (green) in each groups. (E) Percentage of Ki67 positive cells. (F) Cell cycle of AZD2858 treated cells, ICG-001 treated cells and control. (G) Percentage of cells in G₀/G₁ phase, S phase and G2/M phase, 48 hour after cell synchronization. * means <i>p</i> < 0.05; ** means 0.001<<i>p</i> < 0.01; *** means <i>p</i> < 0.001.</p

Time-course of EGFP-U87 glioma cell aggregates invasion into Matrigel.

<p>3D tumor spheroid invasion assay. U87 spheroid 3D invasion into Matrigel was monitored every 24 hours using fluorescence microscope. (A) Non-treated EGFP-U87 cells. (B) ICG-001 treated EGFP-U87 cells. (C) AZD2858 treated EGFP-U87 cells. The white arrows indicate newly formed colonies in control group. Bar: 500um.</p