Extracellular Vesicles RNA Research Articles

Extracellular vesicle RNA signaling in the liver tumor microenvironment

The tumor microenvironment (TME) in liver cancers such as hepatocellular cancer (HCC) consists of a complex milieu of liver tissue-resident cells, infiltrated immune cells, and secreted factors that collectively serve to promote tumor growth and progression. Intercellular crosstalk contributes to tissue homeostasis, and perturbations during injury, inflammation and tumorigenesis that are important for tumor progression. Extracellular vesicle (EV)-mediated transfer of a payload of RNA molecules that serve as an intercellular signaling is an important contributor to tissue homeostasis within the TME. Several types of RNA have been implicated in EV-mediated signaling. Biological processes that can be modulated by EV RNA signaling within the liver include tumor growth, invasion, metastasis, angiogenesis, and modulation of the immune cell activities. This mini-review describes the liver TME, and the biological effects of EV RNA-mediated signaling within the liver to highlight the role of EV RNA in intercellular communication.

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles.

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.

Evaluation of Microvascular Rarefaction in Vascular Cognitive Impairment and Heart Failure (CRUCIAL): Study Protocol for an Observational Study

Introduction: Microvascular rarefaction, the functional reduction in perfused microvessels and structural reduction of microvascular density, seems to be an important mechanism in the pathophysiology of small blood vessel-related disorders including vascular cognitive impairment (VCI) due to cerebral small vessel disease and heart failure with preserved ejection fraction (HFpEF). Both diseases share common risk factors including hypertension, diabetes mellitus, obesity, and ageing; in turn, these comorbidities are associated with microvascular rarefaction. Our consortium aims to investigate novel non-invasive tools to quantify microvascular health and rarefaction in both organs, as well as surrogate biomarkers for cerebral and/or cardiac rarefaction (via sublingual capillary health, vascular density of the retina, and RNA content of circulating extracellular vesicles), and to determine whether microvascular density relates to disease severity. Methods: The clinical research program of CRUCIAL consists of four observational cohort studies. We aim to recruit 75 VCI patients, 60 HFpEF patients, 60 patients with severe aortic stenosis (AS) undergoing surgical aortic valve replacement as a pressure overload HFpEF model, and 200 elderly participants with mixed comorbidities to serve as controls. Data collected will include medical history, physical examination, cognitive testing, advanced brain and cardiac MRI, ECG, echocardiography, sublingual capillary health, optical coherence tomography angiography (OCTa), extracellular vesicles RNA analysis, and myocardial remodelling-related serum biomarkers. The AS cohort undergoing surgery will also have myocardial biopsy for histological microvascular assessment. Discussion: CRUCIAL will examine the pathophysiological role of microvascular rarefaction in VCI and HFpEF using advanced brain and cardiac MRI techniques. Furthermore, we will investigate surrogate biomarkers for non-invasive, faster, easier, and cheaper assessment of microvascular density since these are more likely to be disseminated into widespread clinical practice. If microvascular rarefaction is an early marker of developing small vessel diseases, then measuring rarefaction may allow preclinical diagnosis, with implications for screening, risk stratification, and prevention. Further knowledge of the relevance of microvascular rarefaction and its underlying mechanisms may provide new avenues for research and therapeutic targets.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles reduce inflammation and pyroptosis in acute kidney injury via miR-223-3p/HDAC2/SNRK.

Bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated as a potential therapeutic agent in acute kidney injury (AKI). However, little is known about the mechanisms of action of BMSC-derived EVs in AKI. Based on this, our research was designed to investigate the mechanism behind BMSC-derived EVs controlling inflammation and pyroptosis during AKI. Peripheral blood from AKI patients was used for detection of microRNA (miR)-223-3p, HDAC2, and SNRK expression. An AKI rat model was established, and HK-2 cell injury was induced by lipopolysaccharide (LPS) to establish a cellular model. Co-culture with BMSC-derived EVs and/or gain- and loss-of-function assays were conducted in LPS-treated HK-2 to evaluate the functions of BMSCs-EVs, miR-223-3p, HDAC2, and SNRK. AKI rats were simultaneously injected with EVs and short hairpin RNAs targeting SNRK. The interactions among miR-223-3p, HDAC2, and SNRK were evaluated by RIP, ChIP, and dual-luciferase gene reporter assays. Patients with AKI had low miR-223-3p and SNRK expression and high HDAC2 expression in peripheral blood. Mechanistically, miR-223-3p targeted HDAC2 to accelerate SNRK transcription. In LPS-treated HK-2 cells, BMSCs-EVs overexpressing miR-223-3p increased cell viability and diminished cell apoptosis, KIM-1, LDH, IL-1β, IL-6, TNF-α, NLRP3, ASC, cleaved caspase-1, and IL-18 expression, and GSDMD cleavage, which was nullified by HDAC2 overexpression or SNRK silencing. In AKI rats, BMSCs-EV-shuttled miR-223-3p reduced CRE and BUN levels, apoptosis, inflammation, and pyroptosis, which was abrogated by SNRK silencing. Conclusively, BMSC-derived EV-encapsulated miR-223-3p mitigated AKI-induced inflammation and pyroptosis by targeting HDAC2 and promoting SNRK transcription.

Circular RNAs in extracellular vesicles: Promising candidate biomarkers for schizophrenia.

As one of common and severe mental illnesses, schizophrenia is difficult to be diagnosed exactly. Both its pathogenesis and the causes of its development are still uncertain because of its etiology complexity. At present, the diagnosis of schizophrenia is mainly based on the patient's symptoms and signs, lacking reliable biomarkers that can be used for diagnosis. Circular RNAs in extracellular vesicles (EV circRNAs) can be used as promising candidate biomarkers for schizophrenia and other diseases, for they are not only high stability and disease specificity, but also are rich in contents and easy to be detected. The review is to focus on the research progress of the correlation between circRNAs and schizophrenia, and then to explores the possibility of EV circRNAs as new biomarkers for the schizophrenia diagnosis.

Analysis of Selectively Enriched RNAs in Extracellular Vesicles

Analysis of Selectively Enriched RNAs in Extracellular Vesicles

H19 in Serum Extracellular Vesicles Reflects Resistance to AR Axis-targeted Therapy Among CRPC Patients.

We aimed to evaluate the changes of androgen receptor (AR) signaling-related long non-coding RNAs (lncRNAs) in serum extracellular vesicles (EVs) from prostate cancer (PC) patients, in order to identify novel biomarkers for AR axis-targeted therapy (ARAT)-resistance among castration-resistant PC (CRPC) patients. EVs were isolated from 2 patients before and after acquiring ARAT-resistance. RNA profiling of EVs was performed by RNA-sequencing. The expression levels of selected lncRNAs in EVs were analyzed by digital droplet PCR (ddPCR) in 58 localized and 14 metastatic PC patients at diagnosis, 7 ARAT-naïve and 6 ARAT-resistant CRPC patients. LncRNA H19 expression in PC tissue was examined using published data. In order to analyze the role of H19, the prognosis was analyzed in PC patients and proteomic analysis was performed in 22Rv1 PC cells. RNA-sequencing revealed that AR-regulated RNAs were most enriched in EVs after acquiring ARAT-resistance. Among them, up-regulation of AR signaling-related lncRNAs (PCAT1, H19, HOXA-11AS, ZEB1-AS1, ARLNC1, PART1, CTBP1-AS and PCA3) was confirmed by ddPCR. H19 contained in EVs (EV-H19) was significantly increased among ARAT-resistant patients compared to ARAT-naïve CRPC or metastatic PC patients. In PC tissue, H19 was negatively correlated with AR protein and AR-activity score and up-regulated in neuroendocrine CRPC tissue with low AR expression. Furthermore, EV-H19 expression was significantly associated with worse outcome to androgen-deprivation therapy. Proteomic analysis demonstrated that H19 knockdown enhanced PC-related protein expression. EV-H19 may negatively correlate with AR-signaling activity and could be a marker to diagnose ARAT-resistance among CRPC patients.

Longitudinal analysis of serum-derived extracellular vesicle RNA to monitor dacomitinib treatment response in EGFR-amplified recurrent glioblastoma patients.

Glioblastoma (GBM) is a highly aggressive and invasive brain tumor associated with high patient mortality. A large fraction of GBM tumors have been identified as epidermal growth factor receptor (EGFR) amplified and ~50% also are EGFRvIII mutant positive. In a previously reported multicenter phase II study, we have described the response of recurrent GBM (rGBM) patients to dacomitinib, an EGFR tyrosine kinase inhibitor (TKI). As a continuation of that report, we leverage the tumor cargo-encapsulating extracellular vesicles (EVs) and explore their genetic composition as carriers of tumor biomarker. Serum samples were longitudinally collected from EGFR-amplified rGBM patients who clinically benefitted from dacomitinib therapy (responders) and those who did not (nonresponders), as well as from a healthy cohort of individuals. The serum EV transcriptome was evaluated to map the RNA biotype distribution and distinguish GBM disease. Using long RNA sequencing, we show enriched detection of over 10 000 coding RNAs from serum EVs. The EV transcriptome yielded a unique signature that facilitates differentiation of GBM patients from healthy donors. Further analysis revealed genetic enrichment that enables stratification of responders from nonresponders prior to dacomitinib treatment as well as following administration. This study demonstrates that genetic composition analysis of serum EVs may aid in therapeutic stratification to identify patients with dacomitinib-responsive GBM.

MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology

AbstractExtracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material.

Extracellular Vesicle RNA Contents as Biomarkers for Ocular Diseases.

Extracellular vesicles (EVs) are small vesicles secreted from cells into extracellular space. EVs contain proteins, lipids, and nucleic acids of the cells from which they originate. For this reason, EVs are being studied for use as biomarkers as they can be surrogates for the status of the cell from which they are secreted. Moreover, EVs are found in numerous biofluids and can be taken up by other cells, which allows for transfer of functional cargo, like RNAs, and changes in gene regulation in the recipient cell. Several potential RNA biomarkers have been identified in many diseases, and there is great potential in the vision field for extracellular RNA biomarkers as a diagnostic tool as well as a measure for treatment efficacy.

Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages.

Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment.

The Autocrine Role of Placental Extracellular Vesicles from Missed Miscarriage in Causing Senescence: Possible Pathogenesis of Missed Miscarriage

Placental dysfunction, including senescent changes, is associated with the pathogenesis of missed miscarriage, although the underlying mechanism is unclear. Increasing evidence indicates that placenta-specific miRNAs are packaged in extracellular vesicles (EVs) from placental syncytiotrophoblasts and are released into the maternal circulation. Aberrant cargos including miRNAs in placental EVs have been reported to be associated with the pathogenesis of complicated pregnancies. In this study, we compared the miRNA profiles in EVs derived from missed miscarriage and healthy placentae and investigated possible biological pathways which may be involved in senescence, one cause of missed miscarriage. The total concentration of RNA in placental EVs was not different between the two groups. However, there were 54 and 94 differentially expressed miRNAs in placental large and small EVs from missed miscarriage compared to EVs from healthy controls. The aberrantly expressed miRNAs seen in placental EVs were also observed in missed miscarriage placentae. Gene enrichment analysis showed that some of those differentially expressed miRNAs are involved in cellular senescence, endocytosis, cell cycle and endocrine resistance. Furthermore, transfection of trophoblasts by a single senescence-associated miRNA that was differentially expressed in placental EVs derived from missed miscarriage did not cause trophoblast dysfunction. In contrast, EVs derived from missed miscarriage placenta induced senescent changes in the healthy placenta. Our data suggested that a complex of placental EVs, rather than a few differentially expressed miRNAs in placental EVs derived from missed miscarriage placentae could contribute in an autocrine manner to placental senescence, one of the causes of missed miscarriage.

Effect of chronic intermittent ethanol vapor exposure on RNA content of brain-derived extracellular vesicles.

Extracellular vesicles (EVs) are important players in normal biological function and disease pathogenesis. Of the many biomolecules packaged into EVs, coding and noncoding RNA transcripts are of particular interest for their ability to significantly alter cellular and molecular processes. Here we investigate how chronic ethanol exposure impacts EV RNA cargo and the functional outcomes of these changes. Following chronic intermittent ethanol (CIE) vapor exposure, EVs were isolated from male and female C57BL/6J mouse brain. Total RNA from EVs was analyzed by lncRNA/mRNA microarray to survey changes in RNA cargo following vapor exposure. Differential expression analysis of microarray data revealed a number of lncRNA and mRNA types differentially expressed in CIE compared to control EVs. Weighted gene co-expression network analysis identified multiple male and female specific modules related to neuroinflammation, cell death, demyelination, and synapse organization. To functionally test these changes, whole-cell voltage-clamp recordings were used to assess synaptic transmission. Incubation of nucleus accumbens brain slices with EVs led to a reduction in spontaneous excitatory postsynaptic current amplitude, although no changes in synaptic transmission were observed between control and CIE EV administration. These results indicate that CIE vapor exposure significantly changes the RNA cargo of brain-derived EVs, which have the ability to impact neuronal function.

Extracellular Vesicles in Chronic Demyelinating Diseases: Prospects in Treatment and Diagnosis of Autoimmune Neurological Disorders.

Extracellular vesicles (EVs) represent membrane-enclosed structures that are likely to be secreted by all living cell types in the animal organism, including cells of peripheral (PNS) and central nervous systems (CNS). The ability to cross the blood-brain barrier (BBB) provides the possibility not only for various EV-loaded molecules to be delivered to the brain tissues but also for the CNS-to-periphery transmission of these molecules. Since neural EVs transfer proteins and RNAs are both responsible for functional intercellular communication and involved in the pathogenesis of neurodegenerative diseases, they represent attractive diagnostic and therapeutic targets. Here, we discuss EVs' role in maintaining the living organisms' function and describe deviations in EVs' structure and malfunctioning during various neurodegenerative diseases.

Abstract 12075: Extracellular Vesicle Cargo Long-Noncoding RNAs Are Dynamic Biomarkers in Acute Decompensated Heart Failure

Introduction: Acute decompensation is associated with increased long-term mortality in patients with heart failure (HF). Biomarkers of acute HF that are dynamic during decongestion may provide physiologic insight for this observation. We hypothesized that long-noncoding RNAs (lncRNAs) and mRNAs in extracellular vesicles (EVs) not only mark different types of HF, but also may be dynamically altered with decongestion across physiologically important pathways. Methods: RNA sequencing was performed on plasma-derived EVs in acute HF patients (7 HFpEF; 7 HFrEF) at hospital admission (V1) and at discharge (V2) along with 9 controls without HF. Differential expression analysis was performed using DESeq2 across groups and time-points. Transcripts were prioritized by fold change (±1.5) and statistical significance (<5% FDR). We subsequently validated targets in EVs from 182 additional patients (24 control; 86 HFpEF; 72 HFrEF). Results: Between HF and control patients, 138 lncRNAs and 147 mRNAs were different (Fig a,b). LncRNAs were confirmed to be exclusively present in EVs. Of 13 targets investigated in our validation cohort, 5 lncRNAs and 6 mRNAs were significantly different between control and HF (age, sex-adjusted; Fig c). Pairwise comparisons between EV RNAs at hospital admission versus decongestion revealed a significant change in 4 lncRNAs (Fig d,e ) that was independent of weight change during hospitalization. Notably, distinct fragments derived from these transcripts were noted in HF versus control groups. Conclusions: The plasma EV transcriptome is significantly altered in patients with acute HF. Plasma EV-derived lncRNA fragments demarcate congestion and decongestion better than mRNAs, may be markers for pathophysiologically relevant pathways independent of volume status, and may provide additional prognostic information.

Abstract 15156: Statistical Modeling of Cardiac-Derived Progenitor Cell Extracellular Vesicles to Predict Clinical Trial Outcomes

Introduction: Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). Therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs), carrying beneficial RNA cargo. Despite some successes with pediatric cardiac cell therapy, large variation in cell populations and patient outcomes remains a problem. To investigate these discrepancies and understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. Methods: We isolated CPCs from cardiac biopsies of congenital heart disease patients (n=30) and the lead-in HLHS patients in the CHILD trial (n=7). We cultured CPCs, collected EVs from the media, and sequenced EV miRNA and total RNA. We treated cardiac endothelial cells and fibroblasts with CPC-EVs and measured inflammatory or fibrotic gene expression with qRT-PCR. We treated mesenchymal stromal cells with CPC-EVs and assessed migration in a Boyden chamber system. We treated cardiac endothelial cells seeded on Matrigel with CPC-EVs and measured tube formation. Finally, we put together the CPC-EV sequencing and experimental data in partial least squares and regularized regression models. Results: With our models we determined that the most important CPC-EV RNA signals involved in pro-reparative outcomes had significant fit to cardiac development and signaling pathways. Furthermore, using a model trained on previously collected CPC-EVs, we were able to predict in vitro outcomes for the CHILD clinical samples. Finally, we formed rank-based predictions for the CHILD lead-in patients, based on the in vitro assays. Conclusions: The CHILD phase I trial is set to conclude next year. We will validate our predictions with the release of these clinical data. With this work, we (1) investigated the EV RNA signals involved in pediatric cardiac repair and (2) will use our in vitro results to predict CHILD clinical trial outcomes. Our results highlight the importance of quantitative studies in determining the underlying variance in pediatric cardiac cell therapy.

Serum extracellular vesicle MicroRNAs as candidate biomarkers for acute rejection in patients subjected to liver transplant.

Acute rejection (AR) is a common and grave complication of liver transplantation (LT). The diagnosis of AR is challenging because it has nonspecific clinical features and requires invasive procedures. Since extracellular vesicles (EVs) are promising candidates as indicators for diagnosis of various diseases, this study aimed to identify serum EV microRNAs (miRNAs) as potential biomarkers for AR in patients subjected to LT. We collected clinical information and serum samples from the liver transplant recipients with and without AR (non-AR). EVs from the serum were isolated via ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. EV RNA was extracted and sequenced on an Illumina HiSeq 2500/2000 platform to identify differentially expressed miRNAs between the groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the target gene candidates of the differentially expressed miRNAs to test their functions in biological systems. Then, we validated 12 differentially expressed miRNAs by quantitative real-time PCR. The results demonstrated that 614 EV miRNAs were significantly altered (387 up regulated and 227 down regulated) between non-AR and AR patients. GO enrichment analysis revealed that these target genes were related to cellular processes, single-organism processes, biological regulation, metabolic processes, cells, cell parts, protein-binding processes, nucleoid binding, and catalytic activity. Furthermore, KEGG pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were primarily involved in ubiquitin-mediated proteolysis, lysosomes, and protein processing in the endoplasmic reticulum. miR-223 and let-7e-5p in AR patients were significantly up-regulated compared to those in non-AR patients, whereas miR-199a-3p was significantly down-regulated, which was consistent with sequencing results. The expression of serum EV miRNAs (up-regulated: miR-223 and let-7e-5p and miR-486-3p; down regulated: miR-199a-3p, miR-148a-3p and miR-152-3p) in AR patients was significantly different from that in non-AR patients, and these miRNAs can serve as promising diagnostic biomarkers for AR in patients subjected to liver transplant.

Detection of HPV RNA in Extracellular Vesicles from Neuroendocrine Cervical Cancer Cells.

Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker.

Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells.

Simple SummaryGlioblastoma multiforme (GBM) is the most aggressive and malignant type of central nervous system (CNS) tumour. Although several treatment options are available, patients generally succumb within 14 months after diagnosis. With the rapid progression of exosome bioengineering technologies, novel therapy opportunities are emerging for GBM treatment. The surface of GBM cells is characterised by the overexpression of transmembrane receptor integrins, which are essential for cell interactions with several proteins in the extracellular matrix. Therefore, integrin-binding drug delivery vehicles have been proposed as a potential strategy for glioblastoma therapy. Small extracellular vesicles possess several attractive characteristics for drug delivery: small size, biocompatibility, ability to cross the blood–brain barrier and capacity to be loaded with exogenous materials. Current bioengineering technologies further increase extracellular vesicle capabilities by loading them with anticancer drugs and incorporating targeting ligands. This study explored the capacity of Arginylglycylaspartic acid (RGD, or Arginine–Glycine–Aspartate)-polypeptide-engineered extracellular vesicles to internalise and deliver loaded cargo in GBM cells. The results demonstrate that introducing the RGD ligand to extracellular vesicles could significantly increase their internalisation by GBM cells and hence improve drug delivery efficacy. Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells.

EV-ADD, a database for EV-associated DNA in human liquid biopsy samples.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV‐associated DNA (EV‐DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV‐DNA. To fill this gap, we have manually curated a database of EV‐DNA data derived from human biofluids (liquid biopsy) and in‐vitro studies, called the Extracellular Vesicle‐Associated DNA Database (EV‐ADD). This database contains validated experimental details and data extracted from peer‐reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV‐DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV‐ADD encompasses EV‐DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV‐ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV‐TRACK), to the best of our knowledge, EV‐ADD is the first of its kind to compile all available EV‐DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV‐DNA‐based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV‐DNA field. EV‐ADD will be updated yearly as newly published EV‐DNA data becomes available and it is freely available at www.evdnadatabase.com.