Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells
TL;DR: GBM‐derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor‐supportive phenotypes observed in patients.
Abstract: Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients.
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TL;DR: This review focuses on recent findings and knowledge gaps in the area of EV biogenesis, release, and uptake and highlights examples whereby EV cargoes control basic cellular functions, including motility and polarization, immune responses, and development, and contribute to diseases such as cancer and neurodegeneration.
952 citations
Cites background from "Glioblastoma-derived extracellular ..."
...macrophage differentiation towards a tumor supportive phenotype in culture [121]....
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TL;DR: The ways in which glioblastomas manipulate brain cells and immune cells in their environment to support tumour growth and the opportunities available for new therapies that disrupt these interactions are examined.
Abstract: Glioblastomas are heterogeneous and invariably lethal tumours. They are characterized by genetic and epigenetic variations among tumour cells, which makes the development of therapies that eradicate all tumour cells challenging and currently impossible. An important component of glioblastoma growth is communication with and manipulation of other cells in the brain environs, which supports tumour progression and resistance to therapy. Glioblastoma cells recruit innate immune cells and change their phenotype to support tumour growth. Tumour cells also suppress adaptive immune responses, and our increasing understanding of how T cells access the brain and how the tumour thwarts the immune response offers new strategies for mobilizing an antitumour response. Tumours also subvert normal brain cells - including endothelial cells, neurons and astrocytes - to create a microenviron that favours tumour success. Overall, after glioblastoma-induced phenotypic modifications, normal cells cooperate with tumour cells to promote tumour proliferation, invasion of the brain, immune suppression and angiogenesis. This glioblastoma takeover of the brain involves multiple modes of communication, including soluble factors such as chemokines and cytokines, direct cell-cell contact, extracellular vesicles (including exosomes and microvesicles) and connecting nanotubes and microtubes. Understanding these multidimensional communications between the tumour and the cells in its environs could open new avenues for therapy.
321 citations
TL;DR: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain, and support functional effects of GBM-released EVs following uptake intomicroglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins.
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.
239 citations
Cites methods from "Glioblastoma-derived extracellular ..."
...Variations in the number of EVs released by different GBM/glioma lines have been documented.(19) GBM2 cells which released more EVs than GBM1 cells were used in most experiments....
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TL;DR: This review focuses on the biogenesis and function of microglia-related extracellular vesicles and focus on their putative role in Alzheimer's disease pathology.
228 citations
TL;DR: The aim of this review was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches, and the realization of this ‘uniqueness’ is the first step in the right direction for the complete assessment of EVs.
Abstract: The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation This review comprises the knowledge on EV isolation techniques that are currently available The aim of this review was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches The realization of this ‘uniqueness’ is the first step in the right direction for the complete assessment of EVs
222 citations
Cites background from "Glioblastoma-derived extracellular ..."
...Another study demonstrated that density-gradient isolated glioblastoma-derived EVs modified the phenotype of monocytic cells (56)....
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References
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University Hospital of Lausanne1, University of Toronto2, Erasmus University Rotterdam3, University Medical Center Utrecht4, Queen's University5, European Organisation for Research and Treatment of Cancer6, University of Western Ontario7, Medical University of Vienna8, French Institute of Health and Medical Research9, National Institutes of Health10, University of Tübingen11, University of Calgary12
TL;DR: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up, and a benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years.
Abstract: BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nelia and Amadeo Barletta Foundation, Schering-Plough.
6,161 citations
"Glioblastoma-derived extracellular ..." refers background in this paper
...Despite extensive treatment, the prognosis of patients with glioblastoma multiforme (GBM), the most common primary brain tumor in adults, remains dismal with a median survival of only 15 months.(1) Many factors contribute to the malignant potential of GBMs, including the capacity of GBMs to modulate the immune system....
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Harvard University1, University of North Carolina at Chapel Hill2, University of California, Berkeley3, Washington University in St. Louis4, Lawrence Berkeley National Laboratory5, University of California, San Francisco6, Mayo Clinic7, Memorial Sloan Kettering Cancer Center8, SRA International9, Walter and Eliza Hall Institute of Medical Research10
TL;DR: A robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes is described and multidimensional genomic data is integrated to establish patterns of somatic mutations and DNA copy number.
5,764 citations
"Glioblastoma-derived extracellular ..." refers background or methods in this paper
...Note that classification as either 16/17, 18 or 23 corresponds to a TCGA-based classification as, respectively, neural, classical or mesenchymal.(35) [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary....
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...This corresponds to The Cancer Genome Atlas (TCGA) mesenchymal subtype.(35) The EV-low-associated tumors were classified as 16/17 or 18 (corresponding to the neural or classical subtype(35))....
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TL;DR: This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosomes preparations.
Abstract: Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.
4,492 citations
"Glioblastoma-derived extracellular ..." refers methods in this paper
...Besides the routinely used cell line U87-MG/EGFRvIII, the primary GBM stem cell-like culture GS184 was used as a source of EVs....
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...(b) tRPS-based quantification of GSC-derived EVs....
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...Highly sensitive mass spectrometry (MS) analysis was performed to quantify and compare the proteomic contents of GBM cells and their secreted EVs....
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...Sequential ultracentrifugation (two times at 100,000g for 70 min) was used for the large-panel isolation of GBM and non-GBM EVs.(26) Prior to these ultracentrifugation procedures, cell culture supernatants and Ficoll supernatants were cleared from cellular debris by centrifugation (300g for 10 min, followed by 4,000g for 1 hr) and filtering (0....
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...Despite their overlapping functions,48 macrophages and microglia are highly different cell types, and it is therefore conceivable that they respond differently to EVs....
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Harvard University1, University of North Carolina at Chapel Hill2, University of California, Berkeley3, Washington University in St. Louis4, Lawrence Berkeley National Laboratory5, University of California, San Francisco6, Mayo Clinic7, Memorial Sloan Kettering Cancer Center8, SRA International9, Walter and Eliza Hall Institute of Medical Research10
TL;DR: The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM) and proposed a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes as discussed by the authors.
Abstract: The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.
4,464 citations
TL;DR: Tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test by incorporating an mRNA for a reporter protein into them, and it is demonstrated that messages delivered by microvesicle are translated by recipient cells.
Abstract: Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.
4,118 citations