Circulating cancer-associated extracellular vesicles as early detection and recurrence - PowerPoint Presentation

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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

Extracellular vesicles (

Exosome)Lipid (Ceramide

etc

)

microRNA (

miRNA

)

mRNADNAProtein

Membrane protein

Extracellular vesicles (exosome) as Treasure Chest of Biomarker?

Around 100 nm

Disease

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

Tumor 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

Purification 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

The 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

Healthy;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

GPRC5C

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

GPRC5C 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.

EPS8 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

GPRC5C/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)

ROC Curves between Healthy Donors and Pancreatic Cancer Patients

AUC:0.780.71

These markers indicate moderate accuracy

Age

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

GPRC5C and EPS8

Positive Exosomes were Detected in Serum of Patient with Recurrence

Photon count

(/CD63)

Photon count

(/CD63)

GPRC5C

EPS8

ConclusionWe 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.

Acknowledgement

Tokyo Medical UniversityMolecular and Cellular MedicineMaki AbeAya Shoda

Grants for

National Cancer Center EPOC

Tetsuya

Nakatsura

Yukiko

Kozaki

Nobuhiro

Tsuchiya