Publications by authors named "David G Meckes"

45 Publications

Anticancer and chemosensitization effects of cannabidiol in 2D and 3D cultures of TNBC: involvement of GADD45α, integrin-α5, -β5, -β1, and autophagy.

Drug Deliv Transl Res 2022 Feb 25. Epub 2022 Feb 25.

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, 32307, USA.

To date, promising therapy for triple negative breast cancer (TNBC) remains a serious concern clinically because of poor prognosis, resistance, and recurrence. Herein, anti-cancer potential of synthetic cannabidiol (CBD; Purisys, GA; GMP grade) was explored either alone or as a chemosensitizer followed by post-treatment with doxorubicin (DOX) in TNBC (i.e., MDA-MB-231 and MDA-MB-468) cells. In comparison to 2D cultures, CBD showed greater IC values in 3D (LDP2 hydrogel based) cultures of MDA-MB-231 (6.26-fold higher) and MDA-MB-468 (10.22-fold higher) cells. Next-generation RNA sequencing revealed GADD45A, GADD45G, FASN, LOX, and integrin (i.e., -α5, -β5) genes to be novelly altered by CBD in MDA-MB-231 cells. CIM-16 plate-based migration assay and western blotting disclosed that CBD induces anti-migratory effects in TNBC cells by decreasing fibronectin, vimentin, and integrins-α5, -β5, and -β1. Western blotting, RT-qPCR, and immunocytochemistry revealed that CBD inhibited autophagy (decreased Beclin1, and ATG-5, -7, and -16) of TNBC cells. CBD pre-treatment increased DOX sensitivity in TNBC cells. CBD pre-treatment accompanied by DOX treatment decreased LOX and integrin-α5, and increased caspase 9 protein respectively in MDA-MB-468 cells.
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http://dx.doi.org/10.1007/s13346-022-01137-2DOI Listing
February 2022

Coordination of Zika Virus Infection and Viroplasm Organization by Microtubules and Microtubule-Organizing Centers.

Cells 2021 11 27;10(12). Epub 2021 Nov 27.

Department of Biomedical Sciences, Florida State University, Tallahassee, FL 32306, USA.

Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. Flaviviruses, including ZIKV, reorganize the endoplasmic reticulum (ER) to form a viroplasm, a compartment where virus particles are assembled. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support replication of flaviviruses. Here we investigated the roles of MTs and the cell's MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production.
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http://dx.doi.org/10.3390/cells10123335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699624PMC
November 2021

Extracellular Vesicle Collection from Human Stem Cells Grown in Suspension Bioreactors.

Methods Mol Biol 2022 ;2436:193-204

Department of Chemical & Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA.

Extracellular vesicles (EVs) are particles with 100-1000 nm sizes which are secreted by cells for intercellular communication. Meanwhile, studies have found that EVs secreted by human stem cells carry similar characteristics (microRNAs, proteins, metabolites, etc.) from their cell counterpart. Thus, EVs derived from stem cells, especially human induced pluripotent stem cells (hiPSCs) and human mesenchymal stromal/stem cells (hMSCs) are promising candidates for cell-free therapy. However, conventional planar culture is insufficient to produce a large amount of cells or EVs to satisfy clinical requirements. In this chapter, we described feasible approaches to harvest EVs secreted by lineage-specific hiPSCs and undifferentiated hMSCs in suspension bioreactors. Differentiation of hiPSCs to cortical organoids can be performed in suspension bioreactors and the corresponding EVs can be isolated and purified. This scale-up protocol can be applied to a majority of stem cell types with EV collection thus provides useful information for both experimental and biomanufacturing purposes.
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http://dx.doi.org/10.1007/7651_2021_416DOI Listing
March 2022

Role of Exosomes for Delivery of Chemotherapeutic Drugs.

Crit Rev Ther Drug Carrier Syst 2021 ;38(5):53-97

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.

Exosomes are endogenous extracellular vesicles (30-100 nm) composed with membrane lipid bilayer which carry vesicular proteins, enzymes, mRNA, miRNA and nucleic acids. They act as messengers for intra- and inter-cellular communication. In addition to their physiological roles, exosomes have the potential to encapsulate and deliver small chemotherapeutic drugs and biological molecules such as proteins and nucleic acid-based drugs to the recipient tissue or organs. Due to their biological properties, exosomes have better organotropism, homing capacity, cellular uptake and cargo release ability than other synthetic nano-drug carriers such as liposomes, micelles and nanogels. The secretion of tumor-derived exosomes is increased in the hypoxic and acidic tumor microenvironment, which can be used as a target for nontoxic and nonimmunogenic drug delivery vehicles for various cancers. Moreover, exosomes have the potential to carry both hydrophilic and hydrophobic chemotherapeutic drugs, bypass RES effect and bypass BBB. Exosomes can be isolated from other types of EVs and cell debris based on their size, density and specific surface proteins through ultracentrifugation, density gradient separation, precipitation, immunoaffinity interaction and gel filtration. Drugs can be loaded into exosomes at the biogenesis stage or with the isolated exosomes by incubation, electroporation, extrusion or sonication methods. Finally, exosomal cargo vehicles can be characterized by ultrastructural microscopic analysis. In this review we intend to summarize the inception, structure and function of the exosomes, role of exosomes in immunological regulation and cancer, methods of isolation and characterization of exosomes and products under clinical trials. This review will provide an inclusive insight of exosomes in drug delivery.
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http://dx.doi.org/10.1615/CritRevTherDrugCarrierSyst.2021036301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8691065PMC
October 2021

Mesenchymal stem cell-derived extracellular vesicles ameliorate Alzheimer's disease-like phenotypes in a preclinical mouse model.

Theranostics 2021 13;11(17):8129-8142. Epub 2021 Jul 13.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs. To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aβ) and glial fibrillary acidic protein (GFAP) levels. The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aβ plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aβ plaques was found in the brain of EV-treated mice compared to saline. Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis.
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http://dx.doi.org/10.7150/thno.62069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344012PMC
January 2022

Cannabidiol loaded extracellular vesicles sensitize triple-negative breast cancer to doxorubicin in both in-vitro and in vivo models.

Int J Pharm 2021 Sep 27;607:120943. Epub 2021 Jul 27.

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, USA. Electronic address:

Extracellular Vesicles (EVs) were isolated from human umbilical cord mesenchymal stem cells (hUCMSCs) and were further encapsulated with cannabidiol (CBD) through sonication method (CBD EVs). CBD EVs displayed an average particle size of 114.1 ± 1.02 nm, zeta potential of -30.26 ± 0.12 mV, entrapment efficiency of 92.3 ± 2.21% and stability for several months at 4 °C. CBD release from the EVs was observed as 50.74 ± 2.44% and 53.99 ± 1.4% at pH 6.8 and pH 7.4, respectively after 48 h. Our in-vitro studies demonstrated that CBD either alone or in EVs form significantly sensitized MDA-MB-231 cells to doxorubicin (DOX) (*P < 0.05). Flow cytometry and migration studies revealed that CBD EVs either alone or in combination with DOX induced G1 phase cell cycle arrest and decreased migration of MDA-MB-231 cells, respectively. CBD EVs and DOX combination significantly reduced tumor burden (***P < 0.001) in MDA-MB-231 xenograft tumor model. Western blotting and immunocytochemical analysis demonstrated that CBD EVs and DOX combination decreased the expression of proteins involved in inflammation, metastasis and increased the expression of proteins involved in apoptosis. CBD EVs and DOX combination will have profound clinical significance in not only decreasing the side effects but also increasing the therapeutic efficacy of DOX in TNBC.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8528640PMC
September 2021

Zika Virus Hijacks Extracellular Vesicle Tetraspanin Pathways for Cell-to-Cell Transmission.

mSphere 2021 Jun 30:e0019221. Epub 2021 Jun 30.

Florida State Universitygrid.255986.5 College of Medicine, Department of Biomedical Sciences, Tallahassee, Florida, USA.

Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins, and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the progression of neurological conditions and both the spread and pathogenesis of infectious diseases. It has recently been demonstrated that EVs from hepatitis C virus (HCV)-infected individuals and cells contained replicative-competent viral RNA that was capable of infecting hepatocytes. Being a member of the same viral family, it is likely the Zika virus also hijacks EV pathways to package viral components and secrete vesicles that are infectious and potentially less immunogenic. As EVs have been shown to cross blood-brain and placental barriers, it is possible that Zika virus could usurp normal EV biology to gain access to the brain or developing fetus. Here, we demonstrate that Zika virus-infected cells secrete distinct EV subpopulations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with various sizes and densities compared to those released from noninfected cells. We also show that the EV-enriched tetraspanin CD63 regulates the release of EVs and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika virus infection and virion morphogenesis. Zika virus is a reemerging infectious disease that spread rapidly across the Caribbean and South America. Infection of pregnant women during the first trimester has been linked to microcephaly, a neurological condition where babies are born with smaller heads due to abnormal brain development. Babies born with microcephaly can develop convulsions and suffer disabilities as they age. Despite the significance of Zika virus, little is known about how the virus infects the fetus or causes disease. Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells that are present in all biological fluids. EVs carry signaling factors, proteins, and microRNAs that mediate intercellular communication. EVs have been shown to be a means by which some viruses can alter cellular environments and cross previously unpassable cellular barriers. Thus, gaining a greater understanding of how Zika virus affects EV cargo may aid in the development of better diagnostics, targeted therapeutics, and/or prophylactic treatments.
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http://dx.doi.org/10.1128/mSphere.00192-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265634PMC
June 2021

Multiplex protein profiling method for extracellular vesicle protein detection.

Sci Rep 2021 06 14;11(1):12477. Epub 2021 Jun 14.

Department of Biomedical Sciences, Florida State University College of Medicine, 1115 W. Call Street, Tallahassee, FL, 32306-4300, USA.

Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.
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http://dx.doi.org/10.1038/s41598-021-92012-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203679PMC
June 2021

Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.

Viruses 2021 04 15;13(4). Epub 2021 Apr 15.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.

Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.
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http://dx.doi.org/10.3390/v13040675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071190PMC
April 2021

Biogenesis of Extracellular Vesicles Produced from Human-Stem-Cell-Derived Cortical Spheroids Exposed to Iron Oxides.

ACS Biomater Sci Eng 2021 03 1;7(3):1111-1122. Epub 2021 Feb 1.

Department of Chemical and Biomedical Engineering, Florida State University, Tallahassee, Florida 32306, United States.

Stem-cell-derived extracellular vesicles (EVs) are promising tools for therapeutic delivery and imaging in the medical research fields. EVs that arise from endosomal compartments or plasma membrane budding consist of exosomes and microvesicles, which range between 30 and 200 nm and 100-1000 nm, respectively. Iron oxide nanoparticles can be used to label stem cells or possibly EVs for magnetic resonance imaging. This could be a novel way to visualize areas in the body that are affected by neurological disorders such as stroke. Human induced pluripotent stem cells (iPSK3 cells) were plated on low-attachment plates and treated with SB431542 and LDN193189 during the first week for the induction of cortical spheroid formation and grown with fibroblast growth factor 2 and cyclopamine during the second week for the neural progenitor cell (iNPC) differentiation. iNPCs were then grown on attachment plates and treated with iron oxide (FeO) nanoparticles at different sizes (8, 15, and 30 nm in diameter) and concentrations (0.1, 10, and 100 μM). The spheroids and media collected from these cultures were used for iron oxide detection as well as EV isolation and characterizations, respectively. MTT assay demonstrated that the increased size and concentration of the iron oxide nanoparticles had little effect on the metabolic activity of iNPCs. In addition, the Live/Dead assay showed high viability in all the nanoparticle treated groups and the untreated control. The EVs isolated from these culture groups were analyzed and displayed similar or higher EV counts compared with control. The observed EV size averaged 200-250 nm, and electron microscopy revealed the expected exosome morphology for EVs from all groups. RT-PCR analysis of EV biogenesis markers (CD63, CD81, Alix, TSG101, Syntenin1, ADAM10, RAB27b, and Syndecan) showed differential expression between the iron-oxide-treated cultures and nontreated cultures, as well as between adherent and nonadherent 3D cultures. Iron oxide nanoparticles were detected inside the cortical spheroid cells but not EVs by MRI. The addition of iron oxide nanoparticles does not induce significant cytotoxic effects to cortical spheroids. In addition,, nanoparticles may stimulate the biogenesis of EVs when added to cortical spheroids in vitro.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185622PMC
March 2021

Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells.

PLoS Pathog 2020 12 31;16(12):e1009023. Epub 2020 Dec 31.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, United States of America.

Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins.
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http://dx.doi.org/10.1371/journal.ppat.1009023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774862PMC
December 2020

Alix and Syntenin-1 direct amyloid precursor protein trafficking into extracellular vesicles.

BMC Mol Cell Biol 2020 Jul 30;21(1):58. Epub 2020 Jul 30.

Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306-4300, USA.

Background: Endosomal trafficking and amyloidogenic cleavage of amyloid precursor protein (APP) is believed to play a role in the neurodegeneration observed in Alzheimer's disease (AD). Recent evidence has suggested that packaging and secretion of APP and its amyloidogenic cleaved products into small extracellular vesicles (EVs) may facilitate uptake of these neurotoxic factors during disease progression. However, the molecular mechanisms underlying trafficking of APP into EVs are poorly understood.

