A PATH TO THE STANDARDIZATION OF EXOSOME ISOLATION AND NGS CHARACTERIZATION FOR COMPLEX DISEASE STUDIES

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1 A PATH TO THE STANDARDIZATION OF EXOSOME ISOLATION AND NGS CHARACTERIZATION FOR COMPLEX DISEASE STUDIES CHAD SCHWARTZ, PH.D., ZACH SMITH, M.S. October 9 th, 2015

2 Exosomes: Small Vesicles, HUGE Impact! -Exosomes are small microvesicles, derived from the late endosome, most often described in the literature to be between 40 and 120 nm, and are released by all cell types. -Exosome characterization and analysis comprise a fast, evolving research area even though their biological function has yet to be completely elucidated. Exosomes contain proteins, lipids, and microrna capable of regulating an assortment of target genes. -Recent studies have suggested that exosomes can serve as biomarkers for future clinical and diagnostic use in not only cancer 1-3 but many other human diseases 4. Exciting new findings have implicated exosomes in cardiovascular diseases 5-7, autoimmune syndromes 8, and neurodegenerative disorders such as Alzheimer s 9 and Parkinson s 10 disease, in addition to infectious diseases such as tuberculosis 11, diphtheria 12, and even HIV 13.

3 What are Exosomes? Tetraspanin; e.g. CD63 Microvesicle Cell-specific Receptor Exosome Early endosome Late endosome Small RNA Protein Lipid raft

4 Position Paper An outcry for standardized methods!

5 Sponsored Webinars Many great webinars available for FREE at BeckmanCoulter.com!

6 Exosome-Depleted FBS by Ultracentrifugation Cell Viability 500 Particle Tracking 400 Completed NTA traces Source Ultracentrifuged Commercially depleted Process > 500 ml of FBS in a single spin!

7 Workflow Overview: Exosome Isolation and Characterization Cell Culture

8 Workflow Overview: Exosome Isolation and Characterization Viability Assay Cell Culture

9 Workflow Overview: Exosome Isolation and Characterization Differential Pelleting Viability Assay Cell Culture

10 Workflow Overview: Exosome Isolation and Characterization Automated Density Gradient for Highly Pure Isolation Differential Pelleting Viability Assay Cell Culture

11 Workflow Overview: Exosome Isolation and Characterization Particle Characterization Automated Density Gradient for Highly Pure Isolation Differential Pelleting Viability Assay Cell Culture

12 Workflow Overview: Exosome Isolation and Characterization Particle Characterization Automated Density Gradient for Highly Pure Isolation Small RNA Extraction Differential Pelleting Viability Assay Cell Culture

13 Workflow Overview: Exosome Isolation and Characterization Particle Characterization Small RNA Extraction NGS Library Construction Automated Density Gradient for Highly Pure Isolation Differential Pelleting Viability Assay Cell Culture

Automated Density Gradient for Highly Pure

14 Workflow Overview: Exosome Isolation and Characterization Particle Characterization Small RNA Extraction NGS Library Construction Next Generation Sequencing Automated Density Gradient for Highly Pure Isolation Differential Pelleting Viability Assay Cell Culture

Cell Viability A benign and cancerous colon cell line were maintained in exosomedepleted FBS At 95% confluency, cells were passaged and counted.

15 Cell Viability A benign and cancerous colon cell line were maintained in exosomedepleted FBS At 95% confluency, cells were passaged and counted. Cell media was retained and harvested for exosome isolation Cell Type Description Cell Count Cell Viability HCT 116 Colorectal Carcinoma 1.52 x % CCD 841 CoN Normal Colon 0.82 x %

16 Isolation via an Automated Layering and Fractionation of a Density Gradient Centrifugation Workflow for Exosome Isolation Exosomes are ~ g/ml Fraction 6-9 were pooled based on density and RNA quantification

17 Density Gradient Layering

18 Density Gradient Fractionation

19 Exosome Size Characterization DG Exosome pellets were resuspended in 1X PBS 55 μl samples were analyzed on the DelsaMax Pro 5 sec acq time 20 acquisitions Laser power: 100% Representative Trace

20 A Case Study

21 Extraction Qiagen mirneasy kit was used to extract RNA from all DG fractions. Total RNA was quantified by UV absorbance, Agilent BioAnalyzer mrna Pico Chip, and LifeTech QuantiT- RiboGreen assay Top Left: CCD Density Gradient RNA Top Right: HCT Density Gradient RNA

22 NEBNext Small RNA Library Prep Kit for Illumina 3' Adaptor Ligation RT Primer Hybridization 5' Adaptor Ligation Reverse Transcription PCR Amplification Post PCR Purification and Size Selection

23 NEBNext Small RNA Automation Method

24 Small RNA Characterization Above: Agilent mirna Control library before (red) and after (blue) size selection overlay Top Right: CCD Density Gradient (blue) and Crude (red) overlay Top Left: HCT Density Gradient (blue) and Crude (red) overlay

Libraries were sequenced on an Illumina MiSeq using a 50 cycle SR sequencing run.

