Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

Abstract

Background: To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo.

Methods: Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels.

Results: Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA.

Conclusions: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.

Keywords: brain tumors; exosomes; extracellular vesicles; microRNA; microglia.

Fig. 1.

Exposure of primary mouse microglia to GBM-EVs directs them toward a tumor-associated phenotype. (A) EVs isolated from GBM2 cells expressing palmGFP were incubated for 24 h with primary mouse microglia, followed by confocal fluorescent microscopy using a 20× objective and DAPI staining. (B) Microglia were exposed for 48 h to GBM-EVs (black bars) and compared with untreated cells (white bars). Cytokines increasing over 50% are indicated by black arrowheads, those decreasing by over 50% by white arrowheads. (C) Microglia were exposed to GBM-EVs every 24 h for 5 days. Viability was measured after 7 days and compared with untreated cells (mean ± SEM, *P < .05). (D) Arg-1 mRNA was quantitated in microglia after exposure to GBM-EVs for 24 and 48 h (mean ± SEM, n = 2, *P < .05). Scale bar, 20 μm in (A).

Fig. 2.

EVs secreted by GBM cells are enriched for many miRNAs. (A) MiRNA array analyses of 2 human primary GBM cell lines and EVs secreted by them were performed and a heat map was created using CIMminer. (B) Ratio of intensity in EVs compared with cells is presented in log2 scale for all 1146 miRNAs. (C) Comparison of the 20 most highly enriched miRNAs in EVs from GBM1 and GBM2 cells.

Fig. 3.

GBM cells release EVs enriched for miR-451 and miR-21 compared with microglia. (A and B) Levels of miR-21 (A) and miR-451 (B) in EVs and GBM2 cells were analyzed (mean ± SEM, n = 2). (C) Isolated GBM-EVs were separated on a sucrose density gradient. Levels of miR-21 and miR-451 in the different fractions were analyzed (mean ± SEM, n = 2). Protein levels of ALIX were analyzed by western blotting. (D and E) Levels of miR-451 (D) and miR-21 (E) in GBM cells, GBM-EVs, and primary mouse microglia were analyzed (Ct values presented, n = 2, *P < .05).

Fig. 4.

GBM-derived EVs increase miR-21 and miR-451 levels and decrease c-Myc mRNA levels in primary mouse microglia. (A and B) Primary microglia were exposed to GBM-EVs for 24 and 48 h. Levels of miR-21 (A) and miR-451 (B) were quantitated (mean ± SEM, n = 5, ***P < .001). (C) Levels of c-Myc mRNA were measured (mean ± SEM, n = 6, *P < .05). (D) Schematic representation of 3′UTR of mouse c-Myc mRNA with miR-451 and miR-21 binding sites predicted using computational microRNA target software (http://www.microrna.org). (E and F) Levels of miR-21 (E) and miR-451 (F) in GL261 cells and EVs released from them were analyzed (mean ± SEM, n = 2).

Fig. 5.

In vivo visualization of glioma-derived EVs and uptake by microglia/macrophages. (A) MP-IVM images from a GL261-Fluc-mC-palmtdT in a C57BL/6 mouse implanted with a brain window. Injected i.v. was 150 kDa fluorescein isothiocyanate–dextran to label the blood vasculature. (B) MP-IVM images from a GL261-Fluc-mC-palmtdT tumor in a CX3CR1^GFP/+ mouse brain. Panels on the right show magnified subregions of panel on the left. (C) Individual sections highlighting intracellular localization of red punctae. (D) Frequency distribution of number of discernible red punctae per cell. (E) Tumor size of GL261-Fluc-mC-palmtdT cells implanted intracranially into CX3CR1^+/GFP mice monitored by bioluminescence in vivo Fluc imaging. Cryosections were performed to visualize in vivo EV uptake. Released EV-like entities (palmtdT+) were readily observed around the tumor, as well as within GFP+ cells. Nuclei were visualized by DAPI. Arrows indicate red punctae within GFP+ cells. Bar = 100 000 nm.

Fig. 6.

Microglia isolated from brains of mice with glioma show increased levels of miR-21 and decreased c-Myc mRNA. (A) Representative images of FACS cells from brains of mice without and with gliomas; macrophages and microglia (GFP+, fluorescein isothiocyanate) and tumor cells (mC/palmtdTomato+, PE-A). (B) The % living FACS GFP+ cells from control brains and tumor-implanted brains were determined (mean ± SEM, ***P < .001). (C) The PE-A mean fluorescence of sorted microglia from tumor compared with control brains (**P < .01). (D) Cells sorted based on GFP+ intensity were analyzed for mRNA levels of P2ry12 and Ccr2 (*P < .05, **P < .01), (E) miR-21 levels (*P < .05, **P < .01), and (F) c-Myc levels (***P < .001). (B-F) All graphs include a total of 8 control mice and 9 mice implanted with GBM cells (mean ± SEM, n = 3).