Results: In this study, the mechanism and impact of APP trafficking into extracellular vesicles (EVs) were assessed by a series of inducible gene knockdowns. We demonstrate that vesicle-associated proteins Alix and Syntenin-1 are essential for proper subcellular localization and efficient EV secretion of APP via an endosomal sorting complexes required for transport (ESCRT)-independent pathway. The neurotoxic C-terminal fragment (CTFβ) of APP is similarly secreted in association with small vesicles. These mechanisms are conserved in terminally differentiated neuron-like cells. Furthermore, knockdown of Alix and Syntenin-1 alters the subcellular localization of APP, sequestering the precursor protein to endoplasmic reticulum and endolysosomal compartments, respectively. Finally, transfer of small EVs containing mutant APP confers an increase in reactive oxygen species production and neurotoxicity to human induced pluripotent stem cell-derived cortical neurons and naïve primary neurons, an effect that is ameliorated by Alix and Syntenin-1 depletion.

Conclusions: Altogether these findings elucidate a novel mechanism for understanding the intracellular trafficking of APP and CTFβ into secreted extracellular vesicles, and the resultant potential impact on neurotoxicity in the context of Alzheimer's disease amyloidopathy.
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http://dx.doi.org/10.1186/s12860-020-00302-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392838PMC
July 2020

Epstein-Barr Virus LMP1 Promotes Syntenin-1- and Hrs-Induced Extracellular Vesicle Formation for Its Own Secretion To Increase Cell Proliferation and Migration.

mBio 2020 06 16;11(3). Epub 2020 Jun 16.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment. LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.
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http://dx.doi.org/10.1128/mBio.00589-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298708PMC
June 2020

Extracellular Vesicles in Epstein-Barr Virus Pathogenesis.

Curr Clin Microbiol Rep 2019 Sep 3;6(3):121-131. Epub 2019 Jul 3.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.

Purpose Of Review: Epstein-Barr virus (EBV) is a known determinant for numerous malignancies and may contribute to autoimmune diseases. The underlining mechanisms behind EBV pathologies is not completely understood. Recently, extracellular vesicles (EVs) released from infected cells have been found to produce profound effects on cellular microenvironments. Therefore, in this review we sought to critically evaluate the roles of EVs in EBV pathogenesis and assess their potential therapeutic and diagnostic utility.

Recent Findings: EBV-altered EVs are capable of activating signaling cascades and phenotypic changes in recipient cells through the transfer of viral proteins and RNAs. Moreover, several EV-associated microRNAs have encouraging prognostic or diagnostic potential in EBV-associated cancers.

Summary: Current evidence suggests that EBV-modified EVs affect viral pathogenesis and cancer progression. However, further research is needed to investigate the direct role of both viral and host products on recipient cells and the mechanisms driving viral protein and RNA EV packaging and content modification.
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http://dx.doi.org/10.1007/s40588-019-00123-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015464PMC
September 2019

BioID Combined with Mass Spectrometry to Study Herpesvirus Protein-Protein Interaction Networks.

Methods Mol Biol 2020 ;2060:327-341

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA.

Herpes viruses are important human pathogens that cause a wide range of diseases from skin lesions to malignancies. Protein interactions drive many cellular events and mediate a number of biochemical pathways leading to different physiological outcomes. Protein interactions between viral proteins and host proteins play significant roles in viral entry, replication and suppression of host-immune responses. Therefore, the study of virus-host interactions promises significant advancement in designing therapeutics to control infection and disease. Various approaches are employed in the field to study and identify protein interactions that combine affinity purification along with different detection methods. Advancements in protein purification and high-throughput detection methods have resulted in an unprecedented level of discovery. Here we detail the use of proximity dependent biotinylation (BioID) as a means of affinity purification coupled with the use of LC-MS/MS for the detection and identification of protein-protein interaction networks.
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http://dx.doi.org/10.1007/978-1-4939-9814-2_19DOI Listing
December 2020

Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids.

Cells 2019 08 28;8(9). Epub 2019 Aug 28.

Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, USA.

Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer's-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100-250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by β-tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in Aβ42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating neural degeneration.
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http://dx.doi.org/10.3390/cells8090993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770916PMC
August 2019

Extracellular Vesicle Integrins Distinguish Unique Cancers.

Proteomes 2019 Apr 11;7(2). Epub 2019 Apr 11.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.
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http://dx.doi.org/10.3390/proteomes7020014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630702PMC
April 2019

Extraction of Extracellular Vesicles from Whole Tissue.

