American Society of Hematology

Figure 1

$Figure 1. EVs from CD40/IL-4–stimulated CLL cells are enriched for specific miRNAs. Dynamic light scattering (NanoSight) was used to compare (A) EV size between plasma of patients (CLL) (n = 23) and age-matched healthy donors (HD) (n = 10) and (B) EV particle number/concentration. EV numbers were significantly greater in CLL patients compared with HDs (Mann-Whitney test; P = .03). Horizontal bars represent median values. (C) Comparison of EV size distribution in 2 patients, 1 with high EV particle number (solid line) and the other with lower EV particle numbers (dashed line). (D) Lack of association between total white cell count (WCC; black circles) or platelet count (PLT; gray circles) and CLL-EV numbers. (E) Electron micrographs of CLL-EVs obtained after differential centrifugation of supernatant from CLL cells treated with anti-CD40 antibody (clone EA-5, 1 µg/mL), soluble CD40L (1 µg/mL), and IL-4 (20 ng/mL) for 36 hours. Top and bottom panels show representative CLL-EV particles obtained from 2 individual CLL patients. The size marker is 100 nm. (F) Unsupervised hierarchical clustering of miRNAs with significantly different expression between CLL-EVs and paired parental intracellular fractions from 8 CLL patients following CD40/IL-4 stimulation (Wilcoxon, Mann-Whitney test utilizing an FDR significance criterion limit of 0.05). (G) miRNAs that showed significant differences (Wilcoxon, Mann-Whitney test utilizing an FDR significance criterion limit of 0.01) in expression between CLL-EVs and paired parental intracellular fractions following CD40/IL-4 stimulation. Data are presented using box-and-whisker plots. miR-363, miR-374b, miR-323-3p, and miR-494 demonstrate increased expression in CLL-EVs whereas miR-150, miR-484, and miR-17 show reduced expression compared with parental CLL intracellular miRNA content. (H) Relative expression analysis plot. Cellular miRNA were ranked from the most highly expressed (relative expression arbitrarily set at 1) to the least expressed. The positions of the 4 miRNA whose expression level increased in CLL-EVs compared with parental CLL intracellular miRNA content is shown by the red arrows. The analysis shows that miRNAs that are highly expressed in CLL-EVs are a specific population and do not simply reflect the most highly expressed parental cellular miRNA content. (I) Pie charts showing relative distribution of miRNAs in parental CLL cells and paired CLL-EVs following CD40/IL-4 stimulation. The most highly expressed miRNAs in each compartment are labeled. n.s., not significant.$

EVs from CD40/IL-4–stimulated CLL cells are enriched for specific miRNAs. Dynamic light scattering (NanoSight) was used to compare (A) EV size between plasma of patients (CLL) (n = 23) and age-matched healthy donors (HD) (n = 10) and (B) EV particle number/concentration. EV numbers were significantly greater in CLL patients compared with HDs (Mann-Whitney test; P = .03). Horizontal bars represent median values. (C) Comparison of EV size distribution in 2 patients, 1 with high EV particle number (solid line) and the other with lower EV particle numbers (dashed line). (D) Lack of association between total white cell count (WCC; black circles) or platelet count (PLT; gray circles) and CLL-EV numbers. (E) Electron micrographs of CLL-EVs obtained after differential centrifugation of supernatant from CLL cells treated with anti-CD40 antibody (clone EA-5, 1 µg/mL), soluble CD40L (1 µg/mL), and IL-4 (20 ng/mL) for 36 hours. Top and bottom panels show representative CLL-EV particles obtained from 2 individual CLL patients. The size marker is 100 nm. (F) Unsupervised hierarchical clustering of miRNAs with significantly different expression between CLL-EVs and paired parental intracellular fractions from 8 CLL patients following CD40/IL-4 stimulation (Wilcoxon, Mann-Whitney test utilizing an FDR significance criterion limit of 0.05). (G) miRNAs that showed significant differences (Wilcoxon, Mann-Whitney test utilizing an FDR significance criterion limit of 0.01) in expression between CLL-EVs and paired parental intracellular fractions following CD40/IL-4 stimulation. Data are presented using box-and-whisker plots. miR-363, miR-374b, miR-323-3p, and miR-494 demonstrate increased expression in CLL-EVs whereas miR-150, miR-484, and miR-17 show reduced expression compared with parental CLL intracellular miRNA content. (H) Relative expression analysis plot. Cellular miRNA were ranked from the most highly expressed (relative expression arbitrarily set at 1) to the least expressed. The positions of the 4 miRNA whose expression level increased in CLL-EVs compared with parental CLL intracellular miRNA content is shown by the red arrows. The analysis shows that miRNAs that are highly expressed in CLL-EVs are a specific population and do not simply reflect the most highly expressed parental cellular miRNA content. (I) Pie charts showing relative distribution of miRNAs in parental CLL cells and paired CLL-EVs following CD40/IL-4 stimulation. The most highly expressed miRNAs in each compartment are labeled. n.s., not significant.

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