Standardization of exosome isolation and characterization · A PATH TO THE STANDARDIZATION OF EXOSOME ISOLATION AND NGS CHARACTERIZATION FOR ... Protein Exosome ... Exosome Isolation

A PATH TO THE STANDARDIZATION OF EXOSOME

ISOLATION AND NGS CHARACTERIZATION FOR

COMPLEX DISEASE STUDIES

CHAD SCHWARTZ, PH.D., ZACH SMITH, M.S.

October 9th, 2015

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 cancer1-3 but many other human diseases4.

Exciting new findings have implicated exosomes in cardiovascular diseases5-7,

autoimmune syndromes8, and neurodegenerative disorders such as Alzheimer’s9

and Parkinson’s10 disease, in addition to infectious diseases such as tuberculosis11,

diphtheria12, and even HIV13.

What are Exosomes?

Early

endosome

Late

endosome

Protein

Exosome

Microvesicle

Small RNA

Tetraspanin;

e.g. CD63

Cell-specific

Receptor

Lipid raft

Position Paper

An outcry for standardized methods!

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Exosome-Depleted FBS by Ultracentrifugation

Source Ultracentrifuged Commercially depleted

0

100

200

300

400

500

Co

mp

lete

d N

TA

tra

ce

s

Process > 500 mL of FBS in a single spin!

Cell Viability

Particle Tracking

Workflow Overview: Exosome Isolation and Characterization

Cell Culture


Cell Culture

Viability Assay


Cell Culture

Viability Assay

Differential Pelleting


Cell Culture

Viability Assay


Automated Density Gradient for Highly Pure Isolation


Cell Culture

Viability Assay



Particle Characterization


Cell Culture

Viability Assay



Small

RNA Extraction



Cell Culture

Viability Assay



Small

RNA Extraction

NGS

Library Construction



Cell Culture

Viability Assay



Small

RNA Extraction

NGS

Library Construction

Next Generation Sequencing


Cell Viability

Cell Type Description Cell Count Cell Viability

HCT 116 Colorectal

Carcinoma

1.52 x 108 97.3%

CCD 841 CoN Normal Colon 0.82 x 108 98.4%

• A benign and cancerous colon cell

line were maintained in exosome-

depleted FBS

• At 95% confluency, cells were

passaged and counted.

• Cell media was retained and

harvested for exosome isolation

Isolation via an Automated Layering and Fractionation

of a Density Gradient

Centrifugation Workflow for

Exosome Isolation

Exosomes are

~1.13 – 1.19

g/mL

Fraction 6-9 were

pooled based on

density and RNA

quantification

Density Gradient Layering

Density Gradient Fractionation

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

A Case Study

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

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

NEBNext Small RNA Automation Method

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

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 237.6 1,447,621

HCT_DG 42.1 878,712

CCD_Crude 211.8 1,534,514

HCT_Crude 29.8 1,510,580

Data Analysis: BaseSpace

Libraries were analyzed using the Illumina Small RNA App on BaseSpace

following adaptor trimming.


204 total miRNA

families were

identified,

including 15

families that

were

differentially

expressed.

miRNA Family Mean Count

log2(Fold

Change)

Std. err.

log2(Fold

Change) q value

Regulation

in Colon

Cancer Source

mir-1246 41 3.98 0.733 6.07E-07 Up

Ogata-Kawata, Hiroko et al., 2015

mir-182 29.7 4.87 1.04

0.000018

4 Up Perilli, Lisa et al., 2014

mir-183 66.7 4.61 1.09 0.000114 Up Zhou et. al., 2014


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

References• Vader, P., Breakefield, X.O., Wood, M.J.. Extracellular vesicles: emerging targets for cancer therapy. Trends Mol Med. 2014. 20(7): 385-93.

• El Andaloussi, S., Mager, I., Breakefield, X.O., Wood, M.J.. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug

Discov. 2013. 12(5): 347-57.

• Simpson, R.J., Lim, J.W., Moritz, R.L., Mathivanan, S.. Exosomes: proteomic insights and diagnostic potential. Expert Rev. Proteomics. 2009. 6(3):

267-83.

• 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. 2015. doi: 10.3389/fimmu.2015.00203.

• Amabile, N., Rautou, P-E, Tedgui, A., Boulanger, C.M.. Microparticles: key protagonists in cardiovascular disorders. Semin Thromb. Hemost.

(2010) 36:907–16. doi:10.1055/s-0030-1267044Asdf

• 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) 1:18396. doi:10.3402/jev.v1i0.18396.

• Waldenström, A., Gennebäck, N., Hellman, U., Ronquist, G.. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to

target cells. PLoS One (2012) 7:e34653. doi:10.1371/journal.pone.0034653.

• Robbins, P.D., Morelli, A.E.. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol (2014) 14:195–208. doi:10.1038/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) 103:11172–7. Doi:10.1073/pnas.0603838103.

• 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:10.1186/1750-1326-7-42

• 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:e103811.

doi:10.1371/journal.pone.0103811.

• 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) 177:3757–62. doi:10.4049/jimmunol.177.6.3757.

• Gould S.J., Booth, A.M., Hildreth,J.E.K.. The Trojan exosome hypothesis. Proc Natl Acad Sci USA (2003) 100:10592–7.

doi:10.1073/pnas.1831413100.

• 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. 2014. Doi:10.3402/jev.v3.24783.

• Ogata-Kawata, Hiroko et al. “Circulating Exosomal microRNAs as Biomarkers of Colon Cancer.” Ed. Tadayuki Akagi. PLoS ONE 9.4 (2014):

e92921. PMC. Web. 1 Oct. 2015. Perilli, Lisa et al. “Circulating miR-182 Is a Biomarker of Colorectal Adenocarcinoma

Progression.” Oncotarget 5.16 (2014): 6611–6619. Print.

• Zhou et. al. "Overexpression of microRNA-183 in human colorectal cancer and its clinical significance." Eur J Gastroenterol Hepatol. 2014

Feb;26(2):229-33. doi: 10.1097/MEG.0000000000000002.

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Standardization of exosome isolation and characterization · A PATH TO THE STANDARDIZATION OF EXOSOME ISOLATION AND NGS CHARACTERIZATION FOR ... Protein Exosome ... Exosome Isolation