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.

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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 study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.

The biology, function, and biomedical applications of exosomes

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

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Tumour exosome integrins determine organotropic metastasis

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

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Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

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Isolation of exosomal RNA from serum or plasma using the Qiagen miRNeasy Micro kit

Abstract Liquid biopsy is a minimally invasive alternative to surgical biopsy, encompassing different analytes including extracellular vesicles (EVs), circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), proteins, and metabolites. EVs are released by virtually all cells, but at a higher rate by faster cycling, malignant cells. They encapsulate cargo native to the originating cell and can thus provide a window into the tumour landscape. EVs are often analysed in bulk which hinders the analysis of rare, tumour‐specific EV subpopulations from the large host EV background. Here, we fractionated EV subpopulations in vitro and in vivo and characterized their phenotype and generic cargo. We used 5‐aminolevulinic acid (5‐ALA) to induce release of endogenously fluorescent tumour‐specific EVs (EVPpIX). Analysis of five different subpopulations (EVPpIX, EVCD63, EVCD9, EVEGFR, EVCFDA) from glioblastoma (GBM) cell lines revealed unique transcriptome profiles, with the EVPpIX transcriptome demonstrating closer alignment to tumorigenic processes over the other subpopulations. Similarly, isolation of tumour‐specific EVs from GBM patient plasma showed enrichment in GBM‐associated genes, when compared to bulk EVs from plasma. We propose that fractionation of EV populations facilitates detection and isolation of tumour‐specific EVs for disease monitoring.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12278

Glioblastoma‐derived extracellular vesicle subpopulations following 5‐aminolevulinic acid treatment bear diagnostic implications

L'invention concerne des procedes de dosage d'un echantillon biologique provenant d'un sujet, qui consistent a analyser les composants de fractions de microvesicules a des fins d'evaluation de risque, de diagnostic, de pronostic ou de surveillance d'une maladie ou d'une autre affection medicale chez un sujet, ou de traitement de la maladie ou de l'autre affection medicale chez ce sujet. L'invention concerne aussi des methodes de traitement et d'identification de biomarqueurs au moyen d'une fraction de microvesicules d'un sujet. Des trousses, des compositions pharmaceutiques et des profils associes aux procedes sont egalement decrits.

Matières biologiques liées au cancer dans des microvésicules

BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Patient Serum With Glioma and Cerebrospinal Fluid Extracellular Vesicles


 INTRODUCTION: The utility of photosensitizers (PS), such as 5-Aminolevulinic acid (5-ALA), has been limited to fluorescence-guided surgery (FGS) and photodynamic therapy. The downstream metabolism of 5-ALA results in tumor-specific, highly selective accumulation of PpIX, an endogenous porphyrin, in tumor cells. We have previously reported evidence of PpIX in EVs derived from the plasma of glioblastoma (GBM) patients. Here, we characterize the tumor-specific nature of PpIX fluorescent EVs through quantification of their cargo, demonstrating their utility in the advancement of liquid biopsy for GBM diagnosis and management.
 METHODS: Fluorescence activated cell sorting was adapted for nanoparticle isolation and PpIX fluorescent EVs were sorted from plasma collected from patients (N = 8) diagnosed with GBM, undergoing FGS. RNA cargo was extracted and quantified via low input RNA-Seq. Matching patient tumor and total plasma EVs along with healthy control plasma EVs (N = 8) were sequenced in parallel.
 SUMMARY: Through sequencing analysis, we have demonstrated a distinct landscape of long RNAs (mRNA and lncRNA) in PpIX EVs, with isolation of this EV population resulting in an enrichment of lncRNAs. Considering the emerging diagnostic and prognostic potential of lncRNAs in GBM, exploration of such EVs provides valuable insight into the pathobiology of the tumor of origin. On a broader scale, gene expression analysis of PpIX EV cargo demonstrates at least a ten-fold enrichment of genes that are otherwise buried in the heterogeneous plasma EV background. Furthermore, downstream KEGG orthological analysis of PpIX EV cargo has revealed alignment of 78% of the significant pathways to processes implicated in cancer development and progression. We have identified a panel of genes unique to PpIX EVs, and reflective of the native tumor, that are implicated in GBM pathogenesis including GREM1, MAP4K4, and STAG2. Validation via quantitative and digital PCR of total plasma EVs from patient and healthy cohorts (N = 10, respectively) have yielded similar expression levels, as reflected in sequencing gene expression. Isolation of PpIX EVs, however, insinuates enriched genetic detection.
 CONCLUSION: In summary, this preliminary cohort of GBM patients has demonstrated the utility of 5-ALA induced PpIX fluorescence outside of its original intentions: facilitating tumor-specific EV identification for the progression of liquid biopsy. These findings are an inviting breakthrough in the development of individualized disease detection and monitoring.
 Citation Format: Tiffaney Hsia, Anudeep Yekula, Syeda M. Batool, Bob S. Carter, Leonora Balaj. Novel approach for glioblastoma characterization via tumor specific extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5175.

Leonora Balaj

Papers

Isolation of exosomal RNA from serum or plasma using the Qiagen miRNeasy Micro kit

Glioblastoma‐derived extracellular vesicle subpopulations following 5‐aminolevulinic acid treatment bear diagnostic implications

Matières biologiques liées au cancer dans des microvésicules

BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Patient Serum With Glioma and Cerebrospinal Fluid Extracellular Vesicles

Abstract 5175: Novel approach for glioblastoma characterization via tumor specific extracellular vesicles