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 의학박사 학위논문

Exosomal Protein Profiling for

Predicting the Prognosis of
Intraductal Papillary Mucinous
Neoplasms

췌관내유두상점액종양의 예후 예측을 위한

엑소좀 단백질 프로파일링

2024 년 2 월

서울대학교 대학원

의학과 내과학

박 남 영
Exosomal Protein Profiling for
Predicting the Prognosis of
Intraductal Papillary Mucinous
Neoplasms

지도교수 류 지 곤

이 논문을 의학박사 학위논문으로 제출함

2023 년 10 월

서울대학교 대학원
의학과 내과학
박 남 영

박남영의 박사 학위논문을 인준함

2024 년 1 월

위 원 장 김 용 태 (인)

부위원장 류 지 곤 (인)

위 원 장 진 영 (인)

위 원 김 혜 령 (인)

위 원 이 준 규 (인)
 Abstract

Intraductal papillary mucinous neoplasm (IPMN) is one of the well-known

precancerous lesion. Predicting the malignant risk of IPMN is important but

challenging. This study aimed to discover key exosomal proteins that distinguish

malignant IPMN (mIPMN) from benign IPMN (bIPMN).

Circulating exosomes in plasma from patients were purified using a multi-

dimensional technology comprised of metal-based exosome precipitation and size-

exclusion chromatography. After isolating exosomes in high purity, we performed a

three-step analysis. In the discovery session, we performed protein profiling for 20

patients (6 mIPMN and 14 bIPMN). In the verification session, we enrolled 14

patients (7 mIPMN and 7 bIPMN) and performed parallel reaction monitoring-mass

Spectrometry (PRM-MS) to verify the biomarkers for mIPMN. Survival analysis for

the Cancer Genome Atlas Program (TCGA) pancreas cancer database was performed

to select the biomarker candidates. In the validation session, 24 additional patients

(12 mIPMN and 12 bIPMN) were enrolled independently, and PRM-MS was

performed with blinded patient information. To elucidate the functional relevance of

validated proteins, lentiviral knockdown and overexpression systems were

established in mIPMN cell lines (ASAN-PaCa cell lines). We performed cell titer

glo, transwell, and three-dimensional (3D) dissemination assays for the validated

proteins to investigate their biological functions on cell proliferation, migration, and

invasion in the ASAN-PaCa cell lines.

Size-exclusion chromatographic analysis, transmission electron microscopy, and

nanoparticle tracking analysis have shown that all exosome samples isolated from

i
the plasma of patients are sufficiently pure for downstream quantitative proteomic

analysis. In the discovery session, we identified 75 potential biomarker candidates

through exosomal protein profiling. Based on the results of the PRM-MS and

survival analysis of the TCGA database, we selected PDLIM7, LYPLA1, PDIA6,

and HK1 as the biomarker candidates to be analyzed in the subsequent validation

session. In the independent validation cohort, only PDLIM7 and LYPLA1 showed

significantly different expression. The predictive performance of both candidates

was remarkable with a high value of area under the curve (PDLIM7: 0.875; LYPLA1:

0.885; and combination of both candidates: 0.946). PDLIM7 and LYPLA1 showed

excellent predictive performance even in patients with normal levels of serum

carbohydrate antigen 19-9. In an in vitro study, silencing of PDLIM7 and LYPLA1

expression significantly suppressed the cell proliferation rate and impaired the cell

invasion ability. Notably, the knockdown of PDLIM7 and LYPLA1 expression

resulted in a reduced invasive phenotype and a uniform round shape on 3D spheroids

to mimic the in vivo environment. Similarly, PDLIM7- and LYPLA1-overexpressed

ASAN-PaCa dramatically promoted cell mobility 3D cancer dissemination.

Through exosome proteomic analysis, PDLIM7 and LYPLA1 appear to be

promising biomarkers to predict the malignant potential of IPMN.

Keywords: intraductal papillary mucinous neoplasm, pancreatic cancer, biomarker,

exosome

Student Number: 2021-33889

ii
 Table of Contents

Abstract .................................................................................... i
Table of Contents ................................................................... iii

Chapter 1. Introduction ............................................................ 1

Chapter 2. Methods.................................................................. 4
Chapter 3. Results ................................................................. 14
Chapter 4. Discussion ............................................................ 22
Chapter 5. Conclusions .......................................................... 25

Acknowledgement.................................................................. 26
References ............................................................................. 27
Abstract in Korean ................................................................ 32

List of Table
Table 1................................................................................... 35

List of Figures
Figure 1 ................................................................................. 36
Figure 2 ................................................................................. 37
Figure 3 ................................................................................. 38
Figure 4 ................................................................................. 39
Figure 5 ................................................................................. 40
Figure 6 ................................................................................. 41
Figure 7 ................................................................................. 42
Figure 8 ................................................................................. 43
Figure 9 ................................................................................. 44
iii
 Chapter 1. Introduction

Intraductal papillary mucinous neoplasm (IPMN) is defined as an intraductal

mucin-hypersecreting neoplasm that involves the main or branch pancreatic ducts.1

IPMNs comprise a large proportion of pancreatic cystic neoplasms.2 Although the

incidence of IPMN is reported to be up to 10%, it could have been underestimated

because most of the IPMNs are asymptomatic and found incidentally.3 With

improvement and expanded use of imaging technologies, the incidence of IPMN is

increasing.3-6

IPMNs have a spectrum of neoplastic transformation ranging from low-grade

dysplasia to invasive carcinoma.7,8 Surgical resection is the only curative method for

this precancerous lesion; however, surgery is not recommended for all patients,

considering the low possibility of malignancy and the potential of postoperative

morbidity and mortality.6 The current guidelines rely largely on imaging findings to

evaluate the potential of malignancy, but the levels of evidence are low and

sometimes require bothersome and expensive diagnostic tools.9 Previous studies

reported that the radiographic criteria-based strategies showed limited benefit in

identifying patients with high-grade dysplasia or invasive cancer.10 Although cystic

fluid analysis can provide useful diagnostic information, cystic fluid sampling

requires endoscopic ultrasound procedures which are invasive and not practical for

repeated tests in a follow-up setting.

