Yusuke Yoshioka 1 Tetsuya Nakatsura 2 Takahiro Ochiya 1 Tokyo Medical University National Cancer Center Extracellular vesicles Exosome Lipid Ceramide etc microRNA miRNA ID: 934845
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Slide1
Circulating cancer-associated extracellular vesicles as early detection and recurrence biomarkers for pancreatic ductal adenocarcinoma
Yusuke Yoshioka
1
, Tetsuya Nakatsura
2
, Takahiro Ochiya
1
Tokyo Medical University
National Cancer Center
Slide2Extracellular vesicles (
Exosome)Lipid (Ceramide
etc
)
microRNA (
miRNA
)
mRNADNAProtein
Membrane protein
Extracellular vesicles (exosome) as Treasure Chest of Biomarker?
Around 100 nm
Slide3Disease
Markers
Body fluids
Detection methods
Ref
Colorectal
cancer
CD147
Serum
ExoScreen
(1)
Lung cancer
CD91
SerumELISA(2)Ovarian cancerCD24, EpCAMAscitesnPLEX(3)Prostate cancerDelta-cateninUrineImmunoblotting(4)Pancreatic cancerGPC-1SerumFACS(5)GlioblastomaEGFRvIII,PlasmaμNMR(6)MelanomaCD63, Caveolin-1PlasmaELISA (Exotest)(7)Ovarian cancerCD24, EpCAMPlasmaMicrofluidic device (ExoSearch) (8)Pancreatic cancerEphA2PlasmanPES(9)Lung cancera combined top-30 protein markersPlasmaELISA (EV array)(10)Prostate cancerPSMAUrineEVQuant and TR-FIA(11)
Detection of Cancer-associated Exosomal Proteins in Body Fluids
(1) Yoshioka Y,
et al
: Nat
Commun
, 2014. (2) Ueda K
et al
:
Sci
Rep, 2014. (3)
Im
H
et al
: Nat
Biotechnol
, 2014. (4) Lu Q
et al
: Prostate, 2009.
(5)
Melo
SA
et al
: Nature, 2015. (6) Shao H
, et al
: Nat Med., 2012. (7)
Logozzi
M,
et al
:
PLoS
One, 2009. (8) Zhao
Z
et al
: Lab Chip, 2016. (9) Liang K
et al
: Nat Biomed
Eng
, 2017. (10)
Jakobsen
KR,
et al
: J
Extracell
Vesicles, 2015. (11)
Hartjes
T,
et al
: ISEV 2018
Slide4Tumor Markers for Pancreatic Cancer and Five-year Survival Rate
• Pancreatic cancer is hard to find early stage.• Patients usually have no symptoms until the cancer has already spread to other organs.• CA 19-9 and CEA often cannot be detected until the cancer is already advanced.•Patients with pancreatic cancer suffer the lowest survival rate, with less than 5% surviving past 5 years in Stage IV.•Only 1.7%
of pancreatic cancer patients are initially diagnosed at
stage I
.
Survival rate
(%)
国立病院機構 大阪医療センター
HP(http://www.onh.go.jp/
seisaku/cancer/kakusyu
/suizos.html)より改変
1.7% of patients detected
Five-year survival rate of pancreatic cancer
Slide5Purification of Exosomal Proteins from Patients Sera
1: Exosome derived from serumby ultracentrifugation
2: Purified
exosomal
proteins
Silver Stain
Serum 100
ml substantial amount / lane
Serum
Pancreatic cancer
Healthy donor
Only
ultracentrifuge
is not enough purity for proteomics,,,,
Next step is proteomics analysis for purified exosomal proteins 1MkDa250150100755037252015102
Slide6The Result
of Proteomics Analysis for Exosome
Derived
from
Healthy
Donor Serum Samples
ITIH4 protein [Homo sapiens]
Chain A, The X-ray Crystal Structure Of Full-length Type Ii Human Plasminogen
filamin
-A isoform 1 [Homo sapiens]
talin
1, isoform
CRA_b [Homo sapiens]afamin precursor [Homo sapiens]lectin, galactoside-binding, soluble, 3 binding protein, isoform CRA_a [Homo sapiens]ficolin-3 isoform 1 precursor [Homo sapiens]gelsolin isoform a precursor [Homo sapiens]Radixin [Homo sapiens]ezrin [Homo sapiens]alpha-1-B glycoprotein [Homo sapiens]pregnancy-zone protein, isoform CRA_a [Homo sapiens]Chain A, Crystal Structure Of Recombinant Full Length Human Butyrylcholinesterasemyeloperoxidase, isoform CRA_c [Homo sapiens]eosinophil preperoxidase (AA -127 to 575) [Homo sapiens]Angiotensinogen (serpin peptidase inhibitor, clade A, member 8) [Homo sapiens]Kininogen 1 [Homo sapiens]YWHAZ protein, partial [Homo sapiens]keratin 1 [Homo sapiens]attractin isoform 1 preproprotein [Homo sapiens]attractin isoform 2 preproprotein [Homo sapiens]histidine-rich glycoprotein precursor [Homo sapiens]Insulin-like growth factor binding protein, acid labile subunit [Homo sapieannexin A4 [Homo sapiens]hemoglobin subunit delta [Homo sapiens]hemoglobin subunit epsilon [Homo sapiens]keratin 10 (epidermolytic hyperkeratosis; keratosis palmaris et plantaris), isoform CRA_b [Homo sapiens]heat shock cognate 71 kDa protein isoform 1 [Homo sapiens]extracellular matrix protein 1 isoform 1 precursor [Homo sapiens]extracellular matrix protein 1 isoform 3 precursor [Homo sapiens]
