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.
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 RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute 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.
The establishment of distant metastases depends on the capacity of small numbers of cancer cells to regenerate a tumor after entering a target tissue. The mechanisms that confer this capacity remain to be defined. Here we identify a role for the transcriptional inhibitors of differentiation Id1 and Id3 as selective mediators of lung metastatic colonization in the triple negative [TN, i.e., lacking expression of estrogen receptor and progesterone receptor, and lacking Her2 (human epidermal growth factor receptor 2) amplification] subgroup of human breast cancer. Although broad expression of Id1 has recently been documented in tumors of the rare metaplastic subtype, here we report that rare Id1-expressing cells are also present in the more common TN subset of human breast tumors but not in other subtypes. We also provide evidence that Id1 expression is enriched in clinically obtained hormone receptor negative lung metastases. Functional studies demonstrate that Id1 and its closely related family member Id3 are required for tumor initiating functions, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment. In vivo characterization of lung metastatic progression reveals that Id1 and Id3 facilitate sustained proliferation during the early stages of metastatic colonization, subsequent to extravasation into the lung parenchyma. These results shed light on the proliferative mechanisms that initiate metastatic colonization, and they implicate Id1 and Id3 as mediators of this malignant function in the TN subgroup of breast cancers.proliferation ͉ stem cells ͉ triple negative
The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.
Extracellular Vesicles (EVs) have received considerable attention in recent years, both as mediators of intercellular communication pathways that lead to tumor progression, and as potential sources for discovery of novel cancer biomarkers. For many years, research on EVs has mainly investigated either the mechanism of biogenesis and cargo selection and incorporation, or the methods of EV isolation from available body fluids for biomarker discovery. Recent studies have highlighted the existence of different populations of cancer-derived EVs, with distinct molecular cargo, thus pointing to the possibility that the various EV populations might play diverse roles in cancer and that this does not happen randomly. However, data attributing cancer specific intercellular functions to given populations of EVs are still limited. A deeper functional, biochemical and molecular characterization of the various EV classes might identify more selective clinical markers, and significantly advance our knowledge of the pathogenesis and disease progression of many cancer types.
Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large oncosomes applicable to human platelet-poor plasma, where the presence of caveolin-1-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment.
Severe infection with SARS-CoV-2 is characterized by massive cytokine release and T cell loss. The exaggerated host immune response, incapable of viral clearance, instead aggravates respiratory distress, as well as cardiac and/or damage to other organs. The mortality pattern of SARS-CoV-2 infection, higher in older vs younger adults and almost absent in children, is possibly caused by the effects of age and pre-existing co-morbidities on innate and adaptive immunity. Here, w speculate that the abnormal and excessive immune response to SARS-CoV-2 infection partly depends on T cell immunological memory, that is more pronounced in adults compared to children, and may significantly contribute to immunopathology and massive collateral damage in COVID-19 patients.
The -amyloid precursor protein (APP) 1 is an integral membrane protein from which the -amyloid peptide is generated. The -amyloid peptide forms the extracellular insoluble aggregates characteristic of Alzheimer's disease. The function of APP and the regulation of the proteolytic events generating the -amyloid peptide are still unknown. APP was expected to be involved in signal transduction processes, because of its transmembrane topology. Three main isoforms of APP exist, generated by alternative splicing (APP 770 , APP 751 , and APP 695 ) and all possessing the same intracellular domain (reviewed in Ref.1). Although little is known about the putative extracellular ligand(s) for APP, several results describe the interaction of its intracellular domain with other proteins. These include the interaction with the heterotrimeric G protein Go (2), a 59-kDa ubiquitously expressed protein named APP-BP1 (3), the X11 protein (4), the neuron-abundant Fe65 protein, and an Fe65-like protein (4 -6). It was shown that intact APP binds to oligomeric Go protein and that the intracellular region of APP spanning residues 657-676 activates Go (2, 7). Furthermore, the interaction of APP with a monoclonal antibody directed against its extracellular domain mimics a ligand-receptor binding that triggers Go activation (7). APP-BP1 interacts both in vitro and in vivo with the carboxyl-terminal region of APP, which represents its intracellular domain. This protein is homologous to the product of the Arabidopsis auxin resistance gene AXR1 and to a Caenorabditis elegans protein of unknown function (3).The Fe65 gene is mainly expressed in the neurons of specific regions of the mammalian nervous system (8, 9) and encodes a protein containing two different types of protein-protein interaction domains: the WW domain (reviewed in Ref. 10) and the phosphotyrosine interaction/phosphotyrosine binding (PID/ PTB) domain (reviewed in Ref. 11). The latter was found in the oncoprotein Shc (12, 13), in its relatives , in other apparently unrelated proteins, such as Numb, X11, and Dab (15), and in insulin receptor substrate 1 (IRS-1) and 17). The PID/PTB domains interact with phosphotyrosine residues located in the intracellular domains of growth factor receptors, such as EGF-R, trkA, and plateletderived growth factor receptor in the case of Shc (13) and insulin receptor and interleukin 4 receptor in the case of IRS-1 (16). In contrast, the Fe65 region containing the two PID/PTB domains was demonstrated to interact with the intracellular domain of APP (5).All the PID/PTB domains present in the Shc family, IRS-1, and Fe65 interact with intracellular regions of membrane proteins containing the consensus motif ⌽XNPXY (where ⌽ is hydrophobic and X is any amino acid). However, Fe65 possesses at least two unique characteristics: (i) although all the known members of the PID/PTB family contain only one PID/ PTB element (13), Fe65 is an exception, because its sequence interacting with APP shows two consecutive PID/PTB domains; and (ii) although the Tyr prese...
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