Biomarkers from the blood-based analysis are of great medical interest, especially

when following up a large number of patients. Carbohydrate antigen 19-9 (CA 19-9)

1
is one of the most commonly used and intensively evaluated biomarkers. It is the

only biomarker for pancreatic adenocarcinoma approved by the United States Food

and Drug Administration and included in many international guidelines for patients

with IPMN. However, its diagnostic efficacy for identifying high-grade dysplasia or

invasive carcinoma is unsatisfactory with a sensitivity of 14-81% and specificity of

76-96%.11,12 Carcinoembryonic antigen (CEA) is another well-known biomarker, but

the diagnostic accuracy of serum CEA level is also insufficient.12,13

Exosomes have been actively investigated to discover novel biomarkers for

malignancy such as in pancreatic cancer. Exosomes are small extracellular vesicles

(EVs) that are secreted by all cell types.14-16 The lipid bilayer of the exosome acts as

a protective shield, maintaining the stability of various intracellular cargos, including

DNA, microRNA, and proteins.17 These intracellular cargos play an important role

in local and systemic cell-to-cell communication.18 The cancer-associated exosome

is derived from cancer cells as well as host cells that react to the change by cancer

progression.18-20 In addition, cancer-associated exosomes can promote cell motility,

extracellular matrix remodeling, increased vascular permeability, and enhanced drug

resistance.21 Moreover, exosomes from pancreatic cancer cells can migrate to distant

tissues and may create the pre-metastatic niche.22 Exosomes are secreted at much

higher levels by tumor cells than normal cells and can be easily identified in the

blood sample.18,23 Recently, several studies have reported the usefulness of exosome

analysis in detecting cancers, including pancreatic cancer.17,24,25

Although there has been a lack of effective protein biomarkers that can assess the

malignant risk of IPMN, it could be possible to discover new biomarkers by

analyzing the proteins that are stably enriched in exosomes. A recent study showed

2
the possibility of identifying new biomarkers for IPMN risk assessment with

exosomal protein analysis.26 Therefore, we analyzed exosomal proteins from patients

with malignant IPMN (mIPMN) and benign IPMN (bIPMN) to discover novel

biomarkers that can assess the malignant risk of IPMN.

3
 Chapter 2. Methods

2.1. Definitions and Study Design

Patients with mIPMN or bIPMN were prospectively enrolled from February

2019 to February 2022 and followed up until February 2023. We defined mIPMN

as pathologically proven cancer or high-grade dysplasia. If a lesion could not be

diagnosed by biopsy but malignancy was strongly suspected in a clinicoradiologic

setting, it was also defined as mIPMN. We defined bIPMN as IPMN without high-

risk stigmata or worrisome features on imaging for at least two years of follow-up.

Patients were enrolled in three independent cohorts: discovery, verification, and

validation. In the discovery session, we performed tandem-mass-tag (TMT)-based

exosomal protein profiling analysis to identify differentially expressed proteins

(DEPs) between patients with mIPMN and those with bIPMN. In the verification

session, targeted parallel reaction monitoring-mass spectrometry (PRM-MS)

analysis was performed based on the DEP profile identified in the discovery

session. In the validation session, the diagnostic efficacies of biomarker candidates

identified during the discovery and verification sessions were analyzed in an

independent cohort without revealing their clinical information. The design of this

study is summarized in Figure 1.

We planned to analyze seven patients with mIPMN and 14 with bIPMN for the

discovery cohort, and seven with mIPMN and seven with bIPMN for the

verification cohort. We calculated the sample size of the validation cohort based

on the area under the curve (AUC) values of CA 19-9 and biomarker candidates
4
 found in the verification session, and then enrolled 12 patients with mIPMN and

12 patients with bIPMN. Among the 12 patients with mIPMN in the validation

cohort, eight patients were enrolled at the biobank of Seoul National University

Bundang Hospital. All participants provided their written informed consent before

participating in this study. This study was approved by the Institutional Review

Board of Seoul National University Hospital, Seoul, Korea (1901-147-1007).

2.2. Sample Preparation and Exosome Isolation

By simple venipuncture, blood samples were obtained from patients with

mIPMN and bIPMN following the written informed consent in accordance with

the Helsinki Declaration. To collect 10 mL of venous blood, Vacutainer® Plus

whole blood tubes with K2 EDTA (BD, Franklin Lakes, NJ, U.S.) were used. The

plasma layer was separated from the whole blood sample by centrifugation at 2000

g for 15 min at room temperature, and was carefully retrieved into a Cryogenic

Vial (Corning, Corning, NY, U.S.) using a micropipette without disturbing the

buffy coat. The supernatant was then frozen quickly with liquid nitrogen, and

stored at -80 ℃ or lower.

Exosome isolation was performed using ExoLutE® exosome isolation kits (SL

BIGEN Inc., Incheon, Republic of Korea) according to the manufacturer’s

instructions, as follows. After thawing the frozen plasma samples at room

temperature, a 400 L aliquot of each plasma sample was mixed with 6.8 mL of

RPMI1640 media (Thermo Fisher Scientific, Waltham, MA, U.S.) and 6.05 mL of

proprietary exosome precipitating agent including transition metal ions and

5
 polymer. The precipitated exosomes were recovered at 3000 g for 20 min at room

temperature and then resuspended in a buffer containing chelator. Subsequently,

the crude exosomes were loaded onto a spin-based size exclusion column pre-

equilibrated with 0.22-µm filtered phosphate-buffered saline (PBS), followed by

centrifugation at 300 g for 5 min to purify the exosomes from the contaminating

molecules. The purity of isolated exosome samples was checked with size-

exclusion chromatography (SEC), transmission electron microscope (TEM), and

nanoparticle tracking analysis.

2.3. Protein Profiling for Exosomal Proteins

For exosomal protein profiling, peptides were re-suspended in solvent A and

analyzed using the Q Exactive orbitrap hybrid mass spectrometer (Thermo Fisher

Scientific) coupled with the EASY-nLC 1000 system (Thermo Scientific, Bremen,

Germany). Solvents A and B were 0.1% formic acid in water and 0.1% formic acid

in acetonitrile, respectively. A 180-min gradient (from 5% to 20% solvent B over

135 min, 20% to 35% solvent B over 15 min, 80% solvent B for 10 min, and 5%

solvent B for 20 min) was used. The peptides were loaded onto a trap column (75

μm × 2 cm, 3 μm, C18, 100 Å) and ionized through an EASY-spray column

(50 cm × 75 μm ID) packed with 2-μm C18 particles at an electric potential of

2.0 kV. Full MS scans were acquired at a scan range of 350–1,800 Th and a

resolution of 70,000 at m/z 200. The 12 most abundant ions were fragmented by

data-dependent MS/MS experiments with an isolation window of 1.6 Th and

dynamic exclusion of 30 s and at a normalized collision energy of 30 for higher

energy collisional dissociation. The charge state of 1 was discarded. Maximum ion

6
 injection times were 100 ms for both full MS and MS/MS scans. The automated

gain control target value was set to 1.0 × 106 for both MS and MS/MS scans.

2.4. Protein Identification and Bioinformatic Analysis

For data-dependent analysis, the MS raw files were searched with the Proteome

Discoverer v. 2.1 (Thermo Fisher Scientific) against the Uniprot human database

(released in 2017_10, 20,208). In modification, carbamidomethylation, alkylation

of cysteine (+57.021 Da), TMT 10-plex modification of lysine (+229.163 Da)

were noted as static modifications, and the oxidation of methionine (+15.995 Da)

and N-termination (+229.163 Da) was noted as a variable modification. The false

discovery rate for peptide level was evaluated to 0.01 for removing false positive

data as much as possible. An initial precursor mass deviation up to 10 ppm and a

fragment mass deviation up to 0.02 Da were allowed. To quantify each sample’s

reporter ion, a ‘reporter ion quantifier’ with TMT 10-plex was used. For highly

confident quantifications of protein, the protein ratios were calculated from two or

more unique quantitative peptides in each replicate. All reporter ion intensities

were transformed to log2 values. Proteins that did not display all values in at least

one group were filtered out. A gene ontology (GO) search was performed to

explore the biological processes using gProfiler freeware. GO biological processes

enriched were identified as those with a p-value < 0.05. We selected proteins

involved in enriched biological processes to construct a network depicting the

enriched processes.

7
2.5. Parallel Reaction Monitoring (PRM) Verification

Sample preparation for PRM verification including depletion, tryptic digestion,

and peptide cleanup was carried out similar to the previous profiling sample

preparation. A total of 14 samples from the verification cohort and 24 samples

from the validation cohort were denatured with 8 M urea in 50 mM ABC buffer

(pH 8.0). Protein was reduced with 20 mM DTT for 1 h at 37 °C and alkylated

using 50 mM IAA at room temperature for 30 min in the dark. Trypsin was added

at a 50:1 (w/w) protein-to-trypsin ratio and incubated at 37 ˚C overnight. For

normalization, a retention time kit (iRT kit, Biognosys, Switzerland) was used to

spike samples according to manufacturer’s instructions. 27 The activated trypsin

reaction was quenched with 0.5% trifluoroacetic acid, and peptides were desalted

with a C18 spin column. The eluates were dried and re-suspended in 0.1% formic

acid before liquid chromatography-PRM analysis.

Proteins with adjusted p-values lower than 0.05 in profiling analysis were

classified to 75 target protein candidates for mIPMN. PRM-Transitions of target

proteins were selected with the following criteria: fully tryptic peptides with no

missed cleavages, unique peptide, a 2+ charge-state for precursor ion, and a

peptide length of 6-20 amino acids. The N-terminal signal peptides and peptides

that contained methionine or histidine were excluded. Targeted peptides were

compiled, including 63 unique peptides identified by the individual patient in at

least one of the DEPs in data-dependent acquisition analysis. PRM analyses were

performed on a Q-Exactive HF-X equipped with Dionex Ultimate 3000 RSLC

chromatography system (Thermo Fisher Scientific). The mobile phase A consisted

of 0.1% formic acid in water, the phase B consisted of 0.1% formic acid in
8
 acetonitrile, and the loading phase consisted of 0.1% formic acid in water. The MS

cycle started with a full MS1 scan performed at a resolving power of 45,000 (at

200 m/z), followed by time-scheduled targeted PRM scans acquired at a resolving

power of 15,000 (at 200 m/z) with a normalized collision energy of 28. The

quadrupole isolation window for the PRM events was set to 1.2 Da and the

duration of the time scheduled windows for each pair of endogenous and

isotopically labeled peptides was set to 2 min.

2.6. Cells and Cell Culture

BxPC-3, AsPC-1, HEK293T, and ASAN-PaCa cell lines were obtained from the

American Type Culture Collection (ATCC; Manassas, VA, U.S.). Capan-1, Capan-

2, Panc-1, and MIA PaCa-2 cells were purchased from the Korea Cell Line Bank

(KCLB, Republic of Korea). The BxPC-3, Capan-1, and Capan-2 cells were

cultured in RPMI (Gibco, Carlsbad, CA, U.S.) containing 10% fetal bovine serum

(FBS; Invitrogen, Carlsbad, CA, U.S.) and 1% penicillin/streptomycin (PS;

Gibco). AsPC-1, HEK293T, Panc-1, and ASAN-PaCa cell lines were cultured in

DMEM (Gibco) containing 10% FBS and 1% PS. Cells were maintained at 37 °C

in a humidified atmosphere of 95% air and 5% CO2 and periodically screened for

Mycoplasma contamination.

9
2.7. Stable Transduction of Cells with shRNA or Plasmids

Lentiviral vectors encoding human shPDLIM7 (2 different shRNAs; #1, 5’-

CATCTTCTCATAGTCTCGC-3’; #2, 5’-TGGAGTAGAAGGTCTTTCC-3’),

shLYPLA1 (2 different shRNAs; #1, 5’-TTGATATGTGAACTTCTGA-3’; #2,

5’-AATTCTGTTAGAAGGAATG-3’), and the non-silenced vector (shControl)

were purchased from Thermo Scientific Open Biosystems Human GIPZ Lentiviral

shRNAmir library (Thermo Scientific, Lafayette, CO, U.S.). Generation of the

lentiviruses was co-transfected with pPAX2 and pMD2.G (Addgene, Watertown,

MA, U.S.) into HEK293T cells (ATCC) using Lipofectamine 2000 (Life

Technologies, Waltham, MA, U.S.). Supernatants were collected at 24 h and 48 h

and filtered in 0.45-μm pore syringes. ASAN-PaCa cell lines were infected with

the viral supernatant with 8 μg/ml polybrene, and stable cell lines were selected

with puromycin (range of 2-4 μg/ml).

2.8. Cell Proliferation Assay

Cells were plated in triplicate (3,000 cells/well) and incubated in a medium

containing 10% FBS. Cell growth assessment was performed daily from day 0 to

day 3. Cell proliferation was assessed by measuring intracellular levels of ATP using

the Cell Titer-Glo luminescent cell viability assay kit (Promega, Madison, WI, U.S.).

Luminescence was measured using a luminometer (Glomax® Explore Multimode

Microplate Reader; Promega).

10
2.9. Migration and Invasion Assay

Transwell migration assays were performed in a chamber system, and 1-3 × 105

cells suspended in 100 µL DMEM without serum were added to the upper chamber

of a 6.5 mm (0.8 μm pore size) 24-well transwell (Corning). DMEM with 5% FBS

was placed in the bottom wells. Invasion assays were performed with the same

chamber system using Matrigel (Thermo Fisher Scientific) coating. After 22 h at

37 °C and 5% CO2, the upper chamber was fixed with 4% paraformaldehyde for

10 min and stained with 1% crystal violet. Stained cells per field were calculated

in triplicate using a Nikon (Nikon, Tokyo, Japan) microscope at a magnification

of 100x.

2.10. Three-dimensional (3D) Culture and Immuno-

fluorescence Staining

Stable knockdown and overexpressing cells were seeded on solidified growth

factor-reduced Matrigel (50 μL per well of an 8-well chamber slide) and then

covered with 200 μL of 2% Matrigel-containing medium. When cells were

cultured in 3D for 6 days, they exhibited an invasive growth phenotype

(approximately 4–5 days), and phase contrast images of randomly selected fields

were captured with a Nikon inverted microscope using Nikon Elements software.

Confocal images were captured on a Leica confocal microscope using Leica

software (Leica Microsystems, Wetzlar, Germany).

11
2.11. Western Blotting

Cells were collected and homogenized on ice in RIPA lysis buffer (Thermo

Fisher Scientific). Subsequently, the cell lysates were centrifuged at 4 °C to

separate the proteins. Proteins were quantified using a Bicinchoninic Acid Protein

Assay kit (Thermo Fisher Scientific). Western blotting analysis used anti-LYPLA1

(Proteintech, Wuhan, China) and -PDLIM7 (Proteintech). β-actin (BD Biosciences,

Franklin Lakes, NJ, U.S.) antibodies were used as a loading control.

2.12. Statistical Analysis

Continuous variables are presented as medians with interquartile ranges, and

categorical variables are presented as numbers with percentages. Mann-Whitney

test was performed to assess the differences in exosomal DEP levels between the

two groups. The diagnostic performance of each biomarker candidate and CA 19-

9 was investigated with the receiver operating characteristic (ROC) curve. The

AUC value was calculated according to the method described by DeLong and his

coworkers.28 Confidence intervals (CI) were obtained using the Wilson/Brown

method, and the cutoff values were computed based on Youden’s index. Survival

analysis was performed using the Kaplan-Meier method, and the hazard ratio was

computed with a Mantel-Haenszel method.

The sample size of the validation cohort was calculated based on the AUC

values of biomarker candidates and CA 19-9. PDZ and LIM domain protein 7

(PDLIM7) and Lysophospholipase 1 (LYPLA1) showed higher AUC values

(PDLIM7: 0.96, LYPLA1: 0.95) than CA 19-9 (0.73). A previous meta-analysis

12
reported an AUC value of 0.74 for CA 19-9.12 Based on these results, we

hypothesized that the two exosomal DEPs had an AUC value of 0.95 and

CA 19-9 had an AUC value of 0.74 and calculated the sample size to demonstrate

that the difference would be statistically significant. The statistical analysis was

performed using the R s/w environment (version 4.3.1; The R Foundation for

Statistical Computing, Vienna, Austria) and the Power Analysis and Sample Size

software version 15.0.12. A p-value less than 0.05 was considered statistically

significant.

Statistical analysis of the results of in vitro experiments was performed using

the GraphPad Prism 9.0 program (GraphPad Software Inc., San Diego, CA, U.S.).

Unless otherwise indicated, data are expressed as means ± SE (standard error) of

three independent experiments. Student’s t-test was used to determine the

significance of the results.

13
 Chapter 3. Results

3.1. Baseline Clinical and Pathological Characteristics

A total of 59 participants were enrolled in this study. In the discovery session,

we enrolled seven patients with mIPMN and 14 with bIPMN. However, one of the

mIPMN samples was found to be hemolyzed and unsuitable for further analysis,

and we analyzed six mIPMN and 14 bIPMN samples in the discovery session. The

verification cohort consisted of seven patients with mIPMN and seven with

bIPMN. In the validation session, 12 patients with mIPMN and 12 with bIPMNs

were enrolled. Among 25 patients with mIPMN, 16 had high-risk stigmata and six

showed worrisome features, according to International Consensus Guideline

2017.29 All patients in the mIPMN group were diagnosed with adenocarcinoma or

high-grade dysplasia based on pathologic specimens, except for one patient who

was diagnosed based on clinicoradiologic findings. Because there was only one

patient diagnosed with high-grade dysplasia and the patient’s specimen was

hemolyzed, all patients finally analyzed were confirmed to have invasive

carcinoma. One patient with bIPMN showed elevated CA 19-9, one of the

worrisome features, due to common bile duct sludge at the time of blood sampling,

whereas other patients in the bIPMN group had no high-risk stigmata or worrisome

features. The baseline characteristics of patients in each cohort are summarized in

Table 1.

14
3.2. Purity of Isolated Exosome Samples

The result of SEC-based purity analysis showed that the area of the single peak

corresponding to exosomes in all samples was more than 85%. In addition, particle

analysis using TEM and nanoparticle tracking analyzer exhibited that the

substances isolated from patient plasma were round-shaped nanoparticles ranging

from 30 nm to 300 nm in diameter. These indicate that the substances in all

samples were nanoparticles including exosomes and sufficiently pure for

downstream quantitative proteomic analysis. Figure 2 shows the representative

results of TEM analysis and the size distribution profiles of exosomes isolated

from patient plasma.

3.3. EV Protein Profiling in the Discovery Cohort

We isolated EVs from the plasma of patients with mIPMN and those with

bIPMN. Employing a TMT-based analysis, we acquired quantitative data from

EVs isolated from individual patients, detailing the abundance of each protein. EV

protein profiling was performed using the traditional protein profiling method as

depicted in Figure 1. We identified a total of 1,390 EV proteins, more than 75% of

which were consistent with the ExoCarta EV protein database (Figure 3A). When

compared to the typical profiling method for plasma proteins, analyzing after

isolating the EVs identified approximately 3.6 times more proteins. Plasma is

dominated by a few abundant proteins such as serum albumin, immunoglobulin,

and transferrin, accounting for over 90%, which reduces the overall protein

complexity and hinders the detection of proteins present in small quantities. The

15
EV isolation technique using ExoLutE® (SL BIGEN Inc.) is highly effective in

separating EVs from plasma to reduce abundant proteins such as serum albumin.

This method substantially increases the proportion of low-abundance proteins

from below 10% to well beyond 50%, underscoring its efficacy in mitigating

potential contamination from prevalent proteins. Among the proteins identified

from plasma, known EV markers such as CD9, CD81, and PDCD6IP (ALIX) were

also detected, indicating that the EVs were properly separated from the plasma

(Figure 3B).

To identify EV proteins that significantly differ in mIPMN compared with

bIPMN, a volcano plot analysis was performed, and we selected proteins with a p-

value < 0.05 as DEPs (Figure 3C). In mIPMN-EV, a total of 36 proteins

significantly increased and 39 proteins significantly decreased. Through GO

analysis, we verified the cellular localization of proteins that significantly changed

in mIPMN-EV. The cellular components of both increased and decreased proteins

were primarily extracellular exosomes, and the increased proteins were

specifically distributed in the lysosome, focal adhesion, and actin cytoskeleton.

On the other hand, the decreased proteins were distributed in the endoplasmic

reticulum lumen and integrin complex. Instead of clarifying the role and function

of the proteins included in EV, we highlighted the top five proteins that

demonstrated the most significant changes in mIPMN-EV to develop the proteins

as biomarkers. As a result, tripeptidyl-peptidase 2 (TPP2), microtubule-associated

protein RP/EB family member 1 (MAPRE1), macrophage-stimulating protein

(MST1), PDLIM7, and LYPLA1 were the most significantly increased, while

ceruloplasmin (CP), transforming growth factor beta receptor type 3 (TGFBR3),

16
 insulin-like growth factor II (IGF2), hyaluronan-binding protein 2 (HABP2), and

insulin-like growth factor-binding protein 3 (IGFBP3) were the most significantly

decreased (Figure 4).

3.4. Targeted Analysis in the Verification Cohort

To identify promising biomarkers that can distinguish the mIPMN from bIPMN,

we collected the verification cohort, distinct from the discovery cohort. Due to the

technical challenges associated with PRM-MS in directly assessing protein

biomarkers, we chose to employ peptides derived from the target proteins as

representative markers. From the DEPs identified in the profiling discovery

session, we specifically selected peptides suitable for quantitative analysis in

individual EV proteomic studies for verification purposes. The selection process

included applying exclusion criteria such as peptide length (< 6 or > 24), m/z

values (< 350 or > 1,400), and the presence of miss-cleavage, as well as peptides

containing methionine or histidine.

Subsequently, the 65 peptides were subjected to verification in the experimental

session, leading to the selection of potential biomarker candidates for mIPMN. In

the verification set, we simultaneously analyzed alterations in target proteins

within both plasma proteins and EVs to identify distinctions between these two

sample types. We employed a targeted analysis using PRM-MS, optimized for

minimal protein amounts. Out of the examined proteins, 12 exhibited a noteworthy

increase in mIPMN-EVs compared to bIPMN-EVs. Notably, PDLIM7, LYPLA1,

PDIA6 (Protein disulfide-isomerase A6), and CD5L (CD5 antigen-like)

17
 demonstrated the most significant differences, with p < 0.01, while the remaining

eight proteins showed significance with p < 0.05 (Figure 5). Remarkably, among

the 12 proteins with significant differences in mIPMN-EVs, only four could be

quantitatively analyzed in plasma, and the expression levels for the remaining

eight proteins could not be obtained. Furthermore, the four quantifiable plasma

proteins exhibited no significant differences between mIPMN and bIPMN,

displaying a distinct trend compared to EVs.

3.5. Biomarker Candidates Selection for Validation

We hypothesized that proteins reflecting the characteristics of pancreatic cancer

would be more closely related to the malignant transformation of IPMN and

selected candidates whose high expression was associated with poor survival in

patients with pancreatic cancer. The association between protein expression

changes and the actual survival rate of patients with pancreatic cancer was

analyzed through the cancer genome atlas (TCGA) database. Based on comparing

mRNA expression and the overall survival rate of patients with TCGA pancreatic

cancer, we identified genes that decrease survival rate when their expression

increases (Figure 6). As a result, PDLIM7, LYPLA1, PDIA6, and HK1

(Hexokinase-1) showed hazard ratios of 1.52 (CI: 0.96-2.42, p = 0.072), 2.12 (CI:

1.32-3.09, p = 0.001), 1.67 (CI: 1.09-2.57, p = 0.019), and 1.76 (CI: 1.13-2.73, p

= 0.012), respectively.

Additionally, the four DEPs showed good prognostic efficacy in the verification

cohort. The ROC curve analysis for the four candidates demonstrated high AUC

18
 values: PDLIM7 (0.9592), LYPLA1 (0.9492), PDIA6 (0.9388), and HK1 (0.8878)

(Figure 6B, 6C). With a cutoff value of 37, CA 19-9 showed low sensitivity (0.571),

high specificity (1.000), and lower AUC value (0.7347).

3.6. Validating the Prognostic Efficacy of Candidates in the

Independent Validation Cohort

In the independent validation cohort, PRM-MS was performed to validate the

prognostic efficacy of the four DEPs selected in the verification session. Among

the four DEPs, PDLIM7 and LYPLA1 showed significantly different protein

expressions. The ROC curve analysis in the independent validation cohort

demonstrated the remarkable prognostic efficacy of PDLIM7 and LYPLA1. The

AUC values of PDLIM7 and LYPLA1 were 0.875 and 0.885, respectively. The

combination of both candidates showed improved predictive power with an AUC

value of 0.944. However, the AUC value of CA 19-9 was unusually high (0.976),

and the difference between the AUC values of each candidate and CA 19-9 was

not statistically significant (PDLIM7 vs. CA 19-9: p = 0.1466, LYPLA1 vs. CA

19-9: p = 0.1791, and the combination of PDLIM7 and LYPLA1 vs. CA 19-9: p =

0.473). We did not perform ROC curve analysis for PDIA6 and HK1 because their

expression levels in the validation cohort were not significantly different. Figure

7 demonstrates the expression levels of PDLIM7 and LYPLA1 in the validation

cohort (Figure 7A) and the results of ROC curve analysis of PDLIM7, LYPLA1,

and CA 19-9 (Figure 7B).

19
3.7. Excellent Prognostic Efficacy of PDLIM7 and LYPLA1 in

Patients with Normal CA 19-9 Levels

In patients with CA 19-9 levels of 37 or lower, PDLIM7 and LYPLA1 showed

excellent diagnostic performance. Considering the unusually high AUC value of

CA 19-9 in the validation cohort, we analyzed the diagnostic power of PDLIM7

and LYPLA1 in patients with normal CA 19-9 levels. PDLIM7 showed moderate

sensitivity (0.75) and specificity (0.75) with an AUC value of 0.823. LYPLA1

showed good sensitivity (1.00) and moderate specificity (0.75) with an AUC value

of 0.896. The combination of both candidates showed better sensitivity (1.00) and

specificity (0.833) with an AUC value of 0.917. Figure 8 shows the results of ROC

curve analysis for patients with normal levels of CA 19-9.

