Stephanie N. Hurwitz

ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles.

Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes.

Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers

Packed with biological information, extracellular vesicles (EVs) offer exciting promise for biomarker discovery and applications in therapeutics and non-invasive diagnostics. Currently, our understanding of EV contents is confined by the limited cells from which vesicles have been characterized utilizing the same enrichment method. Using sixty cell lines from the National Cancer Institute (NCI-60), here we provide the largest proteomic profile of EVs in a single study, identifying 6,071 proteins with 213 common to all isolates. Proteins included established EV markers, and vesicular trafficking proteins such as Rab GTPases and tetraspanins. Differentially-expressed proteins offer potential for cancer diagnosis and prognosis. Network analysis of vesicle quantity and proteomes identified EV components associated with vesicle secretion, including CD81, CD63, syntenin-1, VAMP3, Rab GTPases, and integrins. Integration of vesicle proteomes with whole-cell molecular profiles revealed similarities, suggesting EVs provide a reliable reflection of their progenitor cell content, and are therefore excellent indicators of disease.

CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

ABSTRACT Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling. IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitts lymphoma, and Hodgkins lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

The interactome of EBV LMP1 evaluated by proximity-based BioID approach

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.

An Adaptable Polyethylene Glycol-Based Workflow for Proteomic Analysis of Extracellular Vesicles

Extracellular vesicles (EVs), including exosomes are endocytically derived nanovesicles expelled from cells that contain molecular information in the form of lipids, proteins, and nucleic acids. Transfer of this information to other cells in local or distant microenvironments facilitates cell-to-cell communication. Importantly, diseased cells release exosomes containing specific cargo that may contribute to pathology and can be harnessed for diagnostic or prognostic use. The broad potential medical utility of exosomes has fueled rapidly expanding research on understanding the composition and functions of exosomes in normal and pathological conditions. Here, we provide a complete workflow for purifying exosome-sized vesicles from biological fluids for in-depth proteomic analyses. Moreover, this polyethylene glycol-based method is efficient, highly adaptable, and compatible with a variety of downstream applications.

An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease

BACKGROUND Alzheimers disease (AD) is the major cause of dementia that has increased dramatically in prevalence over the past several decades. Yet many questions still surround the etiology of AD. Recently, extracellular vesicles (EVs) that transport protein, lipid, and nucleic acids from cell to cell have been implicated in the clearance and propagation of misfolded proteins. Investigation of EVs in AD progression, and their potential diagnostic utility may contribute to understanding and treating AD. However, the challenges of isolating brain-derived EVs have in part hindered these studies. NEW METHOD Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis. RESULTS We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry. CONCLUSIONS Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.

Extracellular Vesicle Biogenesis in Cancer

Abstract Extracellular vesicles (EVs) represent a broad collection of membrane-enclosed sacs released from cells that contain biologically active cargo and cell type–specific and disease type–specific molecular information. Furthermore, EVs have been demonstrated to perpetuate the pathogenesis and progression of disease through manipulation of cells in the surrounding microenvironment. Based on the unique properties of EVs, there is tremendous interest in developing EV-based diagnostic and therapeutic platforms for cancer and many other diseases. However, before the full medical utility of EVs can be reached, a better understanding of mechanisms driving EV biogenesis must be obtained. Here, we will highlight the current understanding of EV biogenesis in the context of cancer, discuss recent advances in the field, and provide evidence for the utility of human tumor viruses in studying EV biogenesis and trafficking.