J Vis Exp 2019 02 7(144). Epub 2019 Feb 7.

Department of Biomedical Sciences, Florida State University College of Medicine;

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.
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http://dx.doi.org/10.3791/59143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098067PMC
February 2019

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.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

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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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

The Epstein-Barr virus LMP1 interactome: biological implications and therapeutic targets.

Future Virol 2018 Dec 3;13(12):863-887. Epub 2018 Dec 3.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, 32306.

The oncogenic potential of Epstein-Barr virus (EBV) is mostly attributed to latent membrane protein 1 (LMP1), which is essential and sufficient for transformation of fibroblast and primary lymphocytes. LMP1 expression results in the activation of multiple signaling cascades like NF-ΚB and MAP kinases that trigger cell survival and proliferative pathways. LMP1 specific signaling events are mediated through the recruitment of a number of interacting proteins to various signaling domains. Based on these properties, LMP1 is an attractive target to develop effective therapeutics to treat EBV-related malignancies. In this review, we focus on LMP1 interacting proteins, associated signaling events, and potential targets that could be exploited for therapeutic strategies.
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http://dx.doi.org/10.2217/fvl-2018-0120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168621PMC
December 2018

Methodological Approaches to Study Extracellular Vesicle miRNAs in Epstein⁻Barr Virus-Associated Cancers.

Int J Mol Sci 2018 Sep 18;19(9). Epub 2018 Sep 18.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA.

Epstein Barr-virus (EBV) was the first virus identified to be associated with human cancer in 1964 and is found ubiquitously throughout the world's population. It is now established that EBV contributes to the development and progression of multiple human cancers of both lymphoid and epithelial cell origins. EBV encoded miRNAs play an important role in tumor proliferation, angiogenesis, immune escape, tissue invasion, and metastasis. Recently, EBV miRNAs have been found to be released from infected cancer cells in extracellular vesicles (EVs) and regulate gene expression in neighboring uninfected cells present in the tumor microenvironment and possibly at distal sites. As EVs are abundant in many biological fluids, the viral and cellular miRNAs present within EBV-modified EVs may serve as noninvasion markers for cancer diagnosis and prognosis. In this review, we discuss recent advances in EV isolation and miRNA detection, and provide a complete workflow for EV purification from plasma and deep-sequencing for biomarker discovery.
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http://dx.doi.org/10.3390/ijms19092810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164614PMC
September 2018

Transmembrane Domains Mediate Intra- and Extracellular Trafficking of Epstein-Barr Virus Latent Membrane Protein 1.

J Virol 2018 09 16;92(17). Epub 2018 Aug 16.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA

EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63. EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.
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http://dx.doi.org/10.1128/JVI.00280-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096814PMC
September 2018

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

J Neurosci Methods 2018 09 9;307:210-220. Epub 2018 Jun 9.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, 32306, United States. Electronic address:

Background: Alzheimer's 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.
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http://dx.doi.org/10.1016/j.jneumeth.2018.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052957PMC
September 2018

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

Virology 2018 03 9;516:55-70. Epub 2018 Jan 9.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virol.2017.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826876PMC
March 2018

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

J Virol 2018 03 12;92(5). Epub 2018 Feb 12.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication.
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http://dx.doi.org/10.1128/JVI.01969-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809724PMC
March 2018

Erratum to: An Adaptable Polyethylene Glycol-Based Workflow for Proteomic Analysis of Extracellular Vesicles.

Methods Mol Biol 2017 ;1660:E1

Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306, USA.

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http://dx.doi.org/10.1007/978-1-4939-7253-1_36DOI Listing
January 2017

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

Methods Mol Biol 2017 ;1660:303-317

Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-7253-1_25DOI Listing
May 2018

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

J Virol 2017 03 14;91(5). Epub 2017 Feb 14.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA

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. EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's 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.
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http://dx.doi.org/10.1128/JVI.02251-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309960PMC
March 2017

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

Oncotarget 2016 Dec;7(52):86999-87015

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, 32306, USA.

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.
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http://dx.doi.org/10.18632/oncotarget.13569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341331PMC
December 2016

Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis.

J Extracell Vesicles 2016 13;5:31295. Epub 2016 Jul 13.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA;

Background: Extracellular vesicles (EVs) are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown.

Methods: Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion.

Results: Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis.

Conclusion: These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947197PMC
http://dx.doi.org/10.3402/jev.v5.31295DOI Listing
July 2016
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