3.8. PDLIM7 and LYPLA1 Promote Cell Migration and

Invasion in mIPMN Cells

Based on the clinical significance of validated proteins in mIPMN, we first

examined the expression levels of PDLIM7 and LYPLA1 across various

pancreatic cancer cell lines, including the mIPMN cell line. Although there was

no significant difference in the LYPLA1 expression, the expression of the

PDLIM7 protein was markedly higher in ASAN-PaCa mIPMN cells compared to

other pancreatic cancer cell lines (Figure 9A).

To elucidate the functional roles of PDLIM7 and LYPLA1 in mIPMN cell lines,

we established lentiviral knockdown and overexpression cell lines for each of

20
these proteins in ASAN-PaCa cells, respectively (Figure 9B). Silencing of

PDLIM7 and LYPLA1 each significantly suppressed the cell proliferation rate

(Figure 9C). No significant differences were observed in the overexpression cell

line model (Figure 9C). Furthermore, the knockdown of PDLIM7 in ASAN-PaCa

cells significantly impaired cell migration and invasion capabilities, whereas its

overexpression dramatically enhanced these abilities (Figure 9D). While

suppression of LYPLA1 expression had no notable impact on cell migration, it did

lead to a significant decrease in invasion ability (Figure 9D). Conversely, LYPLA1

overexpression resulted in a dramatic increase in cell invasion (Figure 9D).

To further investigate the aggressive phenotype of PDLIM7 and LYPLA1, we

assessed cell dissemination using 3D cell cultures. We found that knockdown of

the expression of PDLIM7 and LYPLA1 led to a diminished invasive phenotype

and resulted in spheroids with a uniform round shape (Figure 9E). In contrast,

overexpression of PDLIM7 and LYPLA1 in ASAN-PaCa cells markedly increased

the cell mobility and 3D cancer dissemination (Figure 9E). These results imply

that PDLIM7 and LYPLA1 possess enhanced dissemination in 3D spheroids,

which more accurately mimic the in vivo environment with characteristics such as

structural organization, nutrient gradients, and hypoxia, compared to monolayer

mIPMN cells.

21
 Chapter 4. Discussion

This study demonstrated the potential of the proteomic analysis of exosome-

derived proteins to assess the malignant risk of IPMN. By analyzing highly

concentrated proteins in exosomes, we discovered biomarker candidates whose

differences were difficult to detect in serum analysis. We found the two novel

biomarker candidates, PDLIM7 and LYPLA1, through comprehensive proteomic

analysis, and their diagnostic power was validated in an independent cohort. In our

in vitro study, we showed that both biomarker candidates had a strong association

with cancer cell proliferation and invasion in mIPMN cell lines.

PDLIM7 and LYPLA1 showed better predictive performance than the sensitivity

and specificity of CA 19-9 reported in previous studies. In particular, the

combination of the two biomarker candidates showed an improved AUC value.

Although this study could not show a difference in the AUC value in the validation

cohort, the predictive power of PDLIM7 and LYPLA1 was considered excellent

considering the extraordinary AUC value of CA 19-9 in the validation cohort.

Because the bIPMN cohort included only patients without worrisome features, the

AUC value of CA 19-9 could be overestimated than the actual prognostic

performance. Additionally, in patients with a CA 19-9 level of 37 or lower, the

combination of the two proteins showed an excellent ability to assess the malignant

risk of IPMN.

PDLIM7 is a protein that contains one amino-terminal PDZ domain and three

carboxy-terminal LIM domains.30 The PDZ domain binds actin filaments, and the

22
LIM domain is known to be involved in mitogenic signaling and insulin-induced

actin cytoskeleton remodeling.31,32 The LIM domain inhibits the autoubiquitination

of murine double minute 2 (MDM2), leading to stabilization of MDM2 and

subsequent degradation of the tumor suppressor p53.33 In pancreatic cancer,

stabilizing MDM2 and subsequent p53 degradation have been reported to increase

tumor cell invasiveness and metastatic activity.34

LYPLA1 is a protein demonstrating both depalmitoylation and lysophospholipase

activities. It is significantly overexpressed in chronic lymphocytic leukemia (CLL)

cells.35,36 Depalmitoylation by LYPLA1 causes post-translational modifications in

CLL cells, affecting localization, mobility, survival signaling, and migration. 35 In

melanoma, the cancer metastasis can be promoted by increased depalmitoylation

activity by phosphorylation of LYPLA1.37 Conversely, inhibition of LYPLA1

reduces cell proliferation, migration, and invasion in non-small cell lung cancer cell

lines.36

In our study, on screening the expression levels of the two biomarkers in various

cell lines, PDLIM7 expression was more elevated in the mIPMN cell line than in

other pancreatic cancer cell lines. This suggests that PDLIM7 could be a

distinguishing marker for mIPMN from other types of pancreatic cancer. To our

knowledge, this study provides the first evidence that PDLIM7 and LYPLA1 play a

crucial role in significantly promoting 3D cell motility and dissemination in mIPMN.

The biomarkers identified in this study are useful because they can be obtained

from blood samples. Repeated radiographic and endosonographic examinations are

burdensome to patients considering long-term radiation exposure, invasive testing,

and financial burden. Additionally, cystic fluid analysis requires invasive procedures
23
to collect the samples and is generally difficult to perform repeatedly. PDLIM7 and

LYPLA1 can be analyzed from blood samples that can be collected in relatively

simple manner; thus, they are expected to be useful for patients with IPMN who

require long-term follow-up.

This study had several limitations. First, because the number of patients meeting

the inclusion criteria was small, a small number of patients with mIPMN were

analyzed. Second, whether the isolated exosomes are derived from cancer cells is

unclear. However, exosomal DEPs from mIPMN and bIPMN patients are thought to

reflect the activity of not only cancer cells, but also cell-to-cell interactions in the

tumor microenvironment and the response of the pre-metastatic niche. Third,

whether PDLIM7 and LYPLA1 are involved in carcinogenesis in patients with

IPMN is unknown. However, in vitro functional analysis has confirmed that

PDLIM7 and LYPLA1 are strongly associated with cell mobility and 3D cancer

dissemination in independent mIPMN cell lines. While these findings suggest an

association of both biomarkers with carcinogenesis, further investigation is

warranted.

24
 Chapter 5. Conclusions

Proteomic analysis of proteins within exosomes may be useful in predicting the

malignant risk of IPMN. We discovered two novel biomarker candidates, PDLIM7

and LYPLA1, and their diagnostic efficacy was validated in an independent cohort.

Knockdown of PDLIM7 or LYPLA1 was also associated with reduced tumor

proliferation and invasion. Further studies with a larger cohort in multiple centers

are required to evaluate the diagnostic efficacy of both candidates.

25
 Acknowledgment

We thank the participating patients and their families; all other researchers and

research center members; Hyeong Min Lee for protein profiling and bioinformatic

analysis; Kyung Min Lee for in vitro functional analysis; and Seoul National

University Hospital Medical Research Collaboration Center for statistical advice.

26
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31
 국문 초록

췌관내유두상점액종양의 예후 예측을 위
한 엑소좀 단백질 프로파일링

서울대학교 대학원
의학과 내과학
박 남 영

췌관내유두상점액종양(Intraductal papillary mucinous neoplasm,

IPMN)은 잘 알려진 췌장암의 전구 병변으로, 그 악성화 가능성을 정확

히 예측하는 것이 매우 중요하다. 그러나 영상의학적 소견에 상당히 의

존하고 있는 현재의 가이드라인들은 그 정확도에 대한 한계가 지속적으

로 지적되고 있다. 본 연구는 엑소좀 단백체 분석을 이용하여 악성

IPMN과 양성 IPMN을 구별할 수 있는 바이오마커를 탐색하고, 그 예측

력을 평가하고자 한다.

혈액 내를 순환하는 엑소좀을 금속 기반 침전과 크기 배제 크로마토그

래피 등의 방법을 이용하여 고순도로 분리한 후, 세 단계의 분석을 진행

하였다. 발견 단계에서는, 20명의 환자(악성 IPMN 6명, 양성 IPMN 14

명)를 모집하여 포괄적인 단백체 분석을 시행하였다. 확인 단계에서는

14명의 환자(악성 IPMN 7명, 양성 IPMN 7명)를 모집하여 병렬반응 모

니터링 질량 분석(Parallel reaction monitoring-mass spectrometry,

32
PRM-MS)을 통해 발견 단계에서 확보한 후보 단백질들에 대해 표적

질량 분석을 진행하였다. 발견 및 확인 단계에서 확인된 후보 단백질 중,

the Cancer Genome Atlas (TCGA) 데이터베이스의 실제 췌장암 환자의

생존율과 연관성이 있는 단백질을 선별하여 다음 검증 과정을 진행하였

다. 선별된 후보 단백질들의 예측력은 독립적인 검증 코호트를 이용하여

검증하였다. 검증 단계에서는 총 24명의 환자(악성 IPMN 12명, 양성

IPMN 12명)를 추가로 모집하였고, 환자 정보를 공개하지 않고 PRM-

MS를 시행하였다. 검증 단계에서 예후 예측력이 확인된 단백질들의 췌

장암과의 기능적 연관성을 확인하기 위해 독립적으로 수립된 IPMN 연

관 췌장암 세포주(ASAN-PaCa 세포주)를 이용하여 녹다운 및 과발현

세포 모델을 수립하였고, 세포 증식, 이동 및 침습 능력을 조사하였다.

크기 배제 크로마토그래피, 전자 현미경 사진, 나노입자 추적 분석 결

과를 종합하였을 때, 엑소좀이 추후 분석을 위해 충분한 정도로 높은 순

도로 분리되었음을 확인할 수 있었다. 발견 단계에서는 총 75 종류의

바이오마커 후보 단백질이 확인되었다. 확인 단계에서는 PRM-MS 결과

와 TCGA 췌장암 데이터베이스의 생존분석 결과를 종합하여, PDLIM7,

LYPLA1, PDIA6, HK1이 유망한 바이오마커 후보일 것으로 판단하였다.

독립적인 검증 코호트에서 엑소좀 단백체 분석을 진행하였을 때,

PDLIM7과 LYPLA1이 유의미하게 발현에 차이를 보였다. PDLIM7과

LYPLA1은 수신자조작특성(Receiver Operating Characteristic) 곡선

분석에서도 우수한 곡선아래면적(Area Under the Curve, AUC) 값을 보

였다. PDLIM7은 0.875, LYPLA1은 0.885, 둘의 조합은 0.946의 높은

33
AUC 값을 보여주었다. 특히, 탄수화물 항원 19-9(Carbohydrate

antigen 19-9)이 정상인 환자에서도 PDLIM7과 LYPLA1은 훌륭한 예

측력을 보여주었다. 세포 실험에서도, PDLIM7과 LYPLA1을 각각 녹다

운하였을 때, 세포 증식 속도가 유의미하게 억제되었고 세포 침입 능력

도 감소되었다. 특히 3차원 배양 모델에서, PDLIM7과 LYPLA1이 녹다

운된 세포는 그 침습적인 표현형이 감소하고 균일한 둥근 모양을 이루었

으나, 두 단백질이 과발현된 세포는 그 이동성과 증식이 촉진되는 모습

을 보였다.

엑소좀 단백체학 분석, 수신자조작특성 곡선 분석 및 세포 실험 결과

를 종합해 볼 때, PDLIM7과 LYPLA1은 IPMN의 악성도를 예측하는 유

용한 바이오마커로 활용될 수 있을 것으로 생각된다.

주요어: 췌관내유두상점액종양, 췌장암, 바이오마커, 엑소좀

학 번: 2021-33889

34
Table 1 The baseline characteristics of patients in each cohort

Discovery Verification Validation

mIPMN bIPMN mIPMN bIPMN mIPMN bIPMN
(n=6)* (n=14) (n=7)† (n=7) (n=12) (n=12)

63.0 65.5 63.0 70.0 67.5 70.0

Age
(58.0-77.0) (58.0-69.0) (59.5-76.0) (66.0-76.5) (58.0-74.0) (66.0-74.0)
Size (cm) 4.5(2..0-6.5) 1.9(1.0-2.2) 4.5(1.8-5.2) 1.1(1.0-1.6) 3.9(2.3-4.5) 1.8(0.4-2.4)
Location
Head 5 5 5 3 6 6
Body, Tail 1 5 2 2 6 6
Multiple 0 4 0 2 0 0
Metastasis 1 N/A 3 N/A 1 N/A
Stage
I 0 N/A 0 N/A 1 N/A
II 3 N/A 3 N/A 7 N/A
III 2 N/A 1 N/A 3 N/A
IV 1 N/A 3 N/A 1 N/A
CA 19-9 ≥ 37 4 1‡ 4 0 8 0
Comorbidities
HTN 3 4 3 5 6 0
DM 2 2 3 3 8 0
DL 1 2 1 0 0 2
CKD 1 1 0 0 0 0
CP 0 1 1 0 0 0

* One patient diagnosed with high-grade dysplasia was excluded due to hemolysis
† One patient was diagnosed by clinicoradiologic findings
‡ One patient with CBD sludge and mild jaundice (total bilirubin 1.2)

mIPMN, malignant intraductal papillary mucinous neoplasm; bIPMN, benign

intraductal papillary mucinous neoplasm; CA 19-9, carbohydrate 19-9; HTN,
hypertension; DM, diabetes mellitus; DL, dyslipidemia; CKD, chronic kidney
disease; CP, chronic pancreatitis

35
Figure 1. Schematic diagram of study

EV, extracellular vesicle; mIPMN, malignant intraductal papillary mucinous

neoplasm; bIPMN, benign intraductal papillary mucinous neoplasm; TMT, tandem-
mass-tag; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PRM-MS,
parallel reaction monitoring mass spectrometry; HR, hazard ratio; PDAC, pancreatic
ductal adenocarcinoma; LYPLA1, Lysophospholipase 1; PDLIM7, PDZ and LIM
domain protein 7; KD, knockdown; OE, overexpression

36
Figure 2. Representative results of isolated exosome samples.

(A) transmission electron microscopy image, (B) result of nanoparticle tracking

analysis.

