Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.microvesicle | circulating

Cell-free circulating microRNAs (miRNAs) in the blood are good diagnostic biomarker candidates for various physiopathological conditions, including cancer, neurodegeneration, diabetes and other diseases. Since their discovery in 2008 as blood biomarkers, the field has expanded rapidly with a number of important findings. Despite the initial optimistic views of their potential for clinical application, there are currently no circulating miRNA-based diagnostics in use. In this article, we review the status of circulating miRNAs, examine different analytical approaches, and address some of the challenges and opportunities.

Exosomes are secreted, membrane-bound vesicles of 50-100 nm known to carry cellular messenger RNAs (mRNA) and microRNAs (miRNA). Several reports have indicated that tumor-derived exosomes are released into the circulation of patients with a variety of cancer types, including epithelial ovarian cancer. The goals of this study are (i) to develop methods for specific capture and molecular profiling of ovarian cancer tumor-derived exosomes from blood plasma and (ii) determine whether information carried in tumor-derived exosomes can serve as a reliable surrogate for tumor tissue for measurement of key miRNA and mRNA biomarkers. In our work to date, we have used exosomes derived from conditioned media of ovarian cancer cell lines to develop and optimize methods for the isolation and physical characterization of exosomes. Using Nanoparticle Tracking Analysis (NTA) technology (NanoSight, Inc.) to directly count and accurately size exosome populations, our initial data indicates that traditional ultracentrifugation protocols may fail to recover a major fraction of exosomes present in conditioned media. The basis for this finding is currently under investigation and the latest results will be presented. In parallel, we are developing affinity capture approaches to isolate specific subpopulations of exosomes using antibodies directed against epithelial cell adhesion molecule (EpCAM) and other exosomal surface proteins. Initial results indicate that a subpopulation of ovarian cancer-derived exosomes can be purified in this manner; molecular characterization of exosomal contents is in progress. Finally, we are analyzing clinical plasma specimens from patients and controls to: (i) test whether bulk properties of exosomes (e.g., size and number) differ between cases and controls, and (ii) perform specific capture of tumor-derived plasma exosomes and compare exosomal mRNA/miRNA profiles to matched tumor tissue to determine the degree of concordance between exosomal and tissue RNA profiles. Latest results from these studies will be presented. Ultimately, we anticipate that this work will lead to methods for molecular phenotyping of epithelial ovarian cancers in individual patients using a blood-based approach. This work was supported in part by a Stand Up To Cancer/AACR Innovative Research Grant to M. Tewari. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3190. doi:1538-7445.AM2012-3190

MicroRNAs (miRNAs) are small regulatory RNA molecules that function in a wide range of normal and pathologic cellular processes. Cell-free miRNAs circulate in blood (i.e. plasma, serum), are stable and can be diagnostic for cancer. Circulating microRNAs in cancer have been reported to exist in multiple biophysical states, including most famously as cargo of 50-100 nm extracellular vesicles known as exosomes. In prior studies of healthy individuals, we have found that a majority of plasma miRNAs are not vesicle-associated and are substantially present as part of a ribonucleoprotein complex containing Argonaute-2 (Ago2). In order to determine the state of miRNAs in the circulation of cancer patients, we quantitatively profiled the distribution of >300 miRNAs in progressive physical and biochemical fractions of plasma from individuals with metastatic prostate cancer and matched, healthy controls. Stepwise differential centrifugation was performed at 12,000g (to collect cell debris, etc.) and 120,000g (to collect exosomes), followed by Ago2-immunoprecipitation. Exosome fractions generally contained little miRNA, both in cases and in controls. Prostate cancer miRNA biomarkers (miR-141, miR-375) were predominantly soluble, partially associated with Ago2 (15-35% of total) and only marginally present in the exosome fraction (<1% of total). In order to quantify the amount of miRNA associated on average with an exosome, we used Nanoparticle Tracking Analysis (NTA) to quantify exosomes isolated from multiple in-vivo and in-vitro sources by differential ultracentrifugation, followed by miRNA extraction and quantification. We found that exosomes contain very few copies on average of their most abundant miRNAs (less than one copy per individual exosome). In contrast, a significant miR-141 biomarker signal was observed in the 12,000xg-pellet from plasma (approximately 30-45% of input), indicating some miRNA could be associated with larger and/or more dense particles (e.g., oncosomes). In summary, these results indicate that (i) prostate cancer-associated miRNAs are not predominantly contained within exosomes, (ii) the majority of exosomes do not carry significant amounts of miRNAs, and (iii) established biomarkers for prostate cancer are present in multiple physical states, including soluble (e.g., Ago2-associated) and large-particle associated (i.e. 12,000xg pellet) forms (possibly as cell debris or oncosomes). A more accurate understanding of the physical state of miRNA biomarkers will help guide their development to fully realize their diagnostic potential. Citation Format: John R. Chevillet, Emily N. Gallichotte, Heather H. Cheng, Lucia N. Vojtech, Jason D. Arroyo, Hilary Briggs, Robert L. Vessella, Celestia S. Higano, Evan Y. Yu, Era L. Pogosova-Agadjanyan, Derek L. Stirewalt, Florian Hladik, Muneesh Tewari. Circulating microRNA biomarkers for cancer exist in multiple biophysical states. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1118. doi:10.1158/1538-7445.AM2013-1118

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