Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3' end adenylated miRNAs are relatively enriched in cells, whereas 3' end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3' end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.

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Most of the current therapies against cancer, and also those against immune diseases or viral infections, consist of empirically designed combination strategies, combining a variety of therapeutic agents. Drug combinations are widely used because multiple drugs affect multiple targets and cell subpopulations. The primary aim is a mutual enhancement of the therapeutic effects, while other benefits may include decreased side effects and the delay or prevention of drug resistance. The large majority of combination regimens are being developed empirically and there are few experimental studies designed to explore thoroughly different drug combinations, using appropriate methods of analysis. However, the study of patterns of possible metabolic and biological interactions in preclinical models, as well as scheduling, should improve the development of most drug combinations. The definition of synergism is that the combination is more effective than each agent separately, e.g., one of the agents augments the actions of the second drug. The definition of antagonism is that the combination is less effective than the single agents, e.g. one of the agents counteracts the actions of the other. A combination can be studied by combining the two agents in various different ways, such as simultaneous or sequential combination schedules. It is essential to test the potency of a combination, before evaluation in the clinic, to prevent antagonistic actions. However, one should realize that an antagonistic action may be desired when toxicity is concerned, i.e. one drug decreases the side effects of another drug. Several attempts have been made to quantitatively measure the dose-effect relationship of each drug alone and its combinations and to determine whether a given combination would gain a synergistic effect. One of the most widely used ways to evaluate whether a combination is effective is the median-drug effect analysis method. Using this method, a combination index (CI) is calculated from drug cytotoxicity or growth inhibition curves. To calculate a CI, the computer software Calcusyn can be used, taking the entire shape of the growth inhibition curve into account for calculating whether a combination is synergistic, additive, or antagonistic. Here, we describe how combinations can be designed in vitro and how to analyze them using Calcusyn or Compusyn. Moreover, pitfalls, limitations, and advantages of using these combinations and Calcusyn/Compusyn are described.

In many cancer types, the expression and function of ∼22 nucleotide-long microRNAs (miRNA) is deregulated. Mature miRNAs can be stably detected in extracellular vesicles (EVs) in biofluids, therefore they are considered to have great potential as biomarkers. In the present study, we investigated whether miRNAs have a distinct expression pattern in urine-EVs of prostate cancer (PCa) patients compared to control males. By next generation sequencing, we determined the miRNA expression in a discovery cohort of 4 control men and 9 PCa patients. miRNAs were validated by using a stemloop RT-PCR in an independent cohort of 74 patients (26 control and 48 PCa-patients). Whereas standard mapping protocols identified > 10 PCa associated miRNAs in urinary EVs, miR-21, miR-375 and miR-204 failed to robustly discriminate for disease in a validation study with RT-PCR-detection of mature miRNA sequences. In contrast, we observed that miRNA isoforms (isomiRs) with 3′ end modifications were highly discriminatory between samples from control men and PCa patients. Highly differentially expressed isomiRs of miR-21, miR-204 and miR-375 were subsequently validated in an independent group of 74 patients. Receiver-operating characteristic analysis was performed to evaluate the diagnostic performance of three isomiRs, resulting in a 72.9% sensitivity with a high (88%) specificity and an area under the curve (AUC) of 0.866. In comparison, prostate specific antigen had an AUC of 0.707 and measuring the mature form of these miRNAs yielded a lower 70.8% sensitivity and 72% specificity (AUC 0.766). We propose that isomiRs may carry discriminatory information which is useful to generate stronger biomarkers.

BackgroundCancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients.MethodExosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS.ResultsBased on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa.ConclusionThese data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner.