Download - UBC Urology Rounds

12-04-04
Exosomes and Cancer Emma (Tomlinson) Guns,
Associate Professor,
Dept. Urologic Sciences,
UBC
Content:
Ø  Cell Biology: Introduction to membrane-derived
cellular vesicles
Ø  Exosomes: Cancer - Exosomes as therapeutics
Ø  Exosomes research at VPC: Novel biomarkers or
therapeutic target ?
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• A membrane-bound
compartment inside
cell.
• 500 nm
• Sorting endocytosed
material before
transport to
lysosome.
Endosome
Thery C. et al., Nature review, 2002
Lysosome
Exocytosis
• Organelles
containing digestive
enzyme
• 0.1-1.2 µm
• Digest excess or
worn-out
organelles, food
particles, viruses
and bacteria.
Recycling / Garbage
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Exosomes
• A membrane-bound
compartment secreted
from normal and
tumor cell.
• Cup-shape
• 30-100 nm
• Elevated secretion in
malignancy effusions.
Prostate. 2009 Feb 1;69(2):159-67.
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Inhibitory effects of exosomes on
immune cells
Functions of exosome in vitro
• Tumor cell derived exosomes have immune
suppressive properties
• induce T cell apoptosis through CD95 ligand or
galectin 9
• Inhibit interleukin-2-induced T cell
proliferation
• Reduce the cytotoxic capacity of natural killer
and CD8 T cell
• Induce the generation of myeloid-derived
suppressor cells
• Impair the differentiation of myeloid
precursors into DCs
• Tumor derived exosomes seem to subvert
antitumor immune response.
Thery C. et al., Nature review, 2009
Exosomes as Therapeutics
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Functions of exosome in vitro
Activating effects of
exosomes on immune cells
• Exosomes as antigen presenting units in
immunotherapy
• Source of exogenous antigens
• Tumor cells: exosome purified from cultured
tumor cells contain tumor antigens which can
induce the activation of antigen-specific T cells
in vitro in the presence of recipient DCs:
•  Epithelial Growth Factor Receptor (EGFR)
•  Carcino-embryonic Antigen (CEA)
•  Melanoma Antigen Recognized by T-cells
(MART1)
Thery C. et al., Nature review, 2009
Exosomes as therapeutics: current research
• Using exosome in anti-tumor immunotherapy as an antigen-presenting unit
• Side effects of exosomal-based therapies? Two Phase I studies
• Mild localized reaction at the site of injection
• Mild fever
(Advanced stage melanoma and non-sc lung)
• Immunogenic properties of DC derived exosomes in vivo: anti-tumor effects
• Injection of DC-derived exosome into mice with established tumors led
to tumor rejection.
• Limitation of DC-based therapies
• Unknown behaviour of DCs after in vivo injection
• Controlling the percentage of live DCs after freeze-thaw protocol
• Limitation of exosome therapies
• Preparation of clinical grade exosome
Thery C. et al., Nature review, 2009
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Guns lab exosome inquiry:
Exosomes in Prostate Cancer
Novel Biomarkers
or
Therapeutic Targets ?
Exosomes/microvesicles released from tumor cells mediate events
essential for tumor growth and progression.
Exosome
Taylor et al 2011: http://
educationbook.aacrjournals.org
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Recycling / Garbage
Exosomes
Recycling / Garbage
Prostate Cancer Progression
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Exosome formation
Thery C. et al., Nature review, 2009
ESCRT + TSG101
Rab11 +
Calcium
Thery C. et al., Nature review, 2009
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Exosomes: Relevance to Cancer
•  Sorting of their cargo is regulated by ESCRT (Endosomal Sorting Complex Required for
Transport) and TSG101 (Tumor Suppressor Gene 101) – released in abundance by tumour
cells.
(Wieckowski and Whiteside, Immunologic Research 2006)
•  Confer changes in surrounding cells via transfer of their cargo.
(Nazarenko et al Can. Res. 2010; Al-Nedawi et al., Cell Cycle 2009)
•  Contain mRNA, miRNA, Proteins and lipids – transfer of exosomes has been shown to
confer a cell survival advantage.
(Nazarenko et al Can. Res. 2010)
•  Transfer signaling hubs, Lipid raft microdomains of cell membrane : Cav-1 mediated Akt
signalling enhancement
(Thompson et al., Prostate Cancer and Prostatic Disease 2010)
•  Guns Lab: Cell-cell communication – Discovery of CYP17 in serum exosomes.
Protein characterization - Therapeutic targeting, biomarker discovery.
(Locke et al., 2009; Hosseini-Beheshti, resubmitted 2012)
Patient
tissues
LNCaP
Locke et al., Can Res 2008.
1st of 6 publications derived from
TFPG 2006-2011
AD N CR StAR
Protein
CYB5A
CYP17A1
LC - radiometric
Analysis:
Acetate and Progesterone
conversion ex-vivo.
LCMS validation
Hydrophobic Lipids
Androgenic Steroids
34.418
AKR1C3
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CYP17A1
TISSUE – secretory profile !
CR
patient
prostate
CYP17
SERUM exosomes
CYP17A1 57 kDa
P450 oxidoreductase 77 kDa
Cytochrome b5 15 kDa
Control
Females
Control
Males
CaP
Patients
H295
Cells
Locke et al., Can Res 2008, Locke et al., Prostate 2009
Cytochrome P450 oxidoreductase,
cytochrome b5 +
CYP17A1 in SERUM exosomes
microvesicular metabolism packages ?
CYP17A1
TISSUE – secretory profile !
CR
patient
prostate
CYP17
SERUM exosomes
CYP17A1 57 kDa
P450 oxidoreductase 77 kDa
Cytochrome b5 15 kDa
Control
Females
Control
Males
CaP
Patients
H295
Cells
Locke et al., Can Res 2008, Locke et al., Prostate 2009
Cytochrome P450 oxidoreductase,
cytochrome b5 +
CYP17A1 in SERUM exosomes
microvesicular metabolism packages ?
Pharmacodynamic readout of CYP17
targeting CR with Abiraterone, TAK-700…..
or development of acquired resistance and +
++CYP17
(DeBono et al 2011)
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Recipient cell
Golgi Apparatus
Nucleus
Golgi Vesicles
Plasma Membrane
(PM) invagination
Intralumenal
Vesicles (ILVs)
MVB
fusion with
PM
CYP17 upregulation
Early Endosome
(EE)
CYP17 rich
Exosomes
Multivesicular
Bodies (MVB)
Nucleus
Abiraterone: acquired resistance +++CYP17
(DeBono et al 2011)
Cell of Origin
Recipient cell
Golgi Apparatus
Nucleus
Golgi Vesicles
Plasma Membrane
(PM) invagination
CYP17
delivery
Intralumenal
Vesicles (ILVs)
MVB
fusion with
PM
Early Endosome
(EE)
Nucleus
Multivesicular
Bodies (MVB)
CYP17 rich
Exosomes
Abiraterone: acquired resistance +++CYP17
Cell of Origin
(DeBono et al 2011)
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Guns lab exosome inquiry:
Isolation and Characterization of Exosomes
Derived from PCa Cell Lines
Observation of exosome
secretion with
immunoelectron microscopy
0 min : Antibody (Ab) binds
to the cell surface
15 min : Ab seen in empty
vesicle (early endosome)
60 min : Ab is found in
Multivesicular bodies (MVB).
180 min : Fusion of some of
MVB with plasma
membrane.
Thery C. et al., Nature review, 2002
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PC3
DU145
LNCaP
C4-2
VCaP
RWPE-1
Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different
androgen independent and androgen sensitive Prostate Cancer cell lines.
PC-3
DU-145
VCaP
LNCaP
C4-2
RWPE-1
Actin
Tubulin
HSP70
HSP90
LAMP2
Rab5
CD9
Western blot Analysis for exosome.
Exosomes have been purified based on
their unique size and density by
ultracentrifugation with 30% sucrosedeuterium. Twenty micrograms of total
protein associated with purified exosomes
were analyzed by Western blot using
different exosome markers in different
prostate cell lines Isolated exosomes from
the prostate cell lines also showed
positive expression of CD63 (data not
shown).