B
Data 1 [4]
8×106

6×106
Particles/mL

4×106

2×106

0
0 2×102 4×102 6×102 8×102 1×103
Size (nm)

37
Figure 3. Results of proteomic analysis in the discovery cohort

(A) A total of 1,390 proteins were quantified in plasma-derived EV. (B) Known EV
markers detected in purified samples indicating proper isolation of exosomes (C)
Volcano plot analysis comparing the EV proteome of mIPMN versus bIPMN.

A B

-1.0 Log2(mIPMN/bIPMN) 1.0

EV, extracellular vesicle; mIPMN, malignant intraductal papillary mucinous

neoplasm; bIPMN, benign intraductal papillary mucinous neoplasm

38
Figure 4. Top five proteins showing the most significant
expression difference in the discovery cohort

(A) Top five increased DEPs and (B) top five decreased DEPs

DEP, differentially expressed proteins; TPP2, tripeptidyl-peptidase 2; MAPRE1,

microtubule-associated protein RP/EB family member 1; MST1, macrophage-
stimulating protein; PDLIM7, PDZ and LIM domain protein 7; LYPLA1,
Lysophospholipase 1; bIPMN, benign intraductal papillary mucinous neoplasm;
mIPMN, malignant intraductal papillary mucinous neoplasm; CP, ceruloplasmin;
TGFBR3, transforming growth factor beta receptor type 3; IGF2, insulin-like growth
factor II; HABP2, hyaluronan-binding protein 2; IGFBP3, insulin-like growth factor-
binding protein 3

39
Figure 5. Twelve proteins with the significant expression
difference in the verification cohort

PDLIM7, PDZ and LIM domain protein 7; LYPLA1, Lysophospholipase 1; PDIA6,

Protein disulfide-isomerase A6; CD5L, CD5 antigen-like; PIGR, Polymeric
immunoglobulin receptor; HK1, Hexokinase-1; C3, Complement C3; SVEP1, Sushi,
von Willebrand factor type A, EGF and pentraxin domain-containing protein 1;
CDSN, Corneodesmosin; CP, Ceruloplasmin; JUP, Junction plakoglobin; DSP,
Desmoplakin; bIPMN, benign intraductal papillary mucinous neoplasm; mIPMN,
malignant intraductal papillary mucinous neoplasm

40
Figure 6. Four biomarker candidates showing differences in
survival rates in TCGA database

(A) Kaplan-Meier curve, (B) ROC curves, and (C) AUC values of four candidates

A PDLIM7 LYPLA1
Probability of Survival

Probability of Survival
100 HR : 1.53 (0.96 - 2.42) 100 HR : 2.02 (1.32 - 3.09)
p-value = 0.072 p-value = 0.001

50 50

High (n=140) High (n=73)

Low (n=36) Low (n=103)
0 0
0 1000 2000 3000 0 1000 2000 3000
Days Days

PDIA6 HK1
Probability of Survival

Probability of Survival
100 HR : 1.67 (1.09 - 2.57) 100 HR : 1.76 (1.13 - 2.73)
p-value = 0.019 p-value = 0.012

50 50

High (n=118) High (n=127)

Low (n=58) Low (n=49)
0 0
0 1000 2000 3000 0 1000 2000 3000
Days Days

B C

TCGA, the Cancer Genome Atlas; ROC, receiver operating characteristic; AUC, area
under the curve; PDLIM7, PDZ and LIM domain protein 7; LYPLA1,
Lysophospholipase 1; PDIA6, Protein disulfide-isomerase A6; HK1, Hexokinase-1;
HR, hazard ratio

41
Figure 7. Result of proteomic analysis in the validation cohort

(A) Expression levels of PDLIM7 and LYPLA1 in validation cohort and (B)
diagnostic efficacy of PDLIM7 and LYPLA1 compared to CA 19-9

PDLIM7, PDZ and LIM domain protein 7; LYPLA1, Lysophospholipase 1; CA 19-9,

carbohydrate antigen 19-9; bIPMN, benign intraductal papillary mucinous neoplasm;
mIPMN, malignant intraductal papillary mucinous neoplasm; AUC, area under the
curve

42
Figure 8. Comparison of AUC values of each biomarker in
patients with normal levels of CA 19-9

(A) ROC curve of PDLIM7, (B) ROC curve of LYPLA1, (C) ROC curve of the
combination of PDLIM7 and LYPLA1

A PDLIM7 B LYPLA1
100 100

80 80
Sensitivity%

Sensitivity%
60 60

40 40

20 20
AUC : 0.82 AUC : 0.90
0 0
0 20 40 60 80 100 0 20 40 60 80 100
100% - Specificity% 100% - Specificity%

AUC, area under the curve; CA 19-9, carbohydrate antigen 19-9; ROC, receiver
operating characteristic; PDLIM7, PDZ and LIM domain protein 7; LYPLA1,
Lysophospholipase 1

43
Figure 9. PDLIM7 and LYPLA1 promote cell migration and
invasion in mIPMN cells

(A) The different expression of PDLIM7 and LYPLA1 in ASAN-PaCa cell line and
various pancreatic cancer cell lines (B) The result of western blot for confirmation
of establishment of KD and OE models of PDLIM7 and LYPLA1 (C) Cell
proliferation analysis of KD and OE models in the 2D-cell culture study (D) The
results of cell migration and invasion ability test for KD and OE models (E) Cell
mobility and 3D cancer dissemination analysis in the 3D-cell culture study

A B

C D

PDLIM7, PDZ and LIM domain protein 7; LYPLA1, Lysophospholipase 1; mIPMN,

malignant intraductal papillary mucinous neoplasm; KD, knockdown; OE,
overexpression; 2D, two-demensional; 3D, three-demensional; Inv, invasion; Mig,
migration

44