insight into cancer research from discovery to...

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Insight into cancer researchfrom discovery to validation

2The world leader in serving science

Exosomes in cancer research:from exosome isolation to biomarker discovery

Zheng Songyue, PhD

Technical Sales Specialists

Life Technologies

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What are Exosomes?

Secreted particles first discovered byTrams et al. in 1981

from: The Scientist, July 2011Exfoliated vesicles from cell

lines with ectoenzyme activity

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What are Exosomes?

Pan et al., (1985) J Cell Biol 942-8

Reticulocytes---“Garbage bags”

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What are Exosomes?

Pu

blic

atio

ns

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• 30-150 nm miVesicles with RNA and protein “cargo”

• Secreted by all cells and found in all body fluids.

• Distinguished in their genesis

• Protein markers: tetraspanins(CD63, CD81, CD9),

Alix, flotillin, TSG101, Rab5b

• Extremely diverse functions, including:

• Cellular communication

• Clean up cell‟s trash

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from: Vlassovet al., BiochimBiophysActa. 2012 1820 (7):

940-948.

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Biological functions of the exosomes

Depending on their cell/tissue of origin many different functions have been

attributed to exosomes. To name a few:

Removal of plasma membrane proteins during the cell maturation process.

Facilitators of the immune response, and antigen presentation.

Roles for exosomes in programmed cell death, angiogenesis, inflammation,

and coagulation were reported

The miRNAs contained within exosomes from breast milk seems to

modulate the development of the infant‟s immune system.

Exosomes play a role in spreading pathogens such as prions and viruses

from one cell to another.

Exosomes mediate the cell-to cell transmission of IFN-α-induced antiviral

activity.

miR-150 within exosomes enhance targeted endothelial cell migration.

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Exosomes and Cancer research

•The spread of oncogenes by exosomes and microvesicles secreted by

tumor cells has been reported.

A small percentage of glioma cells

express a truncated and oncogenic form

of the epidermal growth factor receptor

(EGFRvIII)

EGFRvIII can be „shared‟ between glioma

cells by intercellular, in vivo transfer of

microvesicles.

Activation of transforming signaling

pathways (MAPK and Akt),

Changes in expression of EGFRvIII-

regulated genes (VEGF, Bcl-xL, p27),

Morphological transformation

Increase in anchorage-independent growth

capacity.

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Exosomal cargo: potential in cancer diagnostics

Specific expression patterns of miRNAs or mRNA were identified in serum and

urinary exosomes for lung cancer, colorectal cancer, and prostate cancer, these

exosomal cargos could be used as diagnostic markers.

Biomarkers Location Related disease

miR25, miR223 serumnon-small cell lung cancer

miR141 serum Prostate cancer

mRNA encoded

PCA-3 and

TMPRSS2:ERG

Urine

exosomesProstate cancer

Advantages:Body fluid collection is less painful, easier, quicker and

cheaper way than traditional needle or excision biopsies

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Current method needs for exosome researchers

Needs for exosome researchers

Current methods status for exosome researchers

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NanoSight ®

Light microscopy

Electron Microscopy

Flow Cytometry

Life Technologies‟s Exosome Workflow

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Current methods for exosome isolation

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Ultracentrifugation is the current “Gold

Standard” isolation method for exosomes

Proven method

Produces clean preps

But…

Requires a large amount of

starting material

Yield can be very low

Highly labor intensive and time

consuming

−2-3 days depending on sample

type, sample amount and if

using gradient or cushion

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Exosome Isolation Reagents for Different Body Fluids

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Simple and Reliable Protocol for Exosome Isolation

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Analysis of Exosomes: NanoSight® Analysis Comparison

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Analysis of Exosomes: EM analysis of exosomes

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Analysis of Exosomes: Flow cytometry analysis of exosomes

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Analysis of Exosomes: Exosome tracing by labeling and clean-up

SYTO® RNASelect™

BODIPY® TR Ceramide

Two dyes are suggested to label exosomes

Label RNA in exosomes

Label membrane lipid on exosomes

http://www.lifetechnologies.com/order/catalog/product/S32703







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Analysis of Exosomes: Exosome tracing by labeling and clean-up

Exosome Spin Columns (MW 3000)

• Fast, efficient removal of unincorporated dye from the labeled exosomes

• Buffer exchange, desalting, removal of low molecular weight contaminants

• Replaces more time-consuming purification methods

Measured

by Qubit

Fluorometer

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Purification of Exosome RNA & Protein

Total Exosome RNA & Protein Isolation Kit

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Purification of Exosome RNA & Protein

Total Exosome RNA & Protein Isolation Kit:

Procedural Overview

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RNA Analysis:

qRT-PCR for mRNA and miRNA targets

Analysis of mRNA and miRNA by quantitative RT-PCR using

TaqMan®assays and reagents.

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RNA Analysis:

NGS for the exosomal RNA cargo

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RNA sequencing results for exosomes vs parental HeLa cells

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Protein Analysis:

Western blotting for exosomal marker protein

Bolt gel

iblot 2

Protein separation Dry transfer

ibind

Antibody detection

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Protein Analysis:

Immunopresipitation for exosomal protein

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Summary

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NanoSight ®

Light microscopy

Electron Microscopy

Flow Cytometry

Life Technologies‟s Exosome Workflow

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Life Technologies Tools for Cancer Research

Angiogenesis Apoptosis Cell cycle Cell viability Cell Metabolism

Cell proliferation Autophagy

Qdot® nanocrystals

In vivo imagingCancer cell

analysis

EVOS® Cell Imaging Systems

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Cancer

Epigenetics

Cancer genome &

transcriptome

sequencing

Cancer

genome editing

Cancer cell

RNA analysis

Life Technologies Tools for Cancer Research

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