Monitoring pancreatic carcinogenesis by the molecular imaging of Cathepsin E in vivo using confocal laser endomicroscopy

Article abstract of DOI:10.1371/journal.pone.0106566

The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually increases alo

Full text of DOI:10.1371/journal.pone.0106566

Monitoring Pancreatic Carcinogenesis by the Molecular
Imaging of Cathepsin E In Vivo Using Confocal Laser
Endomicroscopy
Hui Li1., Yongdong Li1., Lei Cui2, Biyuan Wang1, Wenli Cui3, Minghua Li1, Yingsheng Cheng1*
1 Department of Radiology, the Sixth Affiliated People’s Hospital, Shanghai Jiao Tong University, Shanghai, China, 2 College of Science, University of Shanghai for Science
and Technology, Shanghai, China, 3 Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China
Abstract
The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is
specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually
increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene
(DMBA)-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were
consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology
and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE-
overexpressing cells (PANC-1-CTSE) in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the
specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the
fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive
pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we
monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE) in combination with the CTSE-
activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in
diagnosing PDAC and PanINs of all grades (.82.7%). This allowed us to establish effective diagnostic criteria for CLE in
PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations.
Citation: Li H, Li Y, Cui L, Wang B, Cui W, et al. (2014) Monitoring Pancreatic Carcinogenesis by the Molecular Imaging of Cathepsin E In Vivo Using Confocal Laser
Endomicroscopy. PLoS ONE 9(9): e106566. doi:10.1371/journal.pone.0106566
Editor: Matthew Bogyo, Stanford University, United States of America
Received March 5, 2014; Accepted July 31, 2014; Published September 3, 2014
Copyright: ß 2014 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* Email: yingsheng_cheng@163.com
. These authors contributed equally to this work.
and PanIN–III), according to the progression of pathomorpho-
logical changes [6,7].
Clinical research studies [8,9] have found a high discovery rate
Introduction
The onset of pancreatic ductal adenocarcinoma (PDAC) usually
goes undetected. As such, the majority of patients that present with
of PanINs in pancreatic tissue removed from patients at a high risk
advanced-stage disease have a poor prognosis. Invasive PDAC is
of developing PDAC who undergo the partial or total removal of
nearly always a fatal condition, and although it comprises only 3%
the pancreas for an unrelated reason. In addition, for those
of estimated new cancer cases each year, it is the fourth most
patients without invasive PDAC, surgery to remove PanINs has a
common cause of cancer mortality in the United States [1].
high cure rate. Therefore, the monitoring of PDAC in high-risk
Because of its relatively low incidence, current research is
populations is essential to improve the prevention and cure of this
focused on the early detection and screening of patients at high risk
condition. However, despite advances in screening and the early
of developing PDAC [2]. Risk factors for PDAC include a family
history of PDAC; .40 years of age with nonspecific abdominal
discomfort; sudden diabetes, especially atypical forms; chronic
detection of other cancers, such as breast and colon cancer, no
reliable screening test exists for PDAC at present.
Cathepsin E (CTSE) is highly and specifically expressed in
pancreatitis, especially hereditary chronic pancreatitis and chronic
PDAC and PanINs, and its expression gradually increases with
calcifying pancreatitis; patients with familial adenomatous polyp-
disease progression [10]. Various CTSE- optical molecular probes
osis; long-term smoking; heavy alcohol intake; and long-term
have been successively developed in recent years. Tung et al
exposure to hazardous chemicals [3–5].
More than 95% of cases of pancreatic adenocarcinoma are
[10,11]. constructed a smart optical molecular probe for the
specific detection of CTSE. Under the catalysis of CTSE, their
PDAC that originates from pancreatic ductal epithelium, which
probe peptide (a distinct CTSE-selecting substrate) bonds were
develops in a multi-stage process. Pancreatic intraepithelial
hydrolyzed to release fluorophores, which release a fluorescent
neoplasia (PanIN) is the main precancerous lesion of PDAC,
signal. They then succeeded in detecting early PDAC and PanINs
which further develops into early PDAC. These lesions are divided
into low-grade PanIN (PanIN-I) and high-grade PanIN (PanIN-II
in transgenic mice by in vitro imaging for the probe signal [12,13].
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Figure 1. The probe chemical structure and action mechanism.
(a) Chemical structures of optical probes. (b) Action mechanism of CTSE
optical probe.
doi:10.1371/journal.pone.0106566.g001
The United States Food and Drug Administration (FDA)-
approved human immunodeficiency virus (HIV) protease inhibitor
ritonavir (RIT) has a high affinity for CTSE. Therefore,
Weissleder et al. have developed a CTSE optical imaging agent,
ritonavir tetramethyl-BODIPY (RIT-TMB), which can identify
the tumor margin to assess the degree of infiltration during surgery
[14].
In this research, we initially synthetized this specific CTSE-
activatable probe according to the method described by Tung et al
[10]. We then conducted in vivo imaging of heterotopically
implanted tumors generated from the cells that overexpress CTSE
(PANC-1-CTSE) in nude mice. Fluorescence signal changes were
detected in vitro in 7,12-dimethyl-1,2-benzanthracene (DMBA)-
induced rat PanINs of various stages and early PDAC. We further
pioneered the use of in vivo imaging techniques in combination
with confocal laser endomicroscopy (CLE) for the continuous
monitoring of pancreatic carcinogenesis in rats.
Material and Methods
Ethics statement
Animal procedures were performed in accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of
Health. The animal study was approved by the Institutional
Animal Care and Use Committees of the China Medical
Association (Permit Number: CCMA 2012–2397).
Preparation of CTSE-fluorescent probes
All solvents used were of analytical or HPLC grade. Dichloro-
methane (DCM), N-methylpyrrolidone (NMP) and methanol
(MeOH) were purchased from Sinoreagent (Shanghai, China).
N, N-Dimethylformamide (DMF), diethylether, acetonitrile
(MeCN), diisopropylethylamine (DIPEA), DL-dithiothreitol
(DTT), triethylacetate (TEA), dimethylsulfoxide (DMSO), iodoa-
cetic anhydride, piperidine Triisopropylsilane (TIS), triethylamine
(TEA) and 1,2-ethanedithiol (EDT) were purchased from Sigma-
Aldrich (Shanghai, China). HOBt and HBTU were purchased
from Energy Chemical (Shanghai, China). Fmoc-protected amino
acids were custom synthesized by GL Biochem (Shanghai, China).
D-Polylysine was purchased from Sigma-Aldrich (Shanghai,
China).
In order to meet the needs of two different instruments for tests,
Cy7.0 (probe A and probe a) and 6-carboxyl fluorescein
succinimide ester (probe B) were selected as fluorophores, with
excitation wavelengths of near-infrared and 490 nm, respectively.
Peptide
substrates,
Ala-Gly-Phe-Ser-Leu-Pro-Ala-Gly-Cys-
CONH2 (probe A, a selective peptide substrate for CTSE), Gly-
Ser-Pro-Phe-Leu-Ala-Gly-Cys-CONH2 (probe a, a selective pep-
tide substrate for cathepsin D), were synthesized by solid-phase
peptide synthesis (SPPS) on Rink amide Novagel resin (0.1 mmole,
0.61 mmol mg_1) using standard Fmoc chemistry with HBTU/
HOBT coupling on an automatic synthesizer (ABI-433A, Applied
Biosystems). The peptides were purified by HPLC and then
Cy7.0-NHS ester was coupled to the N-termini in anhydrous
DMSO and TEA overnight in the dark. After HPLC purification,
the identity of Cy7.0 labeled peptides was confirmed by LC-MS
using a Thermo Finnigan LCQ Fleet mass spectrometer (Thermo-
Fisher Scientific, West Palm Beach, FL, USA). The results
revealed the high purity (.94%) and confirmed the identities of
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Figure 2. Concrete synthesis procedures of CTSE optical imaging probe A. (a) Synthesis of Cy7.0. (b) Synthesis of Cy7.0-labelled peptide
substrate. (c) Surface modification of poly-lysine and conjugation with Cy7.0-labelled peptide substrate.
doi:10.1371/journal.pone.0106566.g002
the precursor peptides. The probe preparation protocol is similar
to the previously reported protocol for other protease sensitive
probes [15]. Briefly, iodoacetylated- D-Polylysine (DPGC) was
prepared by reacting iodoacetic anhydride with D-Polylysine
(1 mg, MW = 60 KDa) under basic conditions, and then the
prepared DPGC was reacted with Cy7.0-peptide substrates (1 mg)
in sodium acetate buffer (300 ml, 50 mM, pH 6.5) overnight at
room temperature in the dark. The formed imaging probes were
collected by membrane cutoff filtration and quantified by
measuring Cy7.0 molar absorbance at 762 nm. Peptide loadings
on DPGC were calculated using the relative mole ratio of
fluorochrome to D-Polylysine. On average, 21–23 peptides were
attached to each D-Polylysine molecule. The quenching efficiency
of the probes was quantified by measuring the fluorescence signal
intensity of imaging probes to that of free equal-moles of Cy7.0
dye. More than 95% of fluorescent quenching was obtained. For
probe B, the 6-carboxyl fluorescein succinimide ester was used as
the fluorophore polymer, the peptide was similar as probe A, and
was synthesized as described above. (Fig 1, 2, Table 1)
transduction were as follows; PANC-1 was infected with lentivirus
with seven different MOIs (5, 10, 20, 50, 100, 200 or 300) for 72 h.
After determining the optimal condition for infection (MOI = 50),
we examined the effects of the feeder layer on the proficiency of
transduction. The transduction efficiency was evaluated based on
the number of GFP-expressing PANC-1 cells as scored under
fluorescence microscopy.
For whole-cell protein extraction, cells were washed with cold
PBS and subsequently lysed in cold RIPA lysis buffer (50 mM
Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol [DTT],
0.25% sodium deoxycholate and 0.1% NP-40) containing 1 mM
phenylmethysulfonyl fluoride (PMSF), 50 mM sodiumpyropho-
sphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM
EGTA, and a protease inhibitor cocktail (Roche Molecular
Biochemicals, Mannheim, Germany). Cell lysis was performed
on ice for 30 minutes. Clear protein extracts were obtained by
centrifugation for 30 minutes at 4uC. Protein concentrations were
determined by the Bradford method using the Bio-Rad protein
assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) and 20–
40 mg of protein mixed with loading buffer was loaded per lane,
separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-
PAGE). Proteins were transferred to PVDF membrane filters
(Millipore, USA). Nonspecific binding was blocked by incubation
in phosphate-buffered saline (PBS) containing 0.1% Tween 20
(PBS-T) and 5% skim milk. PVDF membranes were blocked with
5% dry milk for 1 hour at 4uC. Membranes were incubated in
anti-cathepsin E primary antibody (Cath E antibody [1:100
dilution; sc-166500; Santa Cruz Biotechnology, Santa Cruz,
California, USA]) overnight at 4uC. The membranes were then
incubated with the corresponding secondary antibody (1:2000,
anti-GAPDH-HRP) in TBST-5% non-fat milk for 1 hour at room
temperature and the immunoreactive bands were visualized using
EZ ECL Chemiluminescence Detection Kit for HRP (Thermo,
US). Images were acquired using the Kodak X-OMAT BT
(Kodak). Membranes were re-probed using b-actin as a loading
control.
Generation of cell line that overexpresses CTSE (PANC-1-
CTSE), Western blot studies and heterotopic implantation
Human CTSE (NM_001910.3) was synthesized (Life Technol-
ogies) and cloned into a NheI/AscI-digested pLenti6.3_MC-
S_IRES2-EGFP plasmid using the In Fusion Cloning Kit
(Clontech, Mountain View, CA). Ligated DNA was transformed
into DH5a cells using the rapid transformation protocol, as
recommended by the manufacturer (Life Technologies). Plasmid
DNA was extracted from ampicillin-resistant colonies grown first
on Luria-Bertani agar plates and then in Luria-Bertani broth, and
plasmids were verified by DNA sequencing (Life Technologies).
Lentiviral particles were generated by transfecting the
HEK293T cell line with packaging vectors (pLP1, pLP2, pLP/
VSVG; obtained from Life Technologies) and pLenti6.3_CH-
SE_IRES2-EGFP for approximately 3 days, at which point
samples of media were collected and filtered through a 0.45-mm
cellulose acetate syringe filter, aliquoted into cryotubes, and snap
frozen in liquid nitrogen.
PANC-1 human pancreatic adenocarcinoma cells (16104 cells/
well, purchased from the Chinese Academy of Sciences cell bank)
with a low expression level of CTSE were seeded in a 12-well plate
and transduced with lentivirus in a 1:1 ratio with growth medium
with 8 mg/ml polybrene. After 72 hours, PANC-1-CTSE cells
were generated, and then trypsinized and split into dishes with
growth medium (DMEM medium with 10% FBS and 1%
glutamine/penicillin/streptomycin).
PANC-1 cells and PANC-1-CTSE cells were cultured in growth
medium, counted with a cytometer, and the cell concentration was
adjusted to 16106 cells/50 ml. PANC-1-CTSE cells and PANC-1
cells were subcutaneously injected into twenty nude mice (male
BALB/C-nu/nu mice, 6 weeks of age, weighing 19–22 g) at the
right thigh root and the right axilla, respectively, for heterotopic
implantation. The mice were inspected daily until tumors were
grown to 3–5 mm, as measured by calipers.
Assessment of CTSE-fluorescent probes
Cultured PANC-1 cells were dissociated into single cells with
trypsin-EDTA and analyzed under fluorescence microscopy to
evaluate transduction efficiency. The conditions for lentiviral
The 20 mice were randomly devided into four groups. Probe a
(4 mg/kg, group 1), probe A (20 mg/kg, group 2), probe A (4 mg/
kg, group 3), and probe A (2 mg/kg, group 4) were respectively
Table 1. Peptide substrates used for imaging probes preparation and their characterization.
Probe
Protease-selective peptide sequence
Calculated mol. wt.
Observed [M+H]+or [M]+
A
a
Cy7.0-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Gly-Cys-CONH2
Cy7.0-Gly-Ser-Pro-Phe-Leu-Ala-Gly-Cys-CONH2
Fluo-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Gly-Cys-CONH2
1567.67
1481.62
1177.48
1567.65
1481.66
1178.52
B
doi:10.1371/journal.pone.0106566.t001
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injected into the mice via the tail vein. After 72 h, mice were
injected intraperitoneally with a mixture of ketamine (100 mg/kg)
and xylazine (10 mg/kg) for deep anesthesia. In vivo NIR imaging
(Kodak-In Vivo Multispectral Imaging System, Carestream
Health Inc., USA) and quantitative determination (software:
Carestream MI) were then conducted.
experimental group, and n = 1 for the control group for each time
point). The tissue surrounding the purse string suture was sampled,
then used for formalin fixation and paraffin embedding, followed
by HE staining, histopathological examination, CTSE immuno-
histochemical analysis and Western blot studies.
CTSE immunohistochemical analysis and Western blot
studies
Preparation and pathological confirmation of in situ
DMBA-induced PDAC in rats
FFPE (formalin-fixed and paraffin-embedded) samples were
collected onto poly-L-lysine coated slides and after a heat-induced
antigen retrieval procedure, with EDTA for pAKT, and citric acid
for other antigens, for 3 min, were processed with a standard
manual streptavidin-peroxidase technique using a biotin-free
detection system (Dako, Carpinteria, CA, USA). Samples were
incubated with primary antibodies at 4uC overnight. For a
negative control, the primary antibody was replaced with an
antibody diluent. A ready-to-use kit (EnVisionTM; Dako) was used
in combination with the primary antibody (Cath E antibody
[1:200 dilution; sc-166500; Santa Cruz Biotechnology, Santa
Cruz, California, USA]), according to the manufacturer’s instruc-
tions.
Rats (SD, male, weighing 200 g, n = 75) were intraperitoneally
injected with a mixture of ketamine (100 mg/kg) and xylazine
(10 mg/kg) to induce a deep anesthesia. The epigastrium was
disinfected and a median incision was made at the abdomen to
expose the pancreas. The pancreatic capsule at the tail of the
pancreas was carefully cut, and a DMBA particle (Sigma
Pharmaceuticals, South Croydon, Victoria, Australia) at a dose
of 5 mg/100 g was placed in it. A purse string suture was
performed around the incision, which was then tightened and
ligated, before the abdominal cavity was closed. For the control
group (n = 5), the purse string suture was carried out without the
DMBA implant. The rats were sacrificed with excess ketamine-
xylazine at 1, 2, 3, 4 and 5 months after surgery (n = 15 for the
Figure 3. Near-infrared in vivo fluorescence imaging of heterotopically transplanted pancreatic adenocarcinoma in nude mice. (a)
Western blot shows the overexpression of CTSE in PANC-1-CTSE cells, which was much higher than that in PANC-1 and HEK293 cells (negative). (b)
Four groups of nude mice (G1-G4) were all subcutaneously injected with PANC-1-CTSE and PANC-1 cell suspensions in the right thigh root (e) and
right axilla (q), respectively. One group of nude mice (G1) was injected with probe a (4 mg/kg), while the others were injected with different doses of
the probe A as follows: G2: 20 mg/kg; G3: 4 mg/kg; G4: 2 mg/kg. Abnormally high signals were observed in the right thigh root of the mice injected
with probe A (G2-M4), with similar values observed in the G2 and G3. These were both significantly higher than that observed in G4. The signals were
absent in the right thigh root of the mice injected with probe a (G1) and in the right axilla of all mice (G1–G4).
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Figure 4. HE staining, immunohistochemical analysis and Western blot studies of CTSE of in situ DMBA-induced PDAC and PanINs.
(a-e) Representative images of histology (HE staining) in pancreatic tissue sections of (a) normal pancreas; (b) PanIN-I; (c) PanIN-II; (d) PanIN-III; and (e)
PDAC. (f-j) Representative images of immunohistochemical localization of CTSE in pancreas tissue sections of (f) normal pancreas; (g) PanIN-I; (h)
PanIN-II; (i) PanIN-III; and (j) PDAC, showing a gradually increasing trend of CTSE expression (brown) with increasing grade of maglignancy. (k)
Western blot shows very low levels in normal pancreatic tissues, but higher levels in PanINs and PDAC tumors. Scale bar, 100 mm.
doi:10.1371/journal.pone.0106566.g004
Proteins were isolated from five tissue types (normal pancreas,
PanIN-I, PanIN-II, PanIN–III, and PDAC) using the Tissue
Protein Extraction Kit (3W0819A, CWBIO, China) according to
the manufacturer’s instruction following by Western blotting
assays as described above.
1. Dynamic in vivo imaging with CLE
During pancreatic carcinogenesis in the DMBA-induced rat
model (n = 5), dynamic in vivo imaging with CLE (Cellvizio Image
Cell, Mauna Kea Technology Inc., France) was carried out at
various stages of pancreatic carcinogenesis (day 0, 30 days, 60
days, 90 days, 120 days, or 150 days after DMBA placement). The
molecular probe (probe B, 2 mg/ml in PBS) was intravenously
injected, then after 72 hours, rats were fixed after inducing a deep
anesthesia; the left upper abdominal wall was disinfected, a small
incision was made, and the handheld micro CLE probe, which
scans and records the signals on the surface of and inside the
tumor layer-by-layer, was inserted. The scanning parameters were
an excitation wavelength of 488 nm, and acquisition bandwidth of
505–585 nm. Finally, the abdomen was closed, animals continued
feeding and observed.
In vitro imaging and pathological comparison of
tumorigenesis of DMBA-induced PDAC in rats injected
with probe A and probe a
During pancreatic carcinogenesis in the DMBA-induced rats
(n = 30), in vitro imaging was carried out at 1, 2, 3, 4, and 5
months after DMBA placement, as well as at various stages of
pancreatic carcinogenesis. The molecular probes (probe A and
probe a, 2 mg/ml in PBS) were randomly injected into the tail
vein of rats (n = 465 for probe A, and n = 265 for probe a). After
72 hours, the animals were sacrificed via excess ketamine-xylazine,
and samples of the tumor tissue, liver, kidney, and muscle were
collected for in vitro imaging and quantitative determination.
Finally, HE staining, histopathological examination and CTSE
immunohistochemical analysis were performed.
2. Prospective, double-blind control study using CLE
DMBA was induced at day 0, 30, 60, 90, and 120 in rats
(n = 865 for the experimental group; n = 265 for the control
group), and at day 150, all animals (n = 47; there were 3 deaths
after day 100) were killed with excess anesthesia after in vivo CLE
imaging (i.e. imaging at the first, second, third, fourth, fifth month
after induction). Samples of PDAC tissue were collected for HE
staining, histopathological examination and CTSE immunohisto-
chemical analysis. CLE researchers (HL and BW) and pathology
researcher (WLC) who evaluated the tumor grade based on CLE
(blinded to the pathological results) and pathological findings
In vivo imaging of rats with DMBA-induced PDAC using
CLE
The handheld micro CLE probe was placed on the surface of
the pancreatic lesions for single-cell-resolution imaging, combined
with the specific CTSE-activatable probe. The CLE images were
then analyzed for intensity of the fluorescence signal, morphology
and size of the CTSE-positive cells, and fluorescent activation
patterns (whether homogeneous or not).
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(blinded to the CLE results), respectively, were not aware of the
induction time.
Image J was used for the quantitative analysis of the fluorescence
intensity of CLE images. For images of every observed lesion and its
surrounding normal pancreatic tissue, researchers selected images
with minimal motion artifact and strong fluorescent signal for
analysis. The average fluorescence intensity was calculated.
Statistical analysis
SPSS statistical software (version 13.0) was used for the statistical
analysis of data. Statistically significant differences were determined
by two-tailed unpaired Student’s t test; the level of significance was
set at p,0.05. Cohen’s kappa value was calculated to assess the
reliability of data collected between the two observers.
Results
Assessment of the validity and specificity of the CTSE-
activatable probe using the heterotopic implantation of
PANC-1-CTSE cells into nude mice
PANC-1-CTSE cells demonstrated immunoreactive bands at
43 KDa, which was the anticipated molecular weight of CTSE.
Densitometry studies showed that the expression levels of CTSE in
PANC-1-CTSE cells were much higher than in PANC-1 cells
(Fig. 3a).
In the nude mice injected with probe A, the probe was
accumulated in the PANC-1-CTSE tumors, whereas the control
PANC-1 tumors showed no probe accumulation. In the nude mice
injected with probe a, both tumor types showed no probe
accumulation (Fig. 3b).
In addition, similar levels of probe accumulation were observed
in the PANC-1-CTSE tumors in mice injected with 20 mg/kg
(signal level: 327623) and 4 mg/kg (signal level: 295618) of probe
A, and both levels were significantly higher (p = 0.005, 0.012) than
those observed in the mice injected with 2 mg/kg (signal level:
116615) of probe A (Fig. 3b). Therefore, we determined that the
optimal injection dose for the probes was 8 mg/kg.
Histopathological, immunohistochemical analysis and
Western blot studies for the assessment of tumorigenesis
and changes of CTSE expression during in situ DMBA-
induced PDAC in rats
In this experiment, histopathological examination of pancreatic
tissue was carried out after the in situ induction of DMBA. A large
number of infiltrating inflammatory cells and interstitial connec-
tive tissue hyperplasia were visible in local pancreatic tissue; and
there was evidence of the pathological changes of chronic
pancreatitis, consistent with the histological features of human
PDAC. After the establishment of the tumor model, a proportion
of rats developed PanIns at 1–2 months, and PDAC at 3–4 months
(Table 2 and Figs. 4a-e).
Immunohistochemical analysis and Western blot studies were
then performed to measure the expression of CTSE (Figs. 2f-k).
The results showed that CTSE expression was very low in normal
pancreatic tissue, but high in PanINs and PDAC tumors, where
CTSE expression level showed an increasing trend along with the
progressive grade.
Assessment of the validity and specificity of the CTSE-
activatable probe using the DMBA-induced PDAC
models
For the near-infrared in vitro imaging, fluorescence signals were
descending in the following normal organs: kidney, liver, spleen,
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Figure 5. In vitro imaging and quantitative determination of PDAC and PanINs in DMBA-induced rats injected with probe A. (a) In
vitro imaging of tissue from tumors (R1 = PanIN-I; R2 = PanIN-II; R3 = PanIN-III; R4 = PDAC), kidney, liver, spleen, muscle and normal pancreas of four
rats. (b) Quantitative analysis of near-infrared fluorescence imaging of tumor tissue and normal pancreatic tissue from four rats. (c-g) Routine
pathological HE staining of (c) normal pancreas; (d) PanIN-I (R1); (e) PanIN-II (R2); (f) PanIN-III (R3); and (g) PDAC (R4). M = muscle; T = tumor; L = liver;
S = spleen; K = kidney; P = pancreas. Scale bar, 100 mm.
doi:10.1371/journal.pone.0106566.g005
normal pancreas, and muscle. In rats injected with probe A, the
fluorescence signals of the pancreatic tumors showed an increasing
trend as the degree of their malignancy increased (Fig. 5).
Through quantitative determination, the ratio of pancreatic
tumor:normal pancreas gradually increased in association with
progressive tumor grade (Fig. 4), except for no significant
difference between the PanIN-II and PanIN-III grades
(p = 0.076). In brief, the low- and high-grade PanINs and PDAC
Figure 6. Quantitative determination curve of the in vitro imaging fluorescence signal of DMBA-induced PDAC and PanINs in rats
injected with probe A and probe a. The results showed that the fluorescent signal ratio of tumor:normal pancreatic parenchyma increased with
progressive pathological grade in rats injected with probe A. On the other hand, the ratio was almost unchanged in rats injected with probe a. Error
bars represent SD. N = normal.
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Figure 7. In vivo CLE imaging of DMBA-induced PDAC in rats and pathological comparison. (a) In vivo CLE imaging of normal pancreas,
low- and high-grade PanIN lesions, and PDAC (blue) with the activated CTSE-sensitive probe. (b) Representative images of histology in pancreas
tissue sections from normal pancreas, low- and high-grade PanIN lesions, and PDAC. (c) Representative images of immunohistochemical localization
of CSTE in pancreas tissue sections from normal pancreas, low- and high-grade PanIN lesions, and PDAC. Scale bar, 100 mm.
doi:10.1371/journal.pone.0106566.g007
could be identified by in vitro fluorescent imaging using probe A.
Comparatively, in rats injected with probe a, the fluorescence
signals of the pancreatic tumors were uniformly faint throughout,
and most were slightly higher than the signals of normal pancreatic
parenchyma (Fig. 6).
Assessing the diagnostic accuracy of CLE combined with
CTSE molecular probe (probe B) on DMBA-induced PDAC
in rats
The pathological data suggested (based on the highest
pathological grade of lumps): of the 40 rats in the experimental
group, except for the four deaths (one rat died at the 82th day after
transplant, one at the 111th day, and two at the 130th day), a total
of three were normal, there were nine low-grade cases (PanIn-I),
sixteen high-grade cases (including PanIn-II and PanIn-III) and
eight of PDAC; while for ten rats in the control group, a total of
nine were normal and there was one low grade case (PanIn-I)
(Fig. 7 and Table 3).
