17 research outputs found
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
Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly
Chronic myeloid leukemia: a model disease of the past, present and future
Chronic myeloid leukemia (CML) has been a “model disease” with a long history. Beginning with the first discovery of leukemia and the description of the Philadelphia Chromosome and ending with the current goal of achieving treatment-free remission after targeted therapies, we describe here the journey of CML, focusing on molecular pathways relating to signaling, metabolism and the bone marrow microenvironment. We highlight current strategies for combination therapies aimed at eradicating the CML stem cell; hopefully the final destination of this long voyage
High-throughput sequencing of two populations of extracellular vesicles provides an mRNA signature that can be detected in the circulation of breast cancer patients
ABSTRACT Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo
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High-throughput sequencing of two populations of extracellular vesicles provides an mRNA signature that can be detected in the circulation of breast cancer patients.
Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo
Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma
Cancer-derived extracellular vesicles (EVs) are membrane-enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. We demonstrate here that large EVs (L-EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L-EVs and small EVs (S-EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L-EVs, despite S-EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L-EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S-EVs from the same patients. Controlled experimental conditions, with spike-ins of L-EVs and S-EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L-EVs. Whole genome sequencing revealed that the DNA in L-EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L-EV-derived DNA reflects the genomic make-up of the tumour of origin. They also support the conclusion that L-EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour-derived genomic alterations
Impact of mesenchymal stromal cell–derived vesicular cargo on B-cell acute lymphoblastic leukemia progression
Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell–derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1− B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment
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Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia.
Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting
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Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles
Large oncosomes (LO) are atypically large (1-10μm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrepTM) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers