Robert Setterquist1, Laura Chapman1, Alex Rai2, Emily Zeringer1, Mu Li1, Tim Barta1, Jeff Schageman1, Susan Magdaleno1, Alexander V. Vlassov1. 1Life Technologies, Austin TX 78744, 2Center for Advanced Laboratory Medicine Columbia University Medical Center & New York Presbyterian Hospital Department of Pathology & Cell Biology.

RESULTS ABSTRACT Exosomes are small vesicles (30-150 nm) found in abundance in human body fluids which function as carriers of different species of RNA and protein between diverse locations in the body. The spectrum of current scientific interest in exosomes is wide and ranges from studying their functions and pathways to utilizing them in diagnostics and therapeutics development. As such, there is a growing need for quick and easy methods for both isolation of exosomes and analysis of their cargo. We present herein a workflow for exosome isolation and analysis which entails: (i) fast and efficient isolation of exosomes from serum, plasma, and urine of both healthy donors and patients with prostate cancer, using Total Exosome Isolation reagents; (ii) characterization of their size distribution and count with Nanosight® LM10 instrument; (iii) extraction of exosome “cargo” with Total Exosome RNA and Protein Isolation kit; (iv) characterization of exosomal RNA content using the Ion Torrent™ PGM™ sequencing and qRT-PCR. The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples. We demonstrate that cancer-specific RNA signatures residing within the exosomes can be delineated from different patient cohorts. This is the first step towards developing a method whereby performance characteristics can be measured and used to optimize a validated assay useful for routine testing of clinical samples.

INTRODUCTION Cells are known to secrete a large variety of vesicles, macromolecular complexes, and smaller molecules like salts and cofactors, into the extracellular space. The types of vesicles secreted are diverse and depend on the origin of the cells and their current state – for example, transformed, differentiated, stimulated, or stressed. Exosomes are a type of nanovesicle that have received increased attention over the past decade. Depending on the cell or tissue of origin, many different roles and functions have been attributed to exosomes, for example: eradication of obsolete molecules, facilitation of the immune response, antigen presentation, programmed cell death, angiogenesis, inflammation, coagulation, dissemination of oncogenes from tumor cells, and spread of pathogens such as prions and viruses from one cell to another. More importantly, exosomes deliver macromolecular messages (RNA and protein) that enable cell-to-cell communication and signaling. Interest towards exosomes, from their function in the body to more practical applications, such as use in diagnostics, biomarkers and therapeutics development, has grown exponentially in the last few years.

MATERIALS AND METHODS Exosomes were recovered from HeLa cell culture media or body fluids with either the Total Exosome Isolation Reagents or differential centrifugation (widely used “homebrew” approach, for comparison). The Total Exosome RNA and Protein Isolation kit was used to purify RNA and isolate protein from the enriched exosome preparations. Exosomal RNA analysis was performed by real-time RT-PCR, using TaqMan® reagents, and by sequencing on the Ion Torrent™ Personal Genome Machine® (PGM). Exosomal protein analysis was performed using Western blots. Optimized Exosome workflow: from isolation to analysis

CONCLUSIONS • Total Exosome Isolation reagents enable fast and efficient extraction of exosomes from cell culture media, blood serum as well as other body fluids, with purity comparable to the ultracentrifugation protocols (based on Nanosight®, EM, qRT-PCR, Western analysis)

• Total Exosome RNA and Protein isolation kit allows fast recovery of exosomal RNA and protein

• Exosomes derived from blood and cell media contain extremely diverse RNA “cargo”: in addition to miRNAs, substantial amounts of mRNA, rRNA, tRNA, and short non-coding RNAs (many of which is unmapped) were found – using PGM sequencing. A number of promising exosomal RNA markers were identified

• The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples.

REFERENCES Vlassov A.V., Magdaleno S., Setterquist R. & Conrad R. (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta. 1820, 940-948. Schageman J., Zeringer E., Li M., Barta T., Lea K., Jian Gu, Magdaleno S., Setterquist R. & Vlassov A.V. (2013) The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research Int. 253957.

ACKNOWLEDGEMENTS Axl Neurauter, Ketil Pedersen, Malin Karlsson, Tine Hildisch, Ivonne Moon, Angie Cheng, Nicholas Dolman, Paul Predki, Jason Johnson

TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic procedures. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agilent is a trademark of Agilent Technologies, Inc. NanoSight is a trademark of NanoSight Ltd. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license.

Development of a standard operating procedure for exosome isolation and analysis using clinical samples: application to cancer biomarker discovery

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•  Similar nanovesicle size distribution in all samples, majority 50-150 nm in size – a typical exosome range •  Reagent recovers more exosomes (shown are different dilutions for NanoSight® analysis) •  The reagent allows recovery of exosomes much faster than ultracentrifugation protocol

NanoSight® Analysis of exosomes: Total Exosome Isolation Reagent vs Ultracentrifugation

Protein Analysis: Western blotting for exosomal marker protein CD63

•  Both ultracentrifugation protocol and reagent recover very clean population of exosomes •  The reagent allows recovery of exosomes much faster than ultracentrifugation protocol

•  Reagent recovers somewhat more exosomes vs ultracentrifugation protocol, resulting in higher RNA yield

RNA Analysis: qRT-PCR for mRNA and miRNA targets

RNA sequencing results: exosomes isolated from cell culture media, serum, urine

• extremely diverse exosomal RNA “cargo”: miRNA, mRNA, rRNA, tRNA, other ncRNA • exosomes found in various body fluids have different cargo • most of the RNAs from cells are incorporated into exosomes • some RNAs are found at the same levels in cells and in exosome preparations -> MDX • some RNAs are found at different levels in cells and in exosome preparations -> exo markers

§ Agilent RNA Pico chip profiles indicate that majority of RNA content is small (< 200 nt), but some full-length RNA species (>1 kb) are present

Initial analysis of RNA cargo of exosomes derived from HeLa cell media

Immunolabeled, negative stain Exosomes ~ 100 nm; Gold particles ~10nm in size

EM analysis of exosomes recovered from cell culture media with Total Exosome Isolation reagent. Immunolabeling with CD63 and CD81 antibodies Simple and efficient recovery of exosomes with Total Exosome Isolation (serum) reagent