25 Sequencing Results Libraries were quantified in triplicate using the Illumina Library Quantification Kit from Kapa BioSystems using manufacturer s specifications. Libraries were sequenced on an Illumina MiSeq using a 50 cycle SR sequencing run. Library Library Concentration (nm) Pass Filter Reads CCD_DG ,447,621 HCT_DG ,712 CCD_Crude ,534,514 HCT_Crude ,510,580

26 Data Analysis: BaseSpace Libraries were analyzed using the Illumina Small RNA App on BaseSpace following adaptor trimming.

Data Analysis: BaseSpace 204 total mirna families were identified, including 15 families that were differentially expressed. log2(fold Change) Std. err.

27 Data Analysis: BaseSpace 204 total mirna families were identified, including 15 families that were differentially expressed. log2(fold Change) Std. err. log2(fold Change) Regulation in Colon Cancer mirna Family Mean Count q value Source Ogata-Kawata, Hiroko et al., mir E-07 Up mir Up Perilli, Lisa et al., 2014 mir Up Zhou et. al., 2014

28 Data Analysis: BaseSpace mirna Family and mirna Precursor expression shows interesting variation between crude and density gradient prepared samples CCD DG CCD Crude HCT DG HCT Crude CCD DG CCD Crude HCT DG HCT Crude

29 References Vader, P., Breakefield, X.O., Wood, M.J.. Extracellular vesicles: emerging targets for cancer therapy. Trends Mol Med (7): El Andaloussi, S., Mager, I., Breakefield, X.O., Wood, M.J.. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov (5): Simpson, R.J., Lim, J.W., Moritz, R.L., Mathivanan, S.. Exosomes: proteomic insights and diagnostic potential. Expert Rev. Proteomics (3): De Toro, J., Herschlik, L., Waldner, C., Mongini, C.. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front. Immunol doi: /fimmu Amabile, N., Rautou, P-E, Tedgui, A., Boulanger, C.M.. Microparticles: key protagonists in cardiovascular disorders. Semin Thromb. Hemost. (2010) 36: doi: /s asdf DeJong O.G., Verhaar, M.C., Chen, Y., Vader, P., Gremmels, H., Posthuma, G., et.al. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell. Vesicles (2012) 1: doi: /jev.v1i Waldenström, A., Gennebäck, N., Hellman, U., Ronquist, G.. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS One (2012) 7:e doi: /journal.pone Robbins, P.D., Morelli, A.E.. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol (2014) 14: doi: /nri3622. Rajendran, L., Honsho,M., Zahn,T.R., Keller,P., Geiger,K.D., Verkade,P., et. al. Alzheimer s disease beta-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. USA (2006) 103: Doi: /pnas Danzer, K.M., Kranich, L.R., Ruf, W.P., Cagsal-Getkin, O., Winslow, A.R., Zhu,L., et. al. Exosomal cell-to-cell transmission of alphasynuclein oligomers. Mol Neurodegener (2012) 7:42. doi: / Kruh-Garcia, N.A., Wolfe, L.M., Chaisson, L.H.,Worodria, W.O., Nahid P., Schorey J.S., et. al. Detection of Mycobacteriumtuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS. PLoS One (2014) 9:e doi: /journal.pone Colino, J., Snapper, C.M. Exosomes from bone marrow dendritic cells pulsed with diphtheria toxoid preferentially induce type1 antigen-specific IgG responses in naïve recipients in the absence of free antigen. J Immunol (2006) 177: doi: /jimmunol Gould S.J., Booth, A.M., Hildreth,J.E.K.. The Trojan exosome hypothesis. Proc Natl Acad Sci USA (2003) 100: doi: /pnas Shelke, G.V., Lasser, C., Gho, Y.S., Lotvall, J.. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles Doi: /jev.v Ogata-Kawata, Hiroko et al. Circulating Exosomal micrornas as Biomarkers of Colon Cancer. Ed. Tadayuki Akagi. PLoS ONE 9.4 (2014): e PMC. Web. 1 Oct Perilli, Lisa et al. Circulating mir-182 Is a Biomarker of Colorectal Adenocarcinoma Progression. Oncotarget 5.16 (2014): Print. Zhou et. al. "Overexpression of microrna-183 in human colorectal cancer and its clinical significance." Eur J Gastroenterol Hepatol Feb;26(2): doi: /MEG

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