Dynamic observation of pancreatic carcinogenesis
through in vivo imaging of in situ DMBA-induced PDAC in
rats using CLE with the CTSE probe (probe B)
CLE continuous dynamic in vivo imaging was used to monitor
the development of DMBA-induced PDAC in rats. We found that
CTSE-positive cells were gradually increased with the delay of
induction time, and the fluorescence signal intensity was increased
and uneven (Videos S1–S6).
The accuracy of the two researchers assessing PanINs grading
was higher than 82.7% (Table 4), and the consistency between the
two observers was 73.8%. We made a retrospective quantitative
analysis of the fluorescent signals of the lesions. The average
Table 3. Comparison between pathological and CLE results.
Normal
Low-grade PanIN
High-grade PanIN
PDAC
Pathology
CLE-1
12
10
16
8
* *
* *
* *
* *
11+1(low)
1(N) *+8+1(high)
* *
9+1(high)
1(low) *+14+1(cancer)
1(low) *+14+1(cancer)
8
CLE-2
12
1(high) *+7
*
* *
The results for CLE-1 and CLE-2 were the assessments of two researchers, respectively. Mistaken assessments are shown in brackets; false negative;
Abbreviations: CLE, confocal laser endomicroscopy; PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma.
doi:10.1371/journal.pone.0106566.t003
false positive.
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Monitoring Pancreatic Carcinogenesis by Imaging CTSE with CLE
Figure 8. Average fluorescence intensity of in vivo CLE imaging of DMBA-induced rat PDAC and PanINs. The average fluorescence
intensity gradually increased with the pathological grade with the exception of no significant difference between PanIN-II and PanIN-III grades
(p = 0.140). Error bars represent SD. N = normal.
doi:10.1371/journal.pone.0106566.g008
fluorescence intensity was 48.0 for PanIN-I; 56.2 for PanIN-II;
61.5 for PanIN-III; and 125.7 for PDAC. In contrast, the
fluorescence intensity of normal pancreatic tissue surrounding
the lesions was 14.2. The difference in fluorescence intensity
between PanIN-II and PanIN-III was not significant (p = 0.140)
(Fig. 8).
membrane molecules in PDAC, some of which are highly
expressed in both PDAC and PanINs, such as CTSE.
CTSE is a type of intracellular hydrolase that cleaves peptide
bonds; it is not expressed in normal pancreatic tissue, but is highly
expressed in PDAC and PanINs. [17–19] Studies have shown that
CTSE expression is up to 28 times elevated in PDAC, compared
to that of the normal pancreas. [20] Uno et al. [21] found that the
CTSE content in pancreatic juice from patients with PDAC was
significantly higher than that from patients with chronic pancre-
atitis. Collectively, these properties identify CTSE as a suitable
biomarker for PDAC. Furthermore, we have confirmed here that
the expression level of CTSE shows an increasing trend with the
development of the tumor in the rat model, which suggests CTSE
levels could potentially be used for the staging of PDAC and
precancerous lesions in the rats.
Discussion
In this study, using the in vivo imaging of heterotopically
implanted CTSE-overexpressing cells in nude mice and the in
vitro imaging of PanINs and PDAC in DMBA-induced rats, we
verified the specificity of a novel synthesized CTSE-activatable
probe. It was determined that this probe could identify low-grade
and high-grade PanINs in addition to PDAC. In combination with
CLE, we further monitored the progressive pancreatic carcino-
genesis of model rats using in vivo imaging. With a double-blind
control study and comparison with pathological data, we
confirmed that CLE combined with the specific CTSE-activatable
probe is a promising modality to monitor in vivo pancreatic
carcinogenesis, and to find and identify PanINs (at low or high
grade) and early PDAC. It is our hope that this approach will one
day become a useful method for the regular screening of high-risk
patients for PDAC.
While it is appropriate to use CTSE for cancer detection, the
development of probes for CTSE-specific detection has greatly
facilitated its use. However, standard fluorescence imaging is
unsuitable for the in vivo imaging of in situ pancreatic tumors, due
to the pancreas being located in the deep abdominal cavity. The
handheld CLE probe [22–25] is an effective tool for in vivo
imaging as the optical fibers can be inserted with minimal trauma.
Using specific CTSE optical molecular probes with CLE, we
carried out pathological prediction and comparison of pancreatic
lesions based on the diagnostic classification criteria, such as the
changes in fluorescence signal intensity of obtained images, cell
Harshe et al. [16] carried out a systemic review of the literature,
and found high expression of 162 secretory molecules and 166
Table 4. Assessment of two researchers on the CLE images, including the average sensitivity, specificity, positive predictive value,
negative predictive value and accuracy.
