Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe
Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near- infrared fluorescent probe
Kuo-Hwa Wang 0 1 2
Yung-Ming Wang 2
Li-Hsuan Chiu 2
Tze-Chien Chen 2
Yu-Hui Tsai 2 4
Chun S. Zuo 2
Kuan-Chou Chen 1 2 3
Chun Austin Changou 2 5
Wen-Fu T. Lai 2 4
0 Department of Obstetrics and Gynecology, Chung Kang branch, Cheng Ching Hospital , Taichung, Taiwan, ROC , 3 Department of Biological Science and Technology, National Chiao Tung University , Hsinchu, Taiwan, ROC , 4 McLean Imaging Center, McLean Hospital, Harvard Medical School , Belmont, MA , United States of America, 5 Department of Obstetrics and Gynecology, Mackay Memorial Hospital , Taipei, ROC
1 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University , Taipei, ROC
2 Editor: Matthew Bogyo, Stanford University , UNITED STATES
3 Department of Urology, Taipei Medical University/Shuang-Ho Hospital , New Taipei City, Taiwan, ROC
4 Department of Research, Taipei Medical University/Shuang-Ho Hospital , New Taipei City, Taiwan, ROC
5 The Ph.D. Program for Cancer Biology and Drug Discovery, Center for Translational Medicine, Taipei Medical University , Taipei, ROC , 9 Department of Dentistry, Taipei Medical University/Shuang-Ho Hospital , New Taipei City, Taiwan, ROC
Epithelial ovarian cancer (EOC) is the seventh most common cancer among women worldwide. The 5-year survival rate for women with EOC is only 30%-50%, which is largely due to the typically late diagnosis of this condition. EOC is difficult to detect in its early stage because of its asymptomatic nature. Recently, near-infrared fluorescent (NIRF) imaging has been developed as a potential tool for detecting EOC at the molecular level. In this study, a NIRF-sensitive probe was designed to detect matrix metalloproteinase (MMP) activity in ovarian cancer cells. A cyanine fluorochrome was conjugated to the amino terminus of a peptide substrate with enzymatic specificity for MMP-3. To analyze the novel MMP3 probe, an in vivo EOC model was established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice. This novel MMP-3-sensitive probe specifically reacted with only the active MMP-3 enzyme, resulting in a significantly enhanced NIRF emission intensity. Histological analysis demonstrated that MMP-3 expression and activity were enhanced in the stromal cells surrounding the ovarian cancer cells. These studies establish a molecular imaging reporter for diagnosing early-stage EOC. Additional studies are required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and prognostic significance.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Competing interests: The authors have declared
that no competing interests exist.
Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related death among
]. Approximately 239,000 cases were recorded in 2012, accounting for nearly 4%
of all new cancer cases among women worldwide (2% overall). The overall 5-year survival rate
for EOC is below 45% , largely because of the considerable difficulty involved in detecting
the disease at early stages. Consequently, approximately 75% of ovarian cancers are diagnosed
at stages III or IV.
Cancer metastasis requires the dissociation of cancer cells from the primary lesion site,
local tissue invasion, angiogenesis, blood or lymphatic system invasion, and seeding at distant
lesion sites [
]. Matrix metalloproteinases (MMPs) play a critical role in facilitating cancer
invasion and metastasis  through the proteolysis of extracellular matrix (ECM) and
nonmatrix components [
]. Specific MMPs such as MMP-3, -7, and -9 promote cancer metastasis [7±
9] and are modulated by WNT signaling [
]. Among them, MMP-3 (stromelysin-1) is
overexpressed in various tumor types [
], and its activation has been reported in ovarian cancer
]. Although several MMPs have been studied in ovarian cancer [
7, 14, 15
], few studies
have focused on MMP-3 in EOC. During tumorigenesis, MMP-3 is a key candidate for
regulating tumor-host interactions , which facilitate early-stage breast carcinogenesis [
However, MMP-3 also inhibits tumor progression in breast cancer and skin squamous cell
carcinoma, studies have reported increased tumor growth rates and higher proliferative indices in
MMP-3-null mice [
]. In addition, MMP-3 activity may reflect a wound-healing response
of the associated tumor stroma as MMP-3 attempts to reconcile the tumor cells back into a
normal tissue structure and to limit cancer cell invasion . Thus, the analysis of early-stage
MMP expression, including MMP-3 expression, may serve as a potential molecular target for
imaging prior to cancer metastasis and progression, especially given the importance of the
tumor stroma in ovarian cancer progression [
Molecular imaging is a novel tool for the in vivo characterization of biologic processes at
the cellular and molecular level [
]. Contrary to traditional diagnostic imaging techniques
such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound,
molecular imaging enables the analysis of molecular mechanisms underlying disease
abnormalities. Various molecular imaging platforms have been employed, including optical imaging
(both fluorescence and bioluminescence), single-photon emission computed tomography
(SPECT), and positron emission tomography (PET), with substrate-based and activity-based
Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer
detection with high sensitivity and multidetection capability. NIRF probes are ideal candidates for
the targeted imaging of cancer tissues, especially given their convenient modification through
conjugation with the moieties of interest [
]. Moreover, NIRF imaging can be combined with
other imaging modalities, and nanoparticles loaded with NIRF dyes and anticancer agents
have the potential to combine; exploit imaging and therapeutic functions to achieve the
ultimate goal of simultaneous diagnosis and treatment [
]. NIRF has been employed to detect
], lysophosphatidic acid [
], and MMP enzymatic activity [
]. Other probes
have been designed to directly bind to HER2 [
] or αvβ3 receptor [
] to target specific cancer
cell types. Activity-based signals can be detected by NIRF-quenched probes, which can shift
their optical properties after protease cleavage. Upon cleavage, the fluorochrome and the
quencher separate, resulting in an enhanced fluorescence signal.
Given that MMP-3 is associated with tumor-activated stromal cells [
], a novel
MMP3-sensitive NIRF probe has been designed in our laboratory to detect stromal cell activation by
early-stage EOC. A NIR fluorochrome was conjugated to the amino terminus of an
MMP3-specific peptide substrate. To analyze the novel MMP-3 probe, an in vivo EOC model was
established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice.
This present study may provide data for delineating the role of MMP-3 expression in EOC
disease progression and its clinical utility in early-stage EOC detection.
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Materials and methods
Methoxy-polyethylene glycol was obtained from Fluka Chemical Co. (Buchs, St. Gallen,
Switzerland) and N-hydroxysuccinimide (NHS) from Aldrich Chemical Co. (St. Louis, MO, USA).
All other reagents and solvents were also purchased from Aldrich. Absorbance and
fluorescence spectra were measured on a U-3010 spectrophotometer (Hitachi, Tokyo, Japan) and an
F-4500 fluorophotometer (Hitachi, Tokyo, Japan), respectively. Proton nuclear magnetic
resonance (NMR) spectra were obtained in CDCl3 in a 400 MHz Fourier-transform-NMR
spectrometer and the functional groups were recorded using a Perkin Elmer FT-IR system 2000
(Waltham, MA, USA).
Synthesis of the MMP-3-sensitive probe
The peptide-protected graft copolymer (PGC) was synthesized from a precursor,
methoxypolyethylene glycol (MPEG) nitrile.
Synthesis of MPEG nitrile. A mixture MPEG of molecular weight 5000 Da (25 g, 5mM),
distilled water (25 mL) and potassium hydroxide (0.5 g) was cooled to 0ÊC-5ÊC in an ice bath.
Acrylonitrile (4.3 mL) was added slowly, and the solution was stirred for 2.5 h at 0ÊC -5ÊC.
The pH of the solution was adjusted to 7 by adding sodium phosphate. The product was
extracted with dichloromethane (200, 70, and 50 mL). The organic layer was dried over
magnesium sulfate. The solution was concentrated, and the product was precipitated through
adding diethylether. The yield of MPEG nitrile was 23.5 g (93%) with the following characteristics:
IR, 2360 cm-1 (-CN); NMR (CDCl3), 2.61 ppm (t, 2H, -CH2CN), 3.3 ppm (s, -OCH3), and 3.5±
3.8 ppm (m, -OCH2CH2O-).
Synthesis of MPEG amide. A mixture MPEG nitrile (23.5 g, 4.7mM) and concentrated
hydrochloric acid (98 mL) was stirred at room temperature for 48 h. The solution was diluted
with 1 L of water and extracted with dichloromethane (200, 150, and 100 mL). The combined
organic layer extracts were washed twice with water, dried over sodium sulfate, filtered, and
concentrated to dryness through rotation evaporation. The yield of MPEG amide was 21.5 g (91%)
with the following characteristics: IR, (3424cm-1, -NH2) and (1638 cm-1, -CONH2); NMR CDCl3,
2.47 ppm (t, 2H, -CH2-CONH2), 3.3 ppm (s, -OCH3), and 3.5±3.8 ppm (m, -OCH2CH2O-).
Synthesis of MPEG propionic acid. MPEG amide (16 g, 3.2mM) was dissolved in 1000
mL of distilled water; 100 g of potassium hydroxide was added, and the solution was stirred for
22 h at room temperature. Sodium chloride (150 g) was added, and the solution was extracted
with dichloromethane (150 mL × 3). The combined organic extracts were washed with 5%
oxalic acid and water (twice), and dried over sodium sulfate. The solution was concentrated,
and the product was precipitated through adding diethylether. The product, MPEG propionic
acid, was collected by filtration and dried over vacuum. The yield of MPEG propionic acid was
14.0 g (87.5%) with the following characteristics: IR, (3453 cm-1, -OH) and NMR (CDCl3),
2.57 ppm (t, 2H, -CH2-COOH), 3.3 ppm (s, -OCH3), and 3.5±3.8 ppm (m, -OCH2CH2O-).
Synthesis of MPEG succinimidyl propionic acid (MPEG-SPA). MPEG propionic acid
(3.4 g, 1mM) was dissolved in dichlorormethane (20 mL), and N-hydroxysuccinimide (0.24 g,
2.1mM) was added. The solution was cooled to 0ÊC, and a solution of
dicyclohexylcarbodiimide (0.43g, 2.1mM) in 4 mL dichloromethane was added dropwise and stirred at room
temperature overnight. The reaction mixture was filtered, concentrated, and precipitated by
addition to diethylether. The yield of MPEG-SPA was 3.3 g (95%) with the following
characteristics: NMR (CDCl3), 2.81 ppm (s, 4H, NHS), 2.92 ppm (t, 2H, -CH2-COO-), 3.3 ppm (s,
-OCH3), and 3.5±3.8 ppm (m, -OCH2CH2O-). (S1 Scheme)
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Synthesis of PGC. PGC was synthesized using MPEG-SPA (397.5 mg, 79.5μM), which
was added slowly to an ice-cold solution of poly-L-lysine hydrobromide (14.6 kDa; 50 mg,
3.4μM) in sodium bicarbonate (0.1M, pH 8.0, 12.5 mL). The pH was adjusted to 7.7 by using
sodium hydroxide, and the mixture was stirred at room temperature for 3 h. The solution was
then extensively ultrafitered (cutoff molecular weight of 3kDa) to remove
small-molecularweight byproducts. PGC was recovered through lyophilization (73.6 kDa; 50.8 mg, 99%), and
the available amino groups for chemical conjugation were quantified using a
2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. There were 51.7 ± 0.2 free amino groups per PGC molecule.
Synthesis of iodoacetylate PGC. PGC (1 mg, 0.1μM) in 200 μL of 50mM NaHCO3 was
iodoacetylated through reaction with an excess of iodoacetic anhydride (3.5 mg, 10μmol) in
100 μL of DMF for 3 h. Ultrafitration (cutoff of 3 kDa) was used to separate the product from
the excess reagent, byproducts, and solvent. No free amino group was detected using the
Synthesis of the MMP-3 peptide. A peptide substrate of MMP-3 (EC 18.104.22.168) was
synthesized on an automatic synthesizer (PS3, Rainin Instrument Co.) through an Fmoc
chemistry by using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro-phosphate
(PyBOP) as the activating agent. The sequences were modified from previous studies [
The sequences are Gly-Pro-Lys-Pro-Tyr-Arg-Ser-Trp-Met-Lys(FITC)-Cys-NH2 (GPKPYRS
WMK(FITC)C-NH2) for the stromelysine-1-sensitive peptide substrate and
Gly-Pro-Lys-ProTyr-Arg-Pro-Trp-Met-Lys(FITC)-Cys-NH2 (GPKPYRPWMK(FITC)C-NH2) for the
scrambled control peptide (S1 Fig). ESI-TOF-MS (M+H)+ analysis revealed the following
characteristics: stromelysine-1 peptide substrate, 1709.8 (calculated) and 1710 (found); control peptide,
1719.7 (calculated) and 1720 (found).
Specificity of the peptide substrate. The stromelysine-1 peptide substrate or control
peptide (1 mg, 0.58 μmol) was incubated with 1 unit of MMP-3 for overnight reaction. MMP-3
was supplied in a 50mM Tris buffer solution (pH 7.4) containing 0.5M sodium chloride,
10mM calcium chloride, 0.05% Brij-35 and 0.02% sodium azide.
Synthesis of peptide-PGC. The iodoacetylated PGC was coupled to 3.2 mg of
stromelysine-1 peptide substrate or 3.2 mg of control peptide through a thiol-specific reaction in
200 μL of acetonitrile and 200 μL of 0.1M sodium acetate (pH 6.5) for 3 h. Excess peptide and
byproducts were separated from the peptide-PGC conjugate through sephadex G-25
sizeexclusion chromatography. The peptide-PGC conjugate was determined by measuring the
fluorescence absorption at 494nm divided by initial available amino groups. A total of
13.1 ± 0.2 stromelysine-1 peptide substrate chains or 13.8 ± 0.2 control peptide chains were
coupled per PGC molecule. To conjugate Cy5.5 dye with the peptide-PGC, 1μM
monoactivated near-infrared Cy5.5 (Amersham-Pharmacia, Piscataway, NJ, USA) was reacted with the
N-terminus of the peptide-PGC (0.5 mg) in 2 mL of 50mM NaHCO3. The reaction mixture
was incubated at room temperature for 1 h, and products were purified using ultrafiltration
with a 3kDa cutoff (S2 Fig). HPLC chromatogram of stromelysine-1 peptide substrate and
control peptide substrate were shown (S3 and S4 Figs), as well as the photochemical data of the
final probes (S5, S6, S7 and S8 Figs).
MMP-3 digestion assay
An MMP-3 digestion assay was applied to examine the in vitro reaction of the
MMP-3-sensitive probe and MMP-3 enzyme through NIRF imaging. Briefly, MMP-3 (Sigma, M1677) was
activated with P-aminophenylmercuric acetate (APMA) for 1 h at 37ÊC. After activation, an
equal volume of the MMP-3-sensitive probe was mixed with the activated MMP-3/APMA
solution. To prepare the solutions for the control groups, equal volumes of the
MMP4 / 17
3-sensitive probe was mixed with an MMP-3 solution without the activation or blank reaction
buffer. All final solutions were incubated at 37ÊC for 30 min before being subjected to NIRF
examination and quantification. For in vitro fluorescence quantification, six samples were
measured in triplicate. Optical imaging of MMP-3 NIRF was performed using an optical
imaging (IVIS-200 Series; Caliper, MA). Excitation and emission bandpass filters of 610-650nm
and 670-700nm, respectively were employed. Both white light and NIRF images were detected
using a Peltier 16-bit charge-coupled device (CCD) camera (PCO GmbH, Gottingen,
Germany, −90ÊC cooled).
Coculture of SKOV3 and WS1 cells
Ovarian cancer cell line, SKOV3, and fibroblast cell line, WS1, were purchased from ATCC
(Manassas, VA, USA). The cells were grown and expanded in McCOY's 5A medium
containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin and
amphotericin B in an incubator at 37ÊC with 5% CO2. The cell model was divided into six
groups: 2 × 104 SKOV3 cells alone, 2 × 104 SKOV3 cells cocultured with 2 × 104 WS1 cells,
2 × 104 SKOV3 cells cocultured with 6 × 104 WS1 cells, 6 × 104 SKOV3 cells alone, 6 × 104
SKOV3 cocultured with 2 × 104 WS1 cells, and 6 × 104 SKOV3 cocultured with 6 × 104 WS1
cells. Cells were cocultured in 24-well dishes in 1 mL of McCOY's 5A medium with 10% FBS
for 3 days.
To examine the specificity of the MMP-3-sensitive probe in detecting enzymatic activity in a
cell culture model, SKOV3 cells, WS1 cells, and cocultured SKOV3 and WS1 cells (3 days)
were treated with the MMP-3 NIRF probes for 30 min prior to imaging and detecting the
enzymatic activity. The cells were washed three times with phosphate-buffered saline (PBS)
and then subjected to confocal fluorescence microscopy (LEXT OLS4100, Olympus, Tokyo,
Japan). The bandpass excitation filter was at 633nm, and the longpass emission filter was
670nm (Omega Optical, Brattleboro, VT, USA). The fluorescence images were digitally
captured using a CCD-SPOT RT digital camera and compiled using Adobe Photoshop™ software
(v7.0) (Softonic International S.A., Barcelona, Spain).
Analysis of MMP-2, MMP-3 and MMP-9 expression in cocultured SKOV3 and WS1 cells
To analyze various MMP expression levels, SKOV3, WS1, and cocultured SKOV3 and WS1
cells (1:1) were seeded at 1.5 × 104/well in 24-well plates. The supernatant was collected from
each group at 1, 2, 4, 6, and 8 days to quantify the total expression levels of MMP-2, MMP-3
and MMP-9 through enzyme-linked immunosorbent assay (ELISA) using Abcam MMP
human ELISA kits (Cambridge, MA, USA). The relative cell number was determined using a
WST-1 cell proliferation reagent (Sigma-Aldrich, 96992).
Imaging of MMP-3 NIRF in an in vivo EOC model
All nude mice were obtained and housed at Taipei Medical University (TMU), under barrier
conditions in a biosafety level III animal laboratory room. All animals were treated according
to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
All animal study procedures were reviewed and approved by the TMU Laboratory Animal
Care Committee. Six-week-old female nude mice were subcutaneously inoculated with 5 × 106
SKOV3 cells suspended in 200 μL of PBS. Experiments with tumor-bearing mice were
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performed 3±11 days after inoculation. In vivo NIRF imaging was performed using an
IVIS200 system with excitation and emission filters at 610±650 nm and 670±700 nm, respectively.
Control and SKOV3 tumor-bearing mice (n = 3 in each group) were intravenously injected
with 200 μL of 2nM MMP-3-sensitive probe via the tail vein after which they were anesthetized
with isoflurane and subjected to imaging analysis. Fluorescent images were taken 2 hours after
the injection at day 3, 5, 7, 11. The tumor size were measured 4mm x 8mm. The fluorescence
intensity was measured in the tumor mass (ROI size = 4mm x 8mm) after adjusting for
background intensity (non-tumor site tissue on the back of the mice) with same size of
measurement. The relative fluorescent intensities were compared between groups. After imaging, the
mice were euthanized by CO2 inhalation according to the Guidelines for the Care and Use of
Laboratory Animals. The tumors, the peritumor areas, and lesion sites, which were analyzed
through NIRF imaging, were excised for pathological confirmation and immunohistochemical
analysis. A stability test was performed using non-tumor inoculated mice to observe baseline
fluorescent intensity and probe distribution. For biodistribution evaluation, Individual organs
were harvested and further evaluated for fluorescent intensity.
Analysis of MMP-3 expression in SKOV3 tumors
Five-micron (5 μm) paraffin-embedded sections were deparaffinized and rehydrated. Antigens
were retrieved by immersion in DECLERE (Cell Marque, Hot Springs, AR, USA) in moist
heat for 15 min. Potential nonspecific binding sites were blocked with 5% normal goat or
rabbit serum in PBS. After blocking, the sections are incubated with antibodies specific for
MMP3 (1:25; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three 5-min washes in PBS,
the sections were incubated with biotin-conjugated IgG (Vector Laboratories, Burlingame,
CA, USA). A vector-ABC streptavidin-peroxidase kit with benzidine substrate was used for
color development. Following counterstaining with diluted hematoxylin, the images were
scanned using Aperio Scanscope Console software (Informer Technologies, Inc., Shingle
Springs, CA, USA). Sections that were not incubated with the primary antibody served as a
negative control. For NIRF imaging, paraffin-embedded sections (without staining) were
examined and photographed using a NIRF microscope with 615±675- and 695±790-nm
bandpass filters at the Cy5.5 channel.
MMP-3 concentrations, relative expression, and signal intensity (SI) were presented as mean
and ±standard deviations (SDs). Independent t tests were performed to compare the
differences between two groups, one-way ANOVA was performed to compare the differences
between three or more groups, and the Bonferroni post-hoc test was used for multiple
comparisons. Statistical analyses were performed using IBM SPSS version 22 for Windows (IBM
Corp., Armond, NY, USA), with two-tailed P<0.05 indicating statistical significance.
Characterization and in vitro imaging of the MMP-3-sensitive NIRF probe
High-performance liquid chromatography (HPLC) elution chromatography confirmed one
major cut of the stromelysine-1 peptide substrate in the presence of MMP-3 and the elution of
two fragments (Fig 1A and 1B). For the specificity test of the peptide substrate, stromelysine-1
peptide substrate or control peptide (1 mg, 0.58 0 mol) was incubated with 1 unit of MMP-3
for overnight reaction. Because the scrambled control peptide was not a substrate of MMP-3,
the peptide remained intact and eluted at the original time. Following the addition of MMP-3,
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Fig 1. Characterization of the novel stromelysine-1 peptide substrate. HPLC chromatogram of the (A)
stromelysine-1 peptide substrate (black) and their digested products by MMP-3(gray) as well as (B) the control peptide
(black) and their digested products by MMP-3 (gray). (C) In vitro activation and inhibition of experimental and
control NIRF probes; the probes (0.009 μM) were incubated with or without MMP-3 (1 unit) in 50 mM Tris buffer
solution. (D) In vitro imaging of the MMP-3 NIRF probe activated by the MMP-3 enzyme. The manifest
orangeyellow color was detected in the tubes containing the MMP-3 probe with activated MMP-3 (3±5) compared with the
blank (1) and MMP-3 only (2) control tubes. A series of MMP-3 probes at 0.045 ng/μL (3), 0.09 ng/μL (4), and 0.18 ng/
μL (5) with MMP-3 (0.1 ng/μL) resulted in increasing SIs of 3.85 ± 0.15, 4.51 ± 0.12, and 4.86 ± 0.17 respectively. Six
samples in triplicate were measured. Optical imaging was performed at 610±650nm excitation and 670±700nm
emission. HPLC, high-performance liquid chromatography; MMP, matrix metalloproteinase; NIRF, near-infrared
the NIRF signal of the stromelysine-1 peptide substrate increased by 4-fold within 40 min,
which was significantly greater than the 1-fold increase in NIRF signals observed for the
control probe (Fig 1C).
To examine the specificity of the synthesized probe in detecting MMP-3 enzymatic activity,
an in vitro enzyme digestion assay was employed. Although a manifest orange-yellow color
was revealed in the tube of activated MMP-3, the other groups showed weak or no NIRF
signals. The SI of the NIRF probes appeared in a dose-dependent manner. The data indicated
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that the MMP-3-sensitive probe can specifically react with only the active MMP-3 enzyme,
resulting in a significantly enhanced intensity of NIRF emissions (Fig 1D).
