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Physical and molecular landscapes of mouse glioma extracellular vesicles define heterogeneity

ABSTRACT: Extracellular vesicles (EVs) comprise a novel mechanism for cellular communication by transferring protein and nucleic acid cargo between cells. By utilizing a sequential filtration protocol, we were able to effectively separate microvesicles (MVs), exosomes and membrane-unbound ribonuclear protein complexes (RNPs) released from EGFR- (Acquaviva, 2011) and PDGFR-driven (Jun, 2018) genetically engineered mouse glioblastoma (GMB) cell lines. Regardless of the oncogenic driver of the tumor, different species of RNA partition into different extracellular compartments, with the mRNA in MVs most closely representing the cellular transcriptome, while miRNAs and tRNA fragments segregate into exosomes and RNPs, respectively. This EV RNA profile, combined with the cognate proteomics data, also serves as a reference for future GBM biomarker discovery.

ORGANISM(S): Mus musculus

PROVIDER: GSE123414 | GEO | 2018/12/07

REPOSITORIES: GEO

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Project description:Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naM-CM-/ve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively. All experiments were done in triplicates. Primary monocytes were collected from buffy coats (BC). The freshly isolated monocytes from three donors (Mono1-3) were either treated with GM-CSF or subjected to RNA isolation. Following treatment, MVs were isolated from the GM-CSF-treated macrophage cultures. RNA was isolated from the remaining cells for profiling (GM1-3). The isolated MVs were then used to treat new BC monocytes for 24 h (BC-GMCSF-MV24). A fraction of the new BC monocytes was subjected to RNA extraction for profiling (BC1-3). For THP1 cells, they were treated with either DMSO or PMA to produce MVs. The MVs were collected and the remaining cells lyzed for RNA extraction and profiling (DMSO1-3 and PMA1-3). The collected MVs from the DMSO or PMA-treated THP1 cells were incubated with new THP1 for 24 h and designated DMSO-MV24 or PMA-MV24. We had a total of 24 samples.

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Physical and molecular landscapes of mouse glioma extracellular vesicles define heterogeneity

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