Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar;102(3):e100-e103.
doi: 10.3324/haematol.2016.153197. Epub 2016 Nov 17.

Extracellular vesicles released from chronic lymphocytic leukemia cells exhibit a disease relevant mRNA signature and transfer mRNA to bystander cells

Katrin S Reiners  1 , Olga Shatnyeva  1 , Elena Vasyutina  1   2 , Teresa Bösl  1 , Hinrich P Hansen  1 , Michael Hallek  1   2   3 , Marco Herling  1   2   3 , Elke Pogge von Strandmann  4   5
Affiliations

Extracellular vesicles released from chronic lymphocytic leukemia cells exhibit a disease relevant mRNA signature and transfer mRNA to bystander cells

Katrin S Reiners et al. Haematologica. 2017 Mar.
No abstract available

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Characterization of extracellular vesicles. (A) Size distribution of CLL-derived vesicles (untreated, left panel, or treated with a TLR9 agonist (CpG, right panel) analyzed by NanoSight Technology (NS300, Malvern). Mean size diameter of vesicles from untreated/treated samples was 106/124 nm for CLL1, 115/99 nm for CLL2 and 97/98 nm for CLL3. All samples were similarly diluted and measured with a sample flow rate of 10 (n=6). (B) Western blot analysis of the exosome-enriched proteins CD63 and CD9 (both antibodies from BioLegend). EV released by 5×109 CLL cells within 38–45 h were resuspended in 40 μL non-reducing lysis buffer; sodium dodecyl sulfate (SDS) gel electrophoresis was performed using 10 μL EV/lane in 12.5% SDS gels and subsequently proteins were transferred to a nitrocellulose membrane. (C) The amount of purified EV protein released by 108 cells was measured with Nanodrop1000 (Thermo Scientific) (n=11). (D) Flow cytometric analysis of CLL cells after 38–45 h culture in Panserin medium with or without 200 nM CpG. Most patients had IgHV mutations (see black circles or squares). Samples without mutations are represented by non-filled circles or non-filled squares. Cell viability was assessed with an apoptosis assay, staining with annexin V-PE and 7-AAD (BioLegend) (PS = phosphatidylserine). Cells were stained with CD69-FITC (BD Biosciences), CD25-FITC (BD Biosciences), purified CD54 and CD82 (both BioLegend), which were detected with goat-anti-mouse-PE (BioLegend). The analysis was performed with a FACS Calibur (BD Biosciences) or FACS Gallios (BeckmanCoulter). Dead cells were excluded by gating for 7AAD (BD Biosciences) negative cells (n=5–14). (E) Flow cytometric analysis of EV: EV were bound to polystyrene beads (Polysciences) and subsequently stained and analyzed as described in (D). Only EV derived from cell samples with a viability of more than 90% were used for further analysis (n=5–9). The IgHV status of one patient analyzed for CD54 expression (left panel) is unknown (labeled with an x). (F) One representative histogram for FACS data depicted in (E).
Figure 2.
Figure 2.
mRNA signature of CLL-derived vesicles. (A) Hierarchical clustering of mRNAseq data (left: all; right: selected for CLL-relevant genes) obtained using next-generation sequencing of RNA isolated from vesicles of normal B cells (obtained from 3 healthy donors) and CLL cells (obtained from 3 patients) cultured in Panserin medium with or without 200 nM CpG (maximal cell density of 2.5×107 cells/mL). Vesicles were purified after 38–45 h from supernatant by serial ultracentrifugation to extract RNA using RNAeasy Mini Kit (Qiagen). B: B-cell derived vesicles, CLL: CLL-derived vesicles, CpG: stimulated with CpG. (B) KEGG pathway analysis of sequencing data to identify top canonical pathways was performed with DAVID 6.7. The functional annotation clustering was performed with the highest classification stringency. The KEGG-pathway function included in DAVID 6.7 was used to interpret the data in the context of biological processes, pathways and networks. Both up- and down-regulated identifiers were defined as value parameters for the analysis. (C–E) Uptake of CLL-vesicles results in TCL1A, LYN and SYK expression in fibroblasts and macrophages. (C) CLL cells were labeled with 5 mM DiO for 10 min and incubated together with HDFn fibroblasts for 16 h. Live cell imaging was performed using a Nikon spinning disc. (D–E) HDFn fibroblasts or THP-1 cells (monocytic cell line) were incubated with purified CLL-EV from two different patients (43 and 64) for 48 h to exclude any contribution or interference with CLL debris. (D) Expression of TCL1A, SYK and LYN in untreated HDFn fibroblasts and THP-1 and in cells treated with CLL-vesicles was measured using quantitative real-time polymerase chain reaction (see also Online Supplementary Table S4). (E) Western blot of cell lysates from THP-1 and HDFn cells untreated (ut) or cultured with CLL-EV (n=2, two independent patients) detecting TCL1A protein.
See this image and copyright information in PMC

References

    1. Guieze R, Wu CJ. Genomic and epigenomic heterogeneity in chronic lymphocytic leukemia. Blood. 2015;126(4):445–453. - PMC - PubMed
    1. Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015;526(7574):525–530. - PMC - PubMed
    1. Ten Hacken E, Burger JA. Microenvironment interactions and B-cell receptor signaling in chronic lymphocytic leukemia: Implications for disease pathogenesis and treatment. Biochim Biophys Acta. 2016;1863(3):401–413. - PMC - PubMed
    1. Farahani M, Rubbi C, Liu L, Slupsky JR, Kalakonda N. CLL Exosomes modulate the transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p. PLoS One. 2015; 10(10):e0141429. - PMC - PubMed
    1. Yeh YY, Ozer HG, Lehman AM, et al. Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling. Blood. 2015;125(21):3297–3305. - PMC - PubMed

Publication types

MeSH terms