Insight into cancer research from discovery to validation 1
Exosomes in cancer research: from exosome isolation to biomarker discovery Zheng Songyue, PhD Technical Sales Specialists Life Technologies The world leader in serving science 2
What are Exosomes? Secreted particles first discovered bytrams et al. in 1981 Exfoliated vesicles from cell lines with ectoenzyme activity from: The Scientist, July 2011 3
What are Exosomes? Reticulocytes--- Garbage bags Pan et al., (1985) J Cell Biol 942-8 4
What are Exosomes? Publications 5
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 6
from: Vlassovet al., BiochimBiophysActa. 2012 1820 (7): 940-948. 7
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 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. The mirnas contained within exosomes from breast milk seems to modulate the development of the infant s immune system. mir-150 within exosomes enhance targeted endothelial cell migration. 8
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 EGFRvIIIregulated genes (VEGF, Bcl-xL, p27), Morphological transformation Increase in anchorage-independent growth capacity. 9
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 serum non-small cell lung cancer mir141 serum Prostate cancer mrna encoded PCA-3 and TMPRSS2:ERG Urine exosomes Prostate cancer Advantages: Body fluid collection is less painful, easier, quicker and cheaper way than traditional needle or excision biopsies 10
Current method needs for exosome researchers Current methods status for exosome researchers Needs for exosome researchers 11
Life Technologies s Exosome Workflow NanoSight Light microscopy Electron Microscopy Flow Cytometry 12
Current methods for exosome isolation 13
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 14
Exosome Isolation Reagents for Different Body Fluids 15
Simple and Reliable Protocol for Exosome Isolation 16
Analysis of Exosomes: NanoSight Analysis Comparison 17
Analysis of Exosomes: NanoSight Analysis Comparison 18
Analysis of Exosomes: NanoSight Analysis Comparison 19
Analysis of Exosomes: EM analysis of exosomes 20
Analysis of Exosomes: Flow cytometry analysis of exosomes 21
Analysis of Exosomes: Flow cytometry analysis of exosomes 22
Analysis of Exosomes: Flow cytometry analysis of exosomes 23
Analysis of Exosomes: Flow cytometry analysis of exosomes 24
Analysis of Exosomes: Exosome tracing by labeling and clean-up Two dyes are suggested to label exosomes SYTO RNASelect BODIPY TR Ceramide Label RNA in exosomes Label membrane lipid on exosomes 25
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 26
Purification of Exosome RNA & Protein Total Exosome RNA & Protein Isolation Kit 27
Purification of Exosome RNA & Protein Total Exosome RNA & Protein Isolation Kit: Procedural Overview 28
RNA Analysis: qrt-pcr for mrna and mirna targets Analysis of mrna and mirna by quantitative RT-PCR using TaqMan assays and reagents. 29
RNA Analysis: NGS for the exosomal RNA cargo 30
RNA sequencing results for exosomes vs parental HeLa cells 31
Protein Analysis: Western blotting for exosomal marker protein Protein separation Dry transfer Antibody detection Bolt gel iblot 2 ibind 32
Protein Analysis: Immunopresipitation for exosomal protein 33
Summary 34
Life Technologies s Exosome Workflow NanoSight Light microscopy Electron Microscopy Flow Cytometry 35
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Life Technologies Tools for Cancer Research Angiogenesis Apoptosis Cell cycle Cell viability Cell Metabolism Cancer cell analysis Cell proliferation Autophagy In vivo imaging Qdot nanocrystals EVOS Cell Imaging Systems 37
Life Technologies Tools for Cancer Research Cancer genome & transcriptome sequencing Cancer genome editing Cancer cell RNA analysis Cancer Epigenetics 38