Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification.

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 noninvasive 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.

A novel assay for exosomal and cell-free miRNA isolation and quantification.

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.

Monitoring Therapy Efficiency in Cancer through Extracellular Vesicles.

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.

Author Correction: Different signatures of miR-16, miR-30b and miR-93 in exosomes from breast cancer and DCIS patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Specific microRNA signatures in exosomes of triple-negative and HER2-positive breast cancer patients undergoing neoadjuvant therapy within the GeparSixto trial.

BACKGROUND: The focus of this study is to identify particular microRNA (miRNA) signatures in exosomes derived from plasma of 435 human epidermal growth factor receptor 2 (HER2)-positive and triple-negative (TN) subtypes of breast cancer (BC). METHODS: First, miRNA expression profiles were determined in exosomes derived from the plasma of 15 TNBC patients before neoadjuvant therapy using a quantitative TaqMan real-time PCR-based microRNA array card containing 384 different miRNAs. Forty-five miRNAs associated with different clinical parameters were then selected and mounted on microRNA array cards that served for the quantification of exosomal miRNAs in 435 BC patients before therapy and 20 healthy women. Confocal microscopy, Western blot, and ELISA were used for exosome characterization. RESULTS: Quantification of 45 exosomal miRNAs showed that compared with healthy women, 10 miRNAs in the entire cohort of BC patients, 13 in the subgroup of 211 HER2-positive BC, and 17 in the subgroup of 224 TNBC were significantly deregulated. Plasma levels of 18 exosomal miRNAs differed between HER2-positive and TNBC subtypes, and 9 miRNAs of them also differed from healthy women. Exosomal miRNAs were significantly associated with the clinicopathological and risk factors. In uni- and multivariate models, miR-155 (p = 0.002, p = 0.003, respectively) and miR-301 (p = 0.002, p = 0.001, respectively) best predicted pathological complete response (pCR). CONCLUSION: Our findings show a network of deregulated exosomal miRNAs with specific expression patterns in exosomes of HER2-positive and TNBC patients that are also associated with clinicopathological parameters and pCR within each BC subtype.

Exosomal microRNAs as tumor markers in epithelial ovarian cancer.

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.

Different signatures of miR-16, miR-30b and miR-93 in exosomes from breast cancer and DCIS patients.

Loading of microRNAs (miRNAs) into exosomes that are involved in cellular communication is a selective process. The current study investigates whether the enrichment of miRNAs in exosomes reflects the pathogenesis of breast cancer (BC) and ductal carcinoma in situ (DCIS). The levels of miRNAs were quantified in exosomes from plasma of 32 BC patients, 8 DCIS patients and 8 healthy women by TaqMan real-time PCR-based miRNA array cards containing 47 different miRNAs. Then, exosomal miR-16, miR-30b and miR-93 that displayed deregulation in the arrays were selected and analyzed in 111 BC patients, 42 DCIS patients and 39 healthy women by TaqMan real-time PCR. Identification of exosomes was performed by Western blot. The levels of exosomal miR-16 were higher in plasma of BC (p = 0.034) and DCIS (p = 0.047) patients than healthy women, and were associated with estrogen (p = 0.004) and progesterone (p = 0.008) receptor status. Particularly, in estrogen-positive patients miR-16 was significantly enriched in exosomes (p = 0.0001). Lower levels of exosomal miR-30b were associated with recurrence (p = 0.034). Exosomal miR-93 was upregulated in DCIS patients (p = 0.001). Our findings suggest that different signatures of miR-16, miR-30b and miR-93 in exosomes from BC and DCIS patients are associated with a particular biology of breast tumors.

Plasma microRNA signature is associated with risk stratification in prostate cancer patients.

The aim of this study was to establish a unique expression profile of circulating cell-free microRNAs (miRNAs) capable of differentiating between prostate cancer (PCa) patients with high-risk and intermediate-risk Gleason scores. MiRNA expression profiles were determined in plasma samples from 79 treatment-naïve PCa patients, 1-2 follow-up samples after radical prostatectomy (RP) from 51 out of the 79 PCa patients, and 33 healthy men, using a quantitative real-time PCR-based array containing 48 selected miRNAs. We identified 27 up- and 2 downregulated plasma miRNAs in PCa patients compared with healthy men. Most of the upregulated miRNA levels were also associated with increasing PSA levels and Gleason scores. Particularly, the levels of miR-16 (p = 0.002), miR-148a (p = 0.006) and miR-195 (p = 0.006) significantly correlated with high-risk Gleason scores, whereby miR-148a (p = 0.003) was also significantly associated with increasing PSA values. The high miRNA levels before RP remained increased in the postsurgical plasma samples. Our findings show a network of deregulated plasma miRNAs. In particular, miR-16, miR-148a and miR-195 are involved in the regulation of the PI3K/Akt signaling pathway. These miRNAs may be promising therapeutic targets for high-risk PCa stratification.