RESUMEN
Glioblastoma (GBM) is an incurable primary malignant brain cancer hallmarked with a substantial protumorigenic immune component. Knowledge of the GBM immune microenvironment during tumor evolution and standard of care treatments is limited. Using single-cell transcriptomics and flow cytometry, we unveiled large-scale comprehensive longitudinal changes in immune cell composition throughout tumor progression in an epidermal growth factor receptor-driven genetic mouse GBM model. We identified subsets of proinflammatory microglia in developing GBMs and anti-inflammatory macrophages and protumorigenic myeloid-derived suppressors cells in end-stage tumors, an evolution that parallels breakdown of the blood-brain barrier and extensive growth of epidermal growth factor receptor+ GBM cells. A similar relationship was found between microglia and macrophages in patient biopsies of low-grade glioma and GBM. Temozolomide decreased the accumulation of myeloid-derived suppressor cells, whereas concomitant temozolomide irradiation increased intratumoral GranzymeB+ CD8+T cells but also increased CD4+ regulatory T cells. These results provide a comprehensive and unbiased immune cellular landscape and its evolutionary changes during GBM progression.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Animales , Neoplasias Encefálicas/metabolismo , Receptores ErbB , Glioblastoma/metabolismo , Humanos , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Temozolomida/uso terapéutico , Microambiente Tumoral/genéticaRESUMEN
Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.
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Ácidos Nucleicos Libres de Células/aislamiento & purificación , MicroARN Circulante/aislamiento & purificación , ARN/aislamiento & purificación , Adulto , Líquidos Corporales/química , Línea Celular , Vesículas Extracelulares/metabolismo , Femenino , Voluntarios Sanos , Humanos , Masculino , MicroARNs/aislamiento & purificación , MicroARNs/metabolismo , ARN/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodosRESUMEN
To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.
Asunto(s)
Comunicación Celular/fisiología , ARN/metabolismo , Adulto , Líquidos Corporales/química , Ácidos Nucleicos Libres de Células/metabolismo , MicroARN Circulante/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Programas InformáticosRESUMEN
In this first-in-human, investigator-initiated, open-label study, three participants with recurrent glioblastoma were treated with CARv3-TEAM-E T cells, which are chimeric antigen receptor (CAR) T cells engineered to target the epidermal growth factor receptor (EGFR) variant III tumor-specific antigen, as well as the wild-type EGFR protein, through secretion of a T-cell-engaging antibody molecule (TEAM). Treatment with CARv3-TEAM-E T cells did not result in adverse events greater than grade 3 or dose-limiting toxic effects. Radiographic tumor regression was dramatic and rapid, occurring within days after receipt of a single intraventricular infusion, but the responses were transient in two of the three participants. (Funded by Gateway for Cancer Research and others; INCIPIENT ClinicalTrials.gov number, NCT05660369.).
Asunto(s)
Receptores ErbB , Glioblastoma , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T CD8-positivos/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/terapia , Glioblastoma/patología , Inmunoterapia Adoptiva/efectos adversos , Recurrencia Local de Neoplasia/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.
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Vesículas Extracelulares , Microscopía/métodos , Animales , Colorantes/química , Epítopos , Vesículas Extracelulares/química , Vesículas Extracelulares/patología , Vesículas Extracelulares/fisiología , Colorantes Fluorescentes/química , HumanosRESUMEN
The past decade has witnessed a rapid growth in the field of extracellular vesicle (EV) based biomarkers for the diagnosis and monitoring of cancer. Several studies have reported novel EV based biomarkers, but the technical and clinical validation phase has been hampered by general challenges common to biomedical research field as well as specific challenges inherent to the nanoparticle field. This has led to more common failures than success stories in the biomarker discovery pipeline. As a result, more attention must be focused on the process of biomarker discovery, verification, and validation to allow for translation and application of novel EV based research to patient care. Herein, we briefly discuss the hurdles and potential solutions in EV biomarker discovery and verification and validation, and clinical translation.
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Biomarcadores de Tumor/sangre , Vesículas Extracelulares/química , Neoplasias/diagnóstico , Juego de Reactivos para Diagnóstico/normas , Investigación Biomédica Traslacional/métodos , Biomarcadores de Tumor/líquido cefalorraquídeo , Biomarcadores de Tumor/orina , Vesículas Extracelulares/metabolismo , Humanos , Cavidad Nasal/química , Neoplasias/sangre , Neoplasias/líquido cefalorraquídeo , Neoplasias/orina , Proyectos de Investigación , Saliva/química , Sensibilidad y Especificidad , Estudios de Validación como AsuntoRESUMEN
The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.
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Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Humanos , Modelos Biológicos , Nanopartículas/química , Neoplasias/patología , Fenómenos ÓpticosRESUMEN
In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1-2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.
