Roles of extracellular vesicles in glioblastoma: foes, friends and informers

Glioblastoma (GB) tumors are one of the most insidious cancers which take over the brain and defy therapy. Over time and in response to treatment the tumor and the brain cells in the tumor microenvironment (TME) undergo many genetic/epigenetic driven changes in their phenotypes and this is reflected in the cellular contents within the extracellular vesicles (EVs) they produce. With the result that some EVs try to subdue the tumor (friends of the brain), while others participate in the glioblastoma takeover (foes of the brain) in a dynamic and ever changing process. Monitoring the contents of these EVs in biofluids can inform decisions based on GB status to guide therapeutic intervention. This review covers primarily recent research describing the different cell types in the brain, as well as the tumor cells, which participate in this EV deluge. This includes EVs produced by the tumor which manipulate the transcriptome of normal cells in their environment in support of tumor growth (foes), as well as responses of normal cells which try to restrict tumor growth and invasion, including traveling to cervical lymph nodes to present tumor neo-antigens to dendritic cells (DCs). In addition EVs released by tumors into biofluids can report on the status of living tumor cells via their cargo and thus serving as biomarkers. However, EVs released by tumor cells and their influence on normal cells in the tumor microenvironment is a major factor in immune suppression and coercion of normal brain cells to join the GB “band wagon”. Efforts are being made to deploy EVs as therapeutic vehicles for drugs and small inhibitory RNAs. Increasing knowledge about EVs in the TME is being utilized to track tumor progression and response to therapy and even to weaponize EVs to fight the tumor

Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines

Background: Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. Methods: We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. Results: Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP-and PC-3-derived MVs highly correlated with prostate cancer progression. Conclusions: Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.Peer reviewe

BRAF(V600) inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF(V600) mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.1114sciescopu

Inhibition of extracellular vesicle-derived miR-146a-5p decreases progression of melanoma brain metastasis via Notch pathway dysregulation in astrocytes

Melanoma has the highest propensity of all cancers to metastasize to the brain with a large percentage of late-stage patients developing metastases in the central nervous system (CNS). It is well known that metastasis establishment, cell survival, and progression are affected by tumour-host cell interactions where changes in the host cellular compartments likely play an important role. In this context, miRNAs transferred by tumour derived extracellular vesicles (EVs) have previously been shown to create a favourable tumour microenvironment. Here, we show that miR-146a-5p is highly expressed in human melanoma brain metastasis (MBM) EVs, both in MBM cell lines as well as in biopsies, thereby modulating the brain metastatic niche. Mechanistically, miR-146a-5p was transferred to astrocytes via EV delivery and inhibited NUMB in the Notch signalling pathway. This resulted in activation of tumour-promoting cytokines (IL-6, IL-8, MCP-1 and CXCL1). Brain metastases were significantly reduced following miR-146a-5p knockdown. Corroborating these findings, miR-146a-5p inhibition led to a reduction of IL-6, IL-8, MCP-1 and CXCL1 in astrocytes. Following molecular docking analysis, deserpidine was identified as a functional miR-146a-5p inhibitor, both in vitro and in vivo. Our results highlight the pro-metastatic function of miR-146a-5p in EVs and identifies deserpidine for targeted adjuvant treatment.publishedVersio

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - An ISEV position paper

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNAencoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolationmethods, optimisation of methodologies to isolate and characteriseminute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Extracellular Vesicles and RNA interference in tumors

Extracellular vesicles (EVs) including apoptotic bodies (ABs), microvesicles (MVs), exosomes (EXOs) and cell derived artificial nanovesicles (NVs) are important mediators of cell-to-cell communication, in part by transferring bioactive molecules such as DNA, mRNA, miRNA, siRNA, proteins, and lipids. These EVs are released by many cell types, including melanoma cells, and are found in many body fluids. EVs derived from various cell types differ in their molecular composition making them as important diagnostic and prognostic markers. The overall aim of this thesis was to use small-RNA sequencing techniques to define the molecular RNA cargo in the EV subsets described above as well as to examine the functional relevance of the EV-associated miRNA and siRNA on recipient cells. Characterization of EVs showed distinct RNA profiles in ABs, MVs, and EXOs, and there were significantly greater amounts of total RNA in EXOs compared to ABs and MVs. Small RNA sequencing revealed distinct repertoires of noncoding RNAs in the EV subsets. EXOs contained unique sets of miRNAs, which were shown to be differentially expressed in melanoma tumors compared with benign naevi in previously published studies, thus making them potentially useful as carriers of therapeutic agents. This study demonstrates that distinct sets of RNA molecules are present in subsets of EVs, and this provides unique insights into the contribution of extracellular RNA in cancer development and progression. The BRAFV600E inhibitor vemurafenib inhibited the growth of in vitro melanoma cell cultures, and EVs isolated from the treated cells had significantly higher RNA and protein contents compared to EVs from non-treated cells. Small RNA sequencing revealed distinct non-coding RNA species with significant alterations in miRNA between treated and non-treated cell-derived EVs. Moreover, treated cells and the EVs derived from them showed significant upregulation of miR-211 in vitro and in vivo. Furthermore, when vemurafenib-treated cell-derived EXOs were transferred to BRAFWT cells, KCNMA1 and IGF2R, genes that are known to play roles in tumor progression, were down-regulated and this resulted in growth attenuation. Overall, miR-211 could be used as a biomarker of response in patients diagnosed with BRAF-mutant melanoma. This study also provides the framework for further investigations into the function of miR-211 in melanoma cells and EVs as well as in cells that might receive miRNA from EVs. Artificial EXO-mimetic NVs were developed by serial extrusion, and they showed similar characteristics as EXOs. Exogenous loading of GFP-siRNA in NVs led to down-regulation of GFP in endothelial cells. Cell-derived NVs carrying endogenously expressed Myc-siRNA showed significant down-regulation of human cMyc both transcriptionally as well as translationally in lymphoma cells. These NVs were efficiently loaded with siRNA and were taken up by recipient cells resulting in the reduction of target gene expression. In conclusion, this study suggests that EXO-mimetic NVs can be a platform for delivering siRNA to cells. Taken together, EVs have significant therapeutic potential. EVs have emerged as a novel and functionally important vehicle of cell-cell communication that can mediate multiple biological effects. In addition, these vesicles might provide unique signatures that can be used as biomarkers of response to drug treatment