Abstract 1118: Circulating microRNA biomarkers for cancer exist in multiple biophysical states.
Authors: Chevillet John R. , Gallichotte Emily N. , Cheng Heather H. , Vojtech Lucia N. , Arroyo Jason D. , Briggs Hilary , Vessella Robert L. , Higano Celestia S. , Yu Evan Y. , Pogosova-Agadjanyan Era L. , Stirewalt Derek L. , Hladik Florian , Tewari Muneesh 
Published: August 2014

Abstract
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 &gt;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 (&lt;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