Asunto(s)
Vesículas Extracelulares/metabolismo , alfa-Sinucleína/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Exosomas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Proteínas tau/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Recent studies suggest that extracellular mitochondria may be involved in the pathophysiology of stroke. In this study, we assessed the functional relevance of endogenous extracellular mitochondria in cerebrospinal fluid (CSF) in rats and humans after subarachnoid hemorrhage (SAH). METHODS: A standard rat model of SAH was used, where an intraluminal suture was used to perforate a cerebral artery, thus leading to blood extravasation into subarachnoid space. At 24 and 72 hours after SAH, neurological outcomes were measured, and the standard JC1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanineiodide) assay was used to quantify mitochondrial membrane potentials in the CSF. To further support the rat model experiments, CSF samples were obtained from 41 patients with SAH and 27 control subjects. Mitochondrial membrane potentials were measured with the JC1 assay, and correlations with clinical outcomes were assessed at 3 months. RESULTS: In the standard rat model of SAH, extracellular mitochondria was detected in CSF at 24 and 72 hours after injury. JC1 assays demonstrated that mitochondrial membrane potentials in CSF were decreased after SAH compared with sham-operated controls. In human CSF samples, extracellular mitochondria were also detected, and JC1 levels were also reduced after SAH. Furthermore, higher mitochondrial membrane potentials in the CSF were correlated with good clinical recovery at 3 months after SAH onset. CONCLUSIONS: This proof-of-concept study suggests that extracellular mitochondria may provide a biomarker-like glimpse into brain integrity and recovery after injury.
Asunto(s)
Líquido Extracelular/metabolismo , Mitocondrias/metabolismo , Recuperación de la Función/fisiología , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Animales , Biomarcadores/líquido cefalorraquídeo , Humanos , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/ultraestructura , Ratas , Ratas Sprague-Dawley , Hemorragia Subaracnoidea/patologíaRESUMEN
Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/metabolismo , Glioma/tratamiento farmacológico , Neurilemoma/tratamiento farmacológico , Enfermedad de Parkinson/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioma/metabolismo , Glioma/patología , Humanos , Neurilemoma/metabolismo , Neurilemoma/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patologíaRESUMEN
Different types of RNAs identified thus far represent a diverse group of macromolecules that are involved in the regulation of different biological processes. RNA is generally thought to be localized primarily in the nucleus and cytoplasm; however, some types of RNA have been detected in the extracellular milieu. These extracellular RNA (exRNA) molecules are protected from degradation and it is now widely accepted that extracellular vesicles and ribonucleoprotein particles serve as transport vehicles for exRNA among cells. The functional consequence of this transfer of genetic information probably encompasses a broad range of normal developmental and physiologic processes in many organisms. This review will focus on the role of exRNA communication in cancer. We will focus on different types of RNA species identified and characterized within tumor-derived extracellular vesicles. Further, we will describe the role of exRNAs in cancer progression, as well as their potential for use as diagnostic biomarkers and therapeutic tools for monitoring and treating cancer, respectively.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/patología , ARN/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Comunicación Celular/fisiología , Progresión de la EnfermedadRESUMEN
The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.
RESUMEN
Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.
Asunto(s)
Vesículas Extracelulares , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Cromatografía en Gel , ARN/análisis , ARN/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismoRESUMEN
Despite the remarkable advances of dermal fillers that reduce wrinkles caused by dermis thickness reduction, they still lack effective hydrogel systems that stimulate collagen generation along with injection convenience. Here, we develop a stem cell-derived extracellular vesicle (EV)-bearing thermosensitive hydrogel (EVTS-Gel) for effective in vivo collagen generation. The TS-Gel undergoes sol-gel transition at 32.6 °C, as demonstrated by the storage and loss moduli crossover. Moreover, the TS-Gel and the EVTS-Gel have comparable rheological properties. Both hydrogels are injected in a sol state; hence, they require lower injection forces than conventional hydrogel-based dermal fillers. When locally administered to mouse skin, the TS-Gel extends the retention time of EVs by 2.23 times. Based on the nature of the controlled EV release, the EVTS-Gel significantly inhibits the dermis thickness reduction caused by aging compared to the bare EV treatment for 24 weeks. After a single treatment, the collagen layer thickness of the EVTS-Gel-treated dermis becomes 2.64-fold thicker than that of the bare EV-treated dermis. Notably, the collagen generation efficacy of the bare EV is poorer than that of the EVTS-Gel of a 10× lesser dose. Overall, the EVTS-Gel shows potential as an antiaging dermal filler for in vivo collagen generation.
Asunto(s)
Colágeno , Dermis , Vesículas Extracelulares , Hidrogeles , Animales , Ratones , Dermis/metabolismo , Dermis/efectos de los fármacos , Colágeno/química , Hidrogeles/química , Hidrogeles/farmacología , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Humanos , Células Madre/citología , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Rellenos Dérmicos/química , Rellenos Dérmicos/farmacologíaRESUMEN
Glioma represents the most common central nervous system neoplasm in adults. Current classification scheme utilizes molecular alterations, particularly IDH1.R132H, to stratify lesions into distinct prognostic groups. Identification of the single nucleotide variant through traditional tissue biopsy assessment poses procedural risks and does not fully reflect the heterogeneous and evolving tumor landscape. Here, we introduce a liquid biopsy assay, mt-IDH1dx. The blood-based test allows minimally invasive detection of tumor-derived extracellular vesicle RNA using only 2 ml plasma volume. We perform rigorous, blinded validation testing across the study population (n = 133), comprising of IDH1.R132H patients (n = 80), IDH1 wild-type gliomas (n = 44), and age matched healthy controls (n = 9). Results from our plasma testing demonstrate an overall sensitivity of 75.0% (95% CI: 64.1%-84.0%), specificity 88.7% (95% CI: 77.0%-95.7%), positive predictive value 90.9%, and negative predictive value 70.1% compared to the tissue gold standard. In addition to fundamental diagnostic applications, the study also highlights the utility of mt-IDH1dx platform for blood-based monitoring and surveillance, offering valuable prognostic information. Finally, the optimized workflow enables rapid and efficient completion of both tumor tissue and plasma testing in under 4 hours from the time of sampling.
Asunto(s)
Neoplasias Encefálicas , Glioma , Isocitrato Deshidrogenasa , Mutación , Humanos , Isocitrato Deshidrogenasa/genética , Glioma/genética , Glioma/sangre , Glioma/diagnóstico , Glioma/patología , Femenino , Masculino , Persona de Mediana Edad , Adulto , Biopsia Líquida/métodos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/diagnóstico , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/sangre , Sensibilidad y Especificidad , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Estudios de Casos y ControlesRESUMEN
Effective diagnosis, prognostication, and management of CNS malignancies traditionally involves invasive brain biopsies that pose significant risk to the patient. Sampling and molecular profiling of cerebrospinal fluid (CSF) is a safer, rapid, and noninvasive alternative that offers a snapshot of the intracranial milieu while overcoming the challenge of sampling error that plagues conventional brain biopsy. Although numerous biomarkers have been identified, translational challenges remain, and standardization of protocols is necessary. Here, we systematically reviewed 141 studies (Medline, SCOPUS, and Biosis databases; between January 2000 and September 29, 2022) that molecularly profiled CSF from adults with brain malignancies including glioma, brain metastasis, and primary and secondary CNS lymphomas. We provide an overview of promising CSF biomarkers, propose CSF reporting guidelines, and discuss the various considerations that go into biomarker discovery, including the influence of blood-brain barrier disruption, cell of origin, and site of CSF acquisition (eg, lumbar and ventricular). We also performed a meta-analysis of proteomic data sets, identifying biomarkers in CNS malignancies and establishing a resource for the research community.
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Biomarcadores de Tumor , Neoplasias Encefálicas , Humanos , Biomarcadores de Tumor/líquido cefalorraquídeo , Neoplasias Encefálicas/líquido cefalorraquídeo , Proteómica/métodos , Proteómica/normas , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Neoplasias del Sistema Nervioso Central/diagnósticoRESUMEN
Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.
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Biología/métodos , Exosomas/química , Biología Celular/tendencias , Centrifugación por Gradiente de Densidad , Microfluídica , Microscopía Electrónica de TransmisiónRESUMEN
Diagnostic platforms providing biomarkers that are highly predictive for diagnosing, monitoring, and stratifying cancer patients are key instruments in the development of personalized medicine. We demonstrate that tumor cells transfer (mutant) RNA into blood platelets in vitro and in vivo, and show that blood platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively. In addition, gene-expression profiling revealed a distinct RNA signature in platelets from glioma patients compared with normal control subjects. Because platelets are easily accessible and isolated, they may form an attractive platform for the companion diagnostics of cancer.
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Biomarcadores de Tumor/metabolismo , Plaquetas/metabolismo , Neoplasias/genética , ARN/metabolismo , Animales , Biomarcadores de Tumor/análisis , Plaquetas/química , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Estudios de Casos y Controles , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Glioma/sangre , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Análisis por Micromatrices , Neoplasias/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN/análisis , ARN/sangre , ARN/genética , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a role.
Asunto(s)
Anticoagulantes/farmacología , Receptores ErbB/metabolismo , Espacio Extracelular/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Heparina/farmacología , Vesículas Transportadoras/efectos de los fármacos , Receptores ErbB/genética , Citometría de Flujo , Glioblastoma/patología , Humanos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.