retinol binding protein 4, plasma, isoform CRA_b [Homo sapiens]
annexin A6 isoform 1 [Homo sapiens]
catalase [Homo sapiens]
tropomyosin alpha-4 chain isoform 2 [Homo sapiens]
tropomyosin alpha-3 chain isoform 3 [Homo sapiens]
mannan-binding lectin serine peptidase 2 [Homo sapiens]
apolipoprotein E mutant E3K
fructose-bisphosphate aldolase A isoform 1 [Homo sapiens]
fructose-bisphosphate aldolase A isoform 2 [Homo sapiens]
aldolase A [Homo sapiens]
Valosin-containing protein [Homo sapiens]
leucine-rich alpha-2-glycoprotein precursor [Homo sapiens]
apolipoprotein A-IV precursor, partial [Homo sapiens]
GDP dissociation inhibitor 2, isoform CRA_a [Homo sapiens]
thrombospondin 1 precursor variant [Homo sapiens]
type I keratin 16 [Homo sapiens]
Keratin 14 [Homo sapiens]
keratin 17, isoform CRA_a [Homo sapiens]
keratin 15 [synthetic construct]
annexin
A1, isoform
CRA_b
[Homo sapiens]
204
exosome
-associated proteins were identified
Slide7Healthy;559, Stage II;512,
Stage III;522, Stage IV;579 different proteins were identified
Identify in any stage of pancreatic cancer and not identify in healthy donor;
182proteins
Select
for
transmembrane proteins;
80 proteins
Nine proteins were selected.
Selected proteins were validated using
exosomes
from serum samples by western blotting
The Results of Proteomics Analysis for Exosome Derived from Serum of Healthy Donor and Pancreatic Cancer Patients
Slide8GPRC5C
EPS8ⅡAⅢ
ⅡA
ⅡB
Healthy Donor
Exosome
Pancreatitis
Exosome
Pancreatic Cancer
Exosome
Western Blotting of GPRC5C and EPS8
CD63
50
kDa
37 kDaPhoton count (/CD63)Photon count (/CD63)Healthy DonorPancreatitisPancreatic CancerHealthy DonorPancreatitisPancreatic Cancer
Slide9GPRC5C is・Official full name is G protein-coupled receptor, class C, group 5, member C
・The protein encoded by this gene is a member of the type 3 G protein-coupled receptor family.・GPRC5C influences cell proliferation and thereby contributes to the appropriate expansion and maintenance of functional
β-
cell mass.
・
GPRC5C5C modulates the renal contribution to systemic pH homeostasis
・
Members of this superfamily are characterized by a signature 7-transmembrane domain motif.The relationship between expression of GPRC5C and cancer progression
has been
not clarified.
Slide10EPS8 is
The expression of EPS8 is increased in pancreatic tumor. ・Official full name is epidermal growth factor receptor pathway substrate 8.
・
It functions as part of the EGFR pathway, though its exact role has not been determined.
・
Indirectly involved in cell cycle progression; its degradation following
ubiquitination
being required during G2 phase to promote cell shape changes.
Ohshima K et al., Oncol Rep, 2019
Slide11GPRC5C/CD63
EPS8/CD63
(n=14)
(n=18)
(n=31)
GPRC5C or EPS8 Positive
Exosomes
were Significantly More Abundant in Pancreatic Cancer Patient Serum than Healthy Donor
P<0.05
P<0.05
(n=14)
(n=18)
(n=31)
Slide12ROC Curves between Healthy Donors and Pancreatic Cancer Patients
AUC:0.780.71
These markers indicate moderate accuracy
Slide13Age
Sex
Stage
CA19-9 (U/mL)
EPS8
GPRC5C
Patient 1
62
F
IIB
19
15.1
10.3
Patient 271FIIA11 4.2 2.3 Patient 351MIII166 1.7 0.9 Patient 466FIIB5 18.4 11.6 Patient 565FIII22 5.6 2.1 Patient 659FIIA1 2.2 0.7 Patient 758FIIB14 4.8 6.5 Patient 856MIIB1 5.5 5.8 Patient 958MIIA11 49.7 58.5 Patient 1071FIIA112 29.3 29.4
EPS8 or GPRC5C-positive patients who measured CA19-9EPS8 or GPRC5C positive exosomes can detect pancreatic cancer that is currently difficult to diagnose and monitor!
GPRC5C or EPS8 Positive
Exosomes
were Significantly More Abundant in Pancreatic Cancer Patient Serum than Healthy Donor
Slide14GPRC5C and EPS8
Positive Exosomes were Detected in Serum of Patient with Recurrence
Photon count
(/CD63)
Photon count
(/CD63)
GPRC5C
EPS8
Slide15ConclusionWe identified new candidate markers for pancreatic cancer.GPRC5C
and EPS8 positive exosomes were detected
in
serum
of patient with recurrence.
We need simple methods for quantification of GPRC5C or EPS8 positive exosomes in patient sera.
Slide16Acknowledgement
Tokyo Medical UniversityMolecular and Cellular MedicineMaki AbeAya Shoda
Grants for
National Cancer Center EPOC
Tetsuya
Nakatsura
Yukiko
Kozaki
Nobuhiro
Tsuchiya