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2D images: PCa cell derived exosomes
Poliakov A. et al., The prostate, 2009
Exosome Cholesterol Content
Cholesterol Content Cell lysate
30 Exosome
25 20 15 10 5 0 PC-­‐3 DU145 VCaP LNCaP C4-­‐2 RWPE Cholesterol concentration of cell lysate.
Cholesterol Content 2 µg Cholesterol/µg Protein µg Cholesterol/µg Protein 35 1.8 1.6 1.4 1.2 1 0.8 0.6 *
0.4 0.2 0 PC3 DU145 VCaP LNCaP C4-­‐2 RWPE-­‐1 Cholesterol concentration of exosomes.
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Exosome Lipid Content
25
Glycerolipid Content
Cell lysate
Glycerolipid %
20
Exosome
15
10
5
0
PC3
DU145
VCaP
C4-2
LNCaP
RWPE-1
Cell lines
Glycerophospholipid %
100
90
80
70
60
50
40
30
20
10
0
Glycerophospholipid Content
PC3
DU145
VCaP
C4-2
LNCaP
RWPE-1
Cell lines
Exosome Lipid Content
Sphingolipid Content
50
Cell lysate
40
Exosome
30
20
10
0
PC3
DU145
VCaP
C4-2
LNCaP
RWPE-1
Cell lines
Glycosphingolipid Content
6
Glycosphingolipid %
Sphingolipidd %
60
5
4
3
2
1
0
PC3
DU145
VCaP
C4-2
LNCaP
RWPE-1
Cell lines
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Exosomes as diagnostic biomarker
Plasma Membrane
(PM) invagination
Intralumenal
Vesicles (ILVs)
MVB
fusion with
PM
Early Endosome
(EE)
Exosomes
Nucleus
Multivesicular
Bodies (MVB)
Cell of Origin
Guns lab exosome inquiry:
Proteomic Characterization of Exosomes
Derived from PCa Cell Lines
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Androgen insensitive PC-3 and DU145 cells
Androgen sensitive
LNCaP and VCaP cells
PC3
DU-145
VCaP
LNCaP
C4-2
RWPE-1
Normal cells (benign RWPE-1)
Exosomes
Protein Profiling
Q-TOF-MS
Functional characterization of proteins found within exosomes following proteomic analysis using Q-TOF mass
Spectrometry in association with ProteinLynx (Waters), Ingenuity characterization and MASCOT computer software programs.
4
0
2
Venn Diagram demonstrating the mutuality of proteins
in exosomes derived from prostate cell lines
* indicates the number of proteins mutual to exosomes derived from both cell lines
() indicates the number of proteins identified in exosomes of either cell line
Hosseini-Beheshti, Adomat, Tomlinson Guns;
Resubmitted to Mol. Cell Prot. 2012
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Biomarker Enrichment in Exosomes
Symbol
PC3 DU145 VCaP C4-2 LNCaP
ANXA2
+
CLSTN1
+
+
+
FASN
+
+
FLNC
+
+
FOLH1
GDF15
+
Type of Cancer(s)
Diagnosis
Cervical Cancer*,
Prostate Cancer*
+
+
Diagnosis
Colon Cancer*,
Prostate Cancer*
+
+
Diagnosis
Prostatic Carcinoma,
Non-Small Cell Lung
Cancer
Diagnosis
Prostate Cancer
Diagnosis
Prostatic Carcinoma
Diagnosis
Colorectal Cancer,
Prostate Cancer,
+
+
Biomarker
Application(s)
Exosome Function: therapeutic targets ?
• Communication between cells without direct cell-cell contact via
bearing combinations of ligands that would engage different cellsurface receptors.
• Recipient cells acquire new adhesion properties upon exosomes
binding to target cells - present new surface molecules
• Exchange cytosol and proteins between two cell type
Thery C. et al., Nature review, 2002
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Exosomes as therapeutic target
Recipient cell
Golgi Apparatus
Nucleus
Golgi Vesicles
Exosomes
Nucleus
Cell of Origin
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J Cell Sci. 2009 Apr 1;122(Pt 7):965-75. Epub 2009 Mar 3.