Sensitivity and specificity of MMP-3 probes in SKOV3 and WS1 cocultures
As shown in Fig 2A±2D, the SIs of the NIRF probes appeared to correlate with the MMP-3
levels in the cell culture media. The 2 × 104 SKOV3 cells cocultured with 2 × 104 WS1 cells, and
2 × 104 SKOV3 cells cocultured with 6 × 104 WS1 cells showed mild and remarkable signal
intensity respectively (Fig 2A and 2C). The mean MMP-3 concentration was significantly
higher in both of the coculture groups, whereas the 2 × 104 SKOV3 cell groups had minimal
florescence signals (p<0.001). The signal was significantly higher in the 1:3 (SKOV3:WS1)
group than in the 1:1 group (0.52 vs. 0.14, respectively, p<0.001; Fig 2E).
Fig 2. Detection of MMP-3 probes in SKOV3 and WS1 cell cocultures. (A±D) Detection of MMP-3 activity through
NIRF imaging. (E, F) Quantification of the signal intensity. Either 1:1 or 3:1 (SKOV3:WS1) showed mild signal intensity (A,
B), whereas 3:1 or 3:3 (SKOV3:WS1) exhibited remarkable signal intensity (C,D). The NIRF SI appeared undetectable in the
culture of 2 × 104, or 6 × 104 SKOV3 cells alone. The mean MMP-3 concentration was significantly higher in cocultures
with increasing WS1 cells than in SKOV3 cells cultured alone (p<0.001). The signal was significantly higher in the 1:3
(SKOV3:WS1) group than in the 1:1 group (0.52 vs. 0.14, respectively, p<0.001) (E). The mean MMP-3 concentration was
significantly higher in the 3:1 and 3:3 (SKOV3:WS1) groups than in 3:0 group (0.24, 0.69 vs. 0, respectively; both p<0.001),
and was significantly higher in the the 3:3 (SKOV3:WS1) cocultures than in the 3:1 cocultures (0.69 vs. 0.24, respectively;
p<0.001) (F). MMP, matrix metalloproteinase; NIRF, near-infrared fluorescence; SI, signal intensity.
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The cell groups of 6 × 104 SKOV3 cocultured with 2 × 104 WS1, and 6 × 104 SKOV3
cocultured with 6 × 104 WS1 exhibited mild and remarkable signal intensity (Fig 2B and 2D). The
mean MMP-3 concentration was significantly higher in the 3:1 and 3:3 (SKOV3:WS1) groups
than in 3:0 group (0.24, 0.69 vs. 0, respectively; both p<0.001), and was significantly higher in
the the 3:3 (SKOV3:WS1) cocultures than in the 3:1 cocultures (0.69 vs. 0.24, respectively;
p<0.001; Fig 2F).
MMP-2, MMP-3, and MMP-9 expression in SKOV3 and WS1 cell cocultures
The MMP-2, MMP-3, and MMP-9 expression levels were next examined in the cocultivation
of SKOV3 and WS1 to mimic the interaction of ovarian cancer cell with stromal cells. The
expression levels were determined in the cocultures and compared with the control group
(SKOV3 alone). The mean relative MMP-3 expression level was significantly higher in the
cocultures than in the SKOV3 control group at days 1 to 8 (all, p<0.001; Fig 3A).
The mean relative MMP-2 expression level was also significantly higher in the cocultures at
days 1 to 8 (p 0.004; Fig 3B). No significant differences were observed in the relative MMP-9
expression among the groups (all p>0.05; Fig 3C).
MMP-3-sensitive probe detects SKOV3 cancer cells in vivo
Nude mice were inoculated with SKOV3 xenografts, which were imaged with the
MMP-3-sensitive probe at days 3, 5, 7, and 11 postimplantation. As shown in Fig 4A, the NIRF signal was
not detected in the tumor until day 5 postimplantation, at which time a 5-fold fluorescence
intensity was observed compared with that detected at day 3 (Fig 4B). The NIRF signal was
slightly decreased at days 7 and 11 (Fig 4C and 4D, respectively), suggesting that the MMP-3
activity peaked at day 5 and gradually decreased thereafter.
MMP-3 activity and expression patterns in the SKOV3 tumor mass
As shown in Fig 5, an increased NIRF signal was obtained in the tumor tissue as compared
with in the control tissue (normal subcutaneous tissue adjacent to the tumor). Hematoxylin
and eosin (H&E) staining of the tumor revealed infiltrating immune cells in the tumor mass
and the surrounding connective tissue (Fig 6A). Immunostaining with an MMP-3 antibody
revealed a predominantly positive-stained region located between the tumor mass and
Fig 3. In vitro MMP expression in SKOV3 and WS1 cell cocultures. (A) MMP-3, (B) MMP-2, and (C) MMP-9 levels in the SKOV3 and WS1 cell cocultures (gray line)
and SKOV3 cells cultured alone (black lines) were analyzed by ELISA. p<0.05, significantly different compared with SKOV3 cell cultures. ELISA, enzyme-linked
immunosorbent assay; MMP, matrix metalloproteinase.
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Fig 4. NIRF imaging of SKOV3 tumor masses in vivo. (A) NIRF imaging of the SKOV3 tumors at days 3, 5, 7, and 11.
(B-D) The mean SI was significantly higher in the tumor than in the adjacent control tissue at days 5 (B), 7 (C), and 11 (D),
p<0.001, significantly different compared with the control tissue. NIRF, near-infrared fluorescence; SI, signal intensity.
surrounding connective tissue, which was colocalized with the positive signal obtained by
using the MMP-3-sensitive probe and NIRF imaging (Fig 6B and 6C, respectively). Taken
together, MMP-3 activity was primarily localized at the boundary of tumor mass and the
adjacent connective tissue, an area that was also infiltrated by inflammatory monocytes.
To further understand the in vivo stability and biodistribution of the probe, stability test
and biodistribution evaluation were perform. As shown in the stability test (S9 Fig), the probe
were mainly distributed to abdomen after 1 hour of injection, and then distributed to kidney
36hr after the injection. For biodistribution evaluation, Individual organs were harvested and
further evaluated for fluorescent intensity. The MMP-3-sensitive probe specifically targeted
the tumor mass, with signals detected also at liver and kidney (S10 Fig). For the control probe,
baseline fluorescent signal was acquired at liver and kidney (S10 Fig), indicating the
biodistribution of the target organs during excretion of the probe from the circulation.
Specific imaging tools are required to detect early-stage EOC to improve the survival outcomes
for these patients. Because MMPs play critical roles in carcinogenesis as well as cancer invasion
and metastasis and are expressed at the early stages of disease progression [
7, 14, 15
synthesized and characterized a novel MMP-3-sensitive probe, and its specificity for MMP-3
activity was examined in both in vitro and animal models of EOC. The MMP-3-sensitive
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Fig 5. Ex vivo NIRF imaging of SKOV3 tumors. NIRF images revealed signals in the dissected tumor masses (right)
as compared with in the adjacent control tissue (left). NIRF, near-infrared fluorescence.
probe could detect MMP-3 activity in SKOV3 and WS1 cocultures as well as in SKOV3 tumors
in vivo, suggesting that it could aid in detecting early-stage EOC.
Lee et al. [
] previously reported the use of NIRF imaging of a gold nanoparticle for the
detection of MMP activity; however, this probe was not specific because it could be cleaved by
MMP-2, MMP-3, MMP-7, and MMP-13. By contrast, our data demonstrated that the
synthesized MMP-3-sensitive probe could specifically detect increased MMP-3 activity not only in
cell cultures but also in tumor-bearing mice in situ. Immunostaining of the tumors revealed
that MMP-3 was clocalized in the probe-binding region, confirming that the MMP-3-sensitive
probe could specifically detect MMP-3 activity in the target region. Studies have confirmed
that many early-stage cancers express elevated levels of MMP-2, MMP-3, and MMP-9 [
]. Specifically, MMP-3 has been associated with tumor initiation and progression [
], and the loss of MMP-3 was shown to suppress lung metastasis in an in vivo model of
mammary carcinoma [
]. MMP-2 and MMP-9 are expressed in the malignant epithelia of
tumors that have undergone an epithelial-to-mesenchymal transformation [
]. These MMPs
are crucial for local invasion of the tumor, angiogenesis, and vascular invasion [
]; thus, they
represent potential therapeutic targets . In the present study, early expression of MMP-3
was observed in the SKOV3 and WS1 cocultures, and MMP-3 activity detected by the novel
probe peaked at day 5 after the inoculation of SKOV3 cells in vivo. Additional studies are
required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and
Cancer-associated MMP expression and activity originates not only in cancer cells but also
in the stromal cells of the surrounding connective tissue. Specifically, fibroblasts and
inflammatory cells in the connective tissue may induce MMPs in response to tumor formation,
which can provoke the invasion of the malignant epithelial cells [
]. In certain epithelial
cancers, host stromal cells expressed most of the up-regulated MMPs during cancer invasion and
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Fig 6. Histological and NIRF analysis of SKOV3 tumor tissues. SKOV3 tumors were analyzed through (A)
hematoxylin and eosin staining, (B) immunohistochemistry using an MMP-3 antibody, and (C) NIRF with the
MMP3-sensitive probe. MMP, matrix metalloproteinase; NIRF, near-infrared fluorescence.
12 / 17
disease progression [
]. Consistent with these studies, our data demonstrated that SKOV3
cells expressed minimal levels of MMP-3 when cultured alone. However, MMP-3 expression
was increased upon cocultivation with WS1 cells. This increase in MMP-3 expression levels
also occurred earlier than that observed for MMP-2 and MMP-9 in cocultures. Our
pathological analysis demonstrated that both MMP-3 expression and activity were increased at the
boundary between the tumor mass and the adjacent connective tissue consisting mainly of
fibroblasts; this finding is consistent with another study reporting MMP-3 expression by
fibroblasts, endothelial cells, and immunocytes exclusively in the tumor stroma. MMP-3 also
exhibited to have a wide range of substrate specificity for various ECM components [
nature of its enzyme activity and expression pattern implies that MMP-3 plays critical role
during an early phase of tumor progression [
] when ovarian cancer cells interact with
Molecular imaging techniques have several advantages over classical imaging techniques as
well as in vitro assays. For example, the analysis of both spatial and temporal distributions of a
protein or its activity enables to permit a more global analysis [
]. Furthermore, because
molecular imaging techniques can be performed in live animals, repeated measurements are
possible, thereby decreasing costs [
]. In addition, these measurements are quantitative in
The present study has some limitations that warrant further discussion. First, the specificity
of the probe was only tested in a subcutaneous EOC model in animals. Therefore, additional
studies are necessary to evaluate its utility in women with EOC. In addition, this particular
model may not reflect the complexities of EOC. Furthermore, additional studies analyzing the
diagnostic and prognostic value of the early detection of MMP-3 activity in EOC are required.
The development of technologies enabling noninvasive and sensitive detection of
earlystage EOC is crucial to improve patient survival. The current study tested a molecular imaging
reporter for detecting EOC disease progression in situ by identifying MMP-3 activity in both a
cell and animal model. The detection of MMP-3 activity at day 5, which subsequently
decreased, implies that this molecule may have a ªhit-and-runº a role during early-stage tumor
growth and suggests that MMP-3 could be a potential molecular target for the early detection
S1 Scheme. Synthesis diagram of stromelysine-1-sensitive peptide substrate and control peptide.
S1 Fig. Synthesis diagram of the MMP-3 peptide and control peptide.
S2 Fig. Synthesis diagram of stromelysine-1-sensitive probe and control probe.
S3 Fig. HPLC chromatogram of stromelysine-1 peptide substrate. The peaks were detected
by UV/Vis = 254 nm. A Superose 6 HR 10/30 column was used, eluted with 0.1% formic acid
(0.4 ml/ min).
S4 Fig. HPLC chromatogram of stromelysine-1 control peptide substrate. The peaks were
detected by UV/Vis = 254 nm. A Superose 6 HR 10/30 column was used, eluted with 0.1%
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formic acid (0.4 ml/ min).
S5 Fig. UV-Vis spectrum of Cy5.5-s1-PGC probe.
S6 Fig. Fluorescence spectrum of Cy5.5-s1-PGC probe 5μM; ex = 670 nm (blue line) and
Cy5.5 1μM; ex = 670 nm (red line).
S7 Fig. HPLC chromatogram of stromelysine-1 peptide substrate (black) and their
digested products by MMP-3 (gray). The peaks were detected by fluorescence detector at ex/
em = 485/530 nm. A Supelco RP-C18 column (5μm, 4.6 × 250 mm) was used, eluted with
MeOH: 0.1% formic acid = 60: 40 (0.6 ml/min).
S8 Fig. HPLC chromatogram of stromelysine-1 control peptide substrate (black) and their
digested products by MMP-3 (gray). The peaks were detected by fluorescence detector at ex/
em = 485/530 nm. A Supelco RP-C18 column (5μm, 4.6 × 250 mm) was used, eluted with
MeOH: 0.1% formic acid = 60: 40 (0.6 ml/min).
S9 Fig. Mice were intravenously injected with 200 μL of 2nM MMP-3-sensitive probe via
tail vein. Fluorescent images were taken after the injection at various time points. The probe
were distributed to abdomen after 1 hour of injection and then distributed to kidney 36hr after
S10 Fig. Biodistribution of MMP-3 sensitive probe and control probe in ovarian
tumorbearing mice. Biodistribution of MMP-3 sensitive probe (A) and the control probe (B) was
investigated at 24 h after injection in ovarian tumor-bearing mice. The ovarian cancer mass
showed significant signal intensity in the group of the MMP-3-sensitive probe, whereas no
signal in the group of control probe. The spleen, liver and kidney showed mild to marked signal
intensity; however, bone, heart, lung, and muscle exhibited no signal. The tissues are oriented
as followings: heart, lung, spleen, tumor (left to right) (Top) kidney, muscle, bone, liver (Left
to Right) (Bottom).
Conceptualization: Kuo-Hwa Wang, Yung-Ming Wang, Yu-Hui Tsai, Chun S. Zuo,
Chou Chen, Wen-Fu T. Lai.
Data curation: Kuo-Hwa Wang, Li-Hsuan Chiu, Tze-Chien Chen, Wen-Fu T. Lai.
Formal analysis: Yung-Ming Wang, Li-Hsuan Chiu, