BACKGROUND Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows non-invasive tumor subtype classification. METHODS DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics. RESULTS Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids. CONCLUSIONS This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV and mutational status of glioblastoma cells and enables their molecular classification.

BACKGROUND: Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. We previously demonstrated that extracellular vesicles (EV) from central nervous system tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. Their implication as biomarkers in tumor disease is under current investigation. It is unclear, however, to what extent cerebrospinal fluid (CSF) EVs from intraspinal tumors are utilizable for diagnostical purposes and how their marker profiles overlap with EVs derived from non tumorous EVs. We analyzed CSF EVs of intraspinal tumors to define CSF EV profiles that allow tumor subtype classification. METHODS: EVs were isolated from CSF of patients suffering from intraspinal meningioma (n=5), ependymoma (n=7) and neurinoma (n=5). Patients suffering from normal pressure hydrocephalus were used as controls (n=5). EVs were analyzed by multiplex bead based assay, immunoblotting, electron microscopy and NTA. RESULTS: CSF EVs were 97.21 ± 3.37nm (intraspinal tumor patients) and 101.6 ± 3.68nm (controls) in sizes and showed vesicular structures by electron microscopy. Particle number were not significantly different between both groups (p = 0.103). Using our 37 protein mutliplex EV profiling kit we found 29 proteins to be expressed in a sufficient manner on CSF EVs. CSF EVs of intraspinal meningioma showed elevated CD62P, HLA-DR, CD40, CD42a and CD45 expression levels, while ependymoma showed decreased levels of CD9, CD63, CD81, whereas neurinomas had elevated levels of SSEA-3 and CD25. CONCLUSION: This is the first comprehensive analysis of CSF EV of intraspinal tumor patients. CSF EV display distinct subpopulations that may allow tumor classification and long-term surveillance. However as tumor-specific EVs may be rare, there is still the need to identify markers that can enrich tumor-specific EVs for molecular profiling.

ABSTRACT The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles made of a phospholipid bilayer and are secreted by all cell types. EVs are present in a variety of body fluids containing proteins, DNA, RNA species, and lipids, and play an important role in cell- to-cell communication and are worth being considered as biomarkers for both early diagnosis of cancer patients and real-time monitoring of treatment response. Recently, emerging evidence verified EVs to have crucial roles in cancer progression and metastasis and a great potential in therapeutic applications. In this review, we discuss the potential of EVs in monitoring the efficacy of cancer therapies.

Exosomes, small membrane vesicles, mediate cell-to-cell communication. Released by a donor cell, they transfer proteins, lipids and nucleic acids to another cell, resulting in modulation of the recipient cell. Tumor-derived exosomes may transform normal, wild type cells into malignant cells. MiRNAs that inhibit post-transcriptionally protein expression of their target mRNAs are assumed to be selectively packaged into exosomes and may be functionally active in the recipient cell. In this study, the first aim was to identify particular miRNA signatures in exosomes derived from plasma of 435 HER2-positive and triple negative BC patients. First, the miRNA expression profiles were determined in exosomes derived from plasma of 15 triple-negative patients before neoadjuvant therapy using quantitative TaqMan real-time PCR-based microRNAs array cards containing 384 different miRNAs. Derived from these results, 45 miRNAs associated with different clinical parameters were then selected and mounted on miRNAs array cards that served for the quantification of exosomal miRNAs in 435 BC patients before therapy and 20 healthy women. A network of deregulated exosomal miRNAs with specific expression patterns that were also associated with clinicopathological parameters was identified in exosomes from HER2-positive and triple-negative patients. In uni- and multivariate models, miR-155 and miR-301 best predicted pathological complete response (pCR). The levels of 4 exosomal miRNAs (miR-27a, miR-155, miR-376a and miR-376c) upregulated before neoadjuvant therapy decreased to normal healthy levels after therapy. These findings may improve the stratification of patients into different risk groups and future targeted therapies. One of these most deregulated miRNAs was miR-376c detected in exosomes from BC patients. Its targets mRNAs were determined by the MirTrap system which locks the miRNA/mRNA pair into the RISC complex which can be pulled down using an antibody against this complex. Using an mRNA array, it was found that 31 mRNAs could be bound by miR-376c. The mRNAs mostly enriched in the RISC complex were the apoptosis inhibitor BCL2, the DNA repair and cell cycle gene BRCA1, the adhesion molecule Cadherin 1 and the cyclin-dependent kinase inhibitor 1A. Functional analyses showed that miR-376c had no impact on cell proliferation and apoptosis. Western blot and ELISA showed significantly higher levels of exosomes in plasma of BC patients than in healthy women. These findings point to an excessive secretion of exosomes in cancer patients. In addition, the size and concentration of the EVs were measured using the Nanoparticle tracking analysis. The measurements revealed that the majority of particles had a size of 120 nm. Finally, EVs were stained by a red fluorescence dye and visualized by confocal microscopy. The videos showed that EVs were released by cells, localized in the cell membrane and uptake by cells. In conclusion, these findings suggest a selective packaging of miRNAs in exosomes from HER2-positive and triple-negative breast cancer patients which is also associated with the different clinical parameters. It is hypothesized that these different miRNA profiles forming the cargo of exosomes may be shuttled from cell to cell. Exosomen, kleine Membranvesikel, vermitteln die Kommunikation von Zelle zu Zelle. Sie werden von einer Donorzelle freigesetzt und transportieren Proteine, Lipide und Nukleinsauren in eine andere Zelle, was zu einer Modulation der Empfangerzelle fuhrt. Vom Tumor befreite Exosomen konnen normale Wildtypzellen in bosartige Zellen umwandeln. Es wird angenommen, dass miRNAs, die die post-transkriptionelle Proteinexpression ihrer ZielmRNAs hemmen, selektiv in Exosomen verpackt werden und in der Empfangerzelle funktionell aktiv sein konnen. In dieser Studie war das erste Ziel, bestimmte miRNA-Signaturen in Exosomen aus dem Plasma von 435 HER2-positiven und triple-negativen Brustkrebspatientinnen zu identifizieren. Zuerst wurden die miRNA-Expressionsprofile in Exosomen aus dem Plasma von 15 triple-negativen Patienten vor einer neoadjuvanten Therapie unter Verwendung quantitativer TaqMan real-time PCR-basierter mikroRNA-Array-Karten mit 384 verschiedenen miRNAs bestimmt. Basierend auf diesen Ergebnissen wurden dann 45 miRNAs, die mit den verschiedenen klinischen Parametern assoziiert waren, ausgewahlt und auf miRNA-Array-Karten angebracht, die zur Quantifizierung exosomaler miRNAs bei 435 Brustkrebspatientinnen vor Therapie und 20 gesunden Frauen dienten. Ein Netzwerk von deregulierten exosomalen miRNAs mit spezifischen Expressionsmustern, die auch mit den klinisch-pathologischen Parametern assoziiert waren, wurde in Exosomen von HER2-positiven und triple-negativen Patientinnen identifiziert. In uni- und multivariaten Modellen prognostizierten miR-155 und miR-301 am besten die pathologische Response (pCR). Die Konzentrationen von 4 exosomalen miRNAs (miR-27a, miR-155, miR-376a und miR-376c), die vor der neoadjuvanten Therapie hochreguliert waren, verkleinerten sich auf normale gesunde Werte nach der Therapie. Diese Ergebnisse konnten die Stratifizierung von Patienten in verschiedene Risikogruppen und zukunftige zielgerichtete Therapien verbessern. Eine dieser am haufigsten deregulierten miRNAs war miR-376c, die in Exosomen von Brustkrebspatientinnen nachgewiesen wurde. Seine Ziel-mRNAs wurden durch das MirTrap-System bestimmt, dass das miRNA/mRNAPaar in den RISC-Komplex aufnimmt, und der dann mithilfe eines Antikorpers gegen diesen Komplex isoliert werden kann. Unter Verwendung eines mRNA-Arrays wurden 31 mRNAs, die durch miR-376c gebunden werden konnten, gefunden. Die im RISC-Komplex meist angereicherten mRNAs waren der Apoptose-Inhibitor BCL2, das DNA-Reparatur- und Zellzyklus-Gen BRCA1, das Adhasionsmolekul Cadherin 1 und der Cyclin-abhangige Kinase- Inhibitor 1A. Funktionelle Analysen zeigten, dass miR-376c keinen Einfluss auf Zellproliferation und Apoptose hatte. Western Blot und ELISA prasentierten signifikant hohere Exosomen-Niveaus im Plasma von Brustkrebspatientinnen als bei gesunden Frauen. Diese Befunde weisen auf eine exzessive Sekretion von Exosomen bei Krebspatienten hin. Zusatzlich wurden die Grose und Konzentration der Exosomen unter Verwendung der Nanopartikel-Tracking-Analyse gemessen. Die Messungen zeigten, dass die Mehrzahl der Partikel eine Grose von 120 nm aufwies. Schlieslich wurden die Exosomen durch einen roten Fluoreszenzfarbstoff gefarbt und durch konfokale Mikroskopie sichtbar gemacht. Die Videos zeigten, dass EVs von Zellen freigesetzt wurden, in der Zellmembran lokalisiert waren und von Zellen aufgenommen wurden. Zusammenfassend schlagen diese Ergebnisse eine selektive Verpackung von miRNAs in Exosomen von HER2-positiven und triple-negativen Brustkrebspatientinnen vor, die ebenfalls mit den unterschiedlichen klinischen Parametern assoziiert ist. Es wird vermutet, dass diese verschiedenen miRNA-Profile, die die Fracht von Exosomen bilden, von Zelle zu Zelle transportiert werden konnen.

Specific microRNAs (miRNAs) are packaged in exosomes that regulate processes in tumor development and progression. The current study focuses on the influence of exosomal miRNAs in the pathogenesis of epithelial ovarian cancer (EOC). MiRNA profiles were determined in exosomes from plasma of 106 EOC patients, eight ovarian cystadenoma patients, and 29 healthy women by TaqMan real‐time PCR‐based miRNA array cards containing 48 different miRNAs. In cell culture experiments, the impact of miR‐200b and miR‐320 was determined on proliferation and apoptosis of ovarian cancer cell lines. We report that miR‐21 (P = 0.0001), miR‐100 (P = 0.034), miR‐200b (P = 0.008), and miR‐320 (P = 0.034) are significantly enriched, whereas miR‐16 (P = 0.009), miR‐93 (P = 0.014), miR‐126 (P = 0.012), and miR‐223 (P = 0.029) are underrepresented in exosomes from plasma of EOC patients as compared to those of healthy women. The levels of exosomal miR‐23a (P = 0.009, 0.008) and miR‐92a (P = 009, 0.034) were lower in ovarian cystadenoma patients than in EOC patients and healthy women, respectively. The exosomal levels of miR‐200b correlated with the tumor marker CA125 (P = 0.002) and patient overall survival (P = 0.019). MiR‐200b influenced cell proliferation (P = 0.0001) and apoptosis (P < 0.008). Our findings reveal specific exosomal miRNA patterns in EOC and ovarian cystadenoma patients, which are indicative of a role of these miRNAs in the pathogenesis of EOC.