Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling

Abstract

Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile.

Figure 1

Characterization of CLL exosomes in plasma. (A) Exosomes were purified by differential centrifugation from CLL patient plasma and subjected to NTA measurement for concentration and size distribution. The representative plot of each CLL sample present here was generated from the average of 5 30-second videos. (B) Dot plot showing the range of exosome concentrations detected in CLL plasma (n = 54). (C) Isolated CLL plasma-derived exosomes were immunoblotted for exosome markers CD63 and CD9. Purified exosomes showed enriched CD63 and CD9 expression compared with plasma samples.

Figure 2

Determination of surface protein expression on CLL exosomes by flow cytometry. (A) Purified CLL exosomes from plasma were first bound to beads with a size that can be detected by direct sorting. Then the exosome-bead complex was stained with fluorophore-conjugated primary antibodies or matched isotype controls and analyzed by flow cytometry. CLL exosomes expressed abundant levels of CD63, CD9, and CD37 but not CD41, CD3, or CD56. (B) The dot plots present a range of expression of each surface antigen on exosomes isolated from 12 CLL patients.

Figure 3

High levels of exosomes are detected in CLL plasma and their concentrations are not correlated with absolute lymphocyte counts. (A) Exosomes were isolated from 40 CLL patients’ plasma and 8 healthy donors’ plasma, and the exosome concentrations were determined by NTA. Plasma from CLL patients showed higher concentrations of exosomes compared with plasma from healthy donors (*P = .026). (B) No significant correlation between plasma exosome concentrations and absolute lymphocyte counts was determined, as calculated by Spearman’s correlation coefficient (n = 39, ρ = 0.04). (C) Plasma exosome concentrations in IGHV-unmutated CLL was not significantly higher than in IGHV-mutated CLL; however, there is a trend in which more unmutated CLL patients exhibit elevated exosome concentrations vs IGHV-mutated CLL patients.

Figure 4

BCR signaling regulates exosome secretion and exosome microRNA expression in CLL. (A) CD19⁺ CLL cells were purified and cultured in AIM V medium with or without α-IgM stimulation for 24 hours. CLL-derived exosomes were isolated from cell culture supernatant, and the exosome concentration was measured by NTA. α-IgM stimulation significantly increased exosome secretion from CLL cells (*P = .013). (B) CD19⁺ CLL cells were isolated and first treated with or without 1 μM ibrutinib for 1 hour and then washed to remove the drug. Cells were then stimulated with α-IgM for 24 hours, and exosomes were isolated from the supernatant for measurement of exosome concentration by NTA. In the 7 of 12 CLL cells that responded to α-IgM stimulation (*P = .011), ibrutinib treatment decreased α-IgM-induced exosome release (P = .064). (C) Exosome microRNA was also isolated from the supernatant of cells incubated with or without α-IgM, and analyzed for miR-150 and miR-155 expression. Both miR-150 and miR-155 showed significantly higher levels of expression after α-IgM stimulation (*P = .042 and *P = .014, respectively).

Figure 5

Exosome concentrations were decreased in CLL patients after receiving ibrutinib therapy. Exosomes were isolated from plasma collected before (cycle 1, day 1) and after ibrutinib treatment (cycle 2, day 1) in a phase 2 clinical trial (OSU-11133; #NCT01589302). Plasma exosome concentration in CLL patients after receiving ibrutinib therapy was significantly reduced compared with pretreatment (P = .05).

Figure 6

Confirmation of exosome and cellular expression of identified microRNAs by quantitative real-time PCR. (A) Exosomes were isolated from 1 mL frozen plasma samples from 69 CLL patients and 15 healthy donors. Isolated exosome microRNA was examined by quantitative real-time PCR to determine the expression of miR29a-c, miR-150, and miR-155. All these microRNAs were expressed at significantly higher levels in CLL patients compared with healthy donors (***P < .001 for all comparisons). The expression data shown here are in a scale of negative CT (cycles required to reach threshold of detection), where a higher number represents higher expression and vice versa. (B) microRNAs were also isolated from the corresponding CD19⁺ CLL or normal B cells and analyzed by quantitative real-time PCR. Cellular levels of miR-150 and miR-155 were significantly higher in tumor cells from CLL patients vs normal B cells from healthy donors (*P = .029 and ***P < .001, respectively). There were no significant differences in the expression of cellular miR-29a-c between CLL patients and healthy donors.