Source
Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University,
Chiba 260-8675, Japan.
Abstract
Src-family tyrosine kinases (SFKs), which participate in a variety of signal transduction events, are
known to localize to the cytoplasmic face of the plasma membrane through lipid modification.
Recently, we showed that Lyn, an SFK member, is exocytosed to the plasma membrane via the Golgi
region along the secretory pathway. We show here that SFK trafficking is specified by the palmitoylation
state. Yet is also a monopalmitoylated SFK and is biosynthetically transported from the Golgi pool of
caveolin to the plasma membrane. This pathway can be inhibited in the trans-Golgi network (TGN)-tocell surface delivery by temperature block at 19 degrees C or dominant-negative Rab11 GTPase. A
large fraction of Fyn, a dually palmitoylated SFK, is directly targeted to the plasma membrane
irrespective of temperature block of TGN exit. Fyn(C6S), which lacks the second palmitoylation site, is
able to traffic in the same way as Lyn and Yes. Moreover, construction of Yes(S6C) and chimeric Lyn or
Yes with the Fyn N-terminus further substantiates the importance of the dual palmitoylation site for
plasma membrane targeting. Taken together with our recent finding that Src, a nonpalmitoylated SFK,
is rapidly exchanged between the plasma membrane and late endosomes/lysosomes, these results
suggest that SFK trafficking is specified by the palmitoylation state in the SH4 domain.
Lyn ac4va4on up-­‐regulated in CRPC BPH Naive pLyn Y397 Recurrent NHT Immunoreac@vity of pLyn (arbital Unit) } 
CRPC 2.5 2.0 1.5 1.0 0.5 0.0 BPH Untreated NHT Recurrent CRPC_TURP Zoubeidi lab, VPC
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Molecular composition of exosome
• Lipid composition
• Cholesterol, sphingolipids enriched
• Phosphatidylserine surface presenting
• Lipid rafts
• Cholesterol-phospholipid ratio in lipid rafts is similar to exosome
• Presence of Cav-1, flotillin (lipid raft markers)
• mRNA and Micro RNA
• Protein composition…..
Thery C. et al., Nature review 2002; Thery C. et al., Nature review 2009; Viaud S. et al., Horm Metab Res
2008; Arienti G. et al., Archives of Biochemistry and Biophysics 1998.
Summary: Molecular composition of exosome
Cell specific
proteins: FOLH1
(prostate)
Thery C. et al., Nature review, 2009
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Guns lab exosome inquiry:
Visualization of exosome transfer
to non-identical cell types
Golgi Apparatus
Nucleus
Recipient cell
Golgi Vesicles
Exosomal Cell-Cell
communication
Plasma Membrane
(PM) invagination
Intracellular
Vesicles (ILVs)
MVB
fusion with
PM
Early Endosome
(EE)
Exosomes
Nucleus
Multi Vesicular
Bodies (MVB)
Cell of Origin
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Confocal Microscopy – Cell tracker data
2-dimensional image:
PC3
VCaP
LNCaP
C4-2
RWPE-1
LNCaP
C4-2
RWPE-1
3-dimensional image:
PC3
VCaP
Confocal Microscopy. Confocal microscopy been used to visualize purified DU145 derived exosomes –
Cell Tracker stained.10,000 PC3, VCaP, LNCaP, C4-2 and RWPE-1 cells were cultured on each chamber slide and
incubated for 12 hours with purified-stained exosomes. Confocal micrograph reveal the fusion of DU145 derived exosomes
with the plasma membrane of all cell lines.
Q.
Are exosomal proteins
transferred intracellularly between
different cell types?
Chaperone proteins in exosomes
PC-3
DU-145
VCaP LNCaP
C4-2
RWPE-1
Clusterin-P
Clusterin-S
HSP27
Western blot analysis of specific chaperones, Clusterin and Hsp27 in exosomes purified from PCa cell lines.
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Chaperone proteins in exosomes
Hsp27
2.5
Protein Concentration
2
1.5
KD Clusterin Exosome
1
KD Mismatch Exosome
0.5
0
KD Clusterin
Exosome
KD Mismatch
Exosome
Protein Concentration (µg/µl)
Protein Concentration (µg/
µl)
Clusterin
2.5
Protein Concentration
2
1.5
KD HSP27 Exosome
1
MisMatch Exosome
0.5
0
KD HSP27 Exosome MisMatch Exosome
Chaperone proteins in exosomes
siRNA Clusterin analysis
PC-3
DU-145
VCaP
LNCaP C4-2
RWPE-1
Clusterin-P
Clusterin-S
PC-3
Clusterin-P
KD Clusterin
Mismatch
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Na Li: Gleave, Zoubeidi labs
Observation of exosome
secretion with
immunoelectron microscopy
0 min : Antibody (Ab) binds
to the cell surface
CLU-GFP
15 min : Ab seen in empty
vesicle (early endosome)
60 min : Ab is found in
Multivesicular bodies (MVB).
180 min : Fusion of some of
MVB with plasma
membrane.
Thery C. et al., Nature review, 2002
Confocal Microscopy – CLU-GFP data
PC3
LNCaP
Confocal microscopy. Confocal microscopy use to visualize exosomes derived
from CLUGFP stably over-expressing LNCaP cell line, which contain CLUGFP .
Tagged exosomes appear to be taken up by PC3 (AR-ve), LNCaP (AR+ve) and
RWPE-1 (benign epithelial) prostate cell lines after incubation overnight. All three
cell lines were further fixed and stained with DAPI (nuclear stain) and E-Cadherin
(plasma membrane) prior to imaging of the cells with confocal microscopy.
RWPE-1
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SUMMARY: WORKING HYPOTHESIS
Exosomes have a pivotal role in cell-cell communication in the local
tumour microenvironment, conferring activation of numerous survival
mechanisms to surrounding cells during prostate cancer progression
and development of therapeutic resistance.
RWPE-1, (normal)
+
Q-TOF
Exosomes
Derived from : LNCaP, DU145 , VCaP, PC-3
cells
Protein profiling
ProteinLynx, MASCOT
Array
Analysis:
+
Advanced Genomics and
Bioinformatics Core:
Transcriptome
profiling
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PC-3, DU145, LNCaP, VCaP, C4-2 and RWPE-1 cells
Casodex, MDV3100, Abiraterone and Dutasteride
Chemo-stress: Taxotere
exosomes
Advanced Genomics and
Bioinformatics Core
Protein Profiling
Q-TOF
Guns lab exosome inquiry:
Validation of in vitro protein profiling work:
Further proteomic profiling of serum from xenograft bearing
mice and PCa patients
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Mice bearing human PCa Xenograft – Cx, OGX-011,
Taxotere, AR + steroidogenesis inhibitors
SERUM
exosomes
PCa patients: OGX-011 (CLU targeting), Abiraterone
and Taxotere
Advanced Genomics and
Bioinformatics Core
Sample Enrichment:
Affinity
Chromatography:
Human FOLH-1, PSMA
Protein profiling
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Q-TOF
Elham Hosseini-Beheshti
Hans Adomat
Jennifer Locke
Steven Pham
Amina Zoubeidi
Ka Mun Nip, Na Li
Susan Barr
Ladan Fazli
Martin Gleave
Kim Chi
Anthony Joshua
Buck Hales
UBC TEM facility
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Supplementary Data
Important factors in exosome secretion in
normal and cancer cell
• Normal Cells
• Ca2+ Concentration
• e.g. Blood Cell
• Cell Type
• e.g. Reticulocytes and T cell, vs. DCs and macrophages
• Cell Cycle
• Mature DCs vs. Immature DCs
•  Tumor cell
• Radiation
• 
Stage of disease
Thery C. et al., Nature review, 2009
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Exosome purification
100-200 ml of
conditioned media
Remove cell
debris
Centrifugation
(6000 rpm, 4°C, 10 min)
Pre-cleared media
(100 KDa MWCO filter capsule)
Purifying
exosome
Remove Protein
aggregate
Ultracentrifugation
(30%sucrose-deuterium oxide)
Exosome
30