Sensitivity
Specificity
PPV
NPV
Accuracy
Normal
92.1
90.3
88.2
87.5
94.6
92.1
88.7
92.1
92.4
82.6
82.9
91.7
94.5
95.5
92.3
95.3
92.2
82.7
82.7
91.2
Low-grade
High-grade
Cancer
Abbreviations: CLE, confocal laser endomicroscopy; PPV, positive predictive value; NPV, negative predictive value.
doi:10.1371/journal.pone.0106566.t004
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Monitoring Pancreatic Carcinogenesis by Imaging CTSE with CLE
size and morphology, and the heterogeneous pancreatic tissue
structure. The diagnosis accuracy was above 82.7%, confirming
the suitability of using CLE images for determining the staging of
PanINs (low or high grade) and PDAC.
PDAC. It is our hope that an effective CLE diagnosis system for
PanINs and PDAC will be established through this and more in-
depth studies in the future, to facilitate the regular screening of
PDAC in high-risk populations, and also the early detection and
early treatment of PDAC.
The use of CLE optical molecular imaging in this study was
appropriate for recording the actual CTSE expression level of
diseased tissue in the pancreas. While CLE has advantages, such as
minimal trauma to recipients and high sensitivity, some problems
with the technique still need to be resolved. These include (1)
intravenous injection/local application: the local application of a
molecular probe is more suitable for clinical endoscopy, as it has
significantly reduced side effects when compared with systemic
application [26–28]. However, its local application only stains the
tumor surface, whereas the use of CLE allows the observation of
the epithelial surface structure and the deep structure of the tumor;
tomography can also be performed when compared with other
optical technologies. Therefore, in this research, we injected the
probes intravenously. (2) Due to the limited CLE equipment
available for this study, i.e. only having one excitation wavelength
of 488 nm, we had to modify our molecular probes to label it with
fluorescein, which was a limitation of this study as improved
penetration could have been achieved with near-infrared fluores-
cence imaging. This issue could be overcome by using a different
instrument. (3) Although there are many research projects looking
at the clinical application of CLE for human gastrointestinal
diseases, it is much more complex to implement CLE imaging of
the pancreas. However, this problem could be solved with the
development of endoscopic techniques such as endoscopic
ultrasonography-guided fine needle puncture [29–31], which
negates the need for open surgery.
Supporting Information
Text S1
(DOCX)
Video S1 Imaging of normal pancreatic tissue on a
microscopic level. Just a few fluorescence signals in the
darkness can be visualized.
(AVI)
Video S2 Imaging of 30th days, speculated as low grade
PanINs. CTSE-positive cells are shown.
(AVI)
Video S3 Imaging of 60th days, speculated as high
grade PanINs. More CTSE-positive cells are shown than before
and the fluorescence signal intensity is increased.
(AVI)
Video S4 Imaging of 90th days, speculated as high
grade PanINs. More and more CTSE-positive cells are shown
and the fluorescence signal intensity is further increased.
(AVI)
Video S5 Imaging of 120th days, speculated as early
PDAC. CTSE-positive cells were diffusive, and the fluorescence
signal intensity was increased and uneven.
(AVI)
The in situ DMBA-induced PDAC model in rats used in this
study was limited compared with the transgenic PDAC mouse
model because the etiology of our model is inconsistent with that of
human PDAC. But, the model used was relatively consistent with
the histopathological features of human PDAC [32,33]; PanIN
grading and the formation of PDAC showed an increasing trend
with the extension of DMBA induction time; and CTSE
expression level was also gradually increased with the development
of lesions. For the transgenic PDAC mouse model, the PDAC and
PanINs often show multiple lesions and lack connective tissue
hyperplasia; Some genes abnormally expressed in human PDAC
are not expressed in the transgenic PDAC mouse model. [34,35]
Therefore, we performed this study using the chemical-induced
model in rats on the basis of careful observation of the pathological
features.
Video S6 Imaging of 150th days, speculated as PDAC.
CTSE-positive cells were strewn, and the fluorescence signal
intensity was increased and uneven.
(AVI)
Acknowledgments
We thank Hecheng Bao, Yi Lian and Xin Wang for advice on technical
assistance, Jiacheng Li and Xin Liang for critically reading manuscript.
Author Contributions
Conceived and designed the experiments: HL YC ML. Performed the
experiments: HL YL LC WC. Analyzed the data: HL BW. Wrote the
paper: YL HL.
Our research has opened a new avenue for the early imaging
diagnosis of PDAC, and confirmed CTSE as a biomarker for
monitoring pancreatic carcinogenesis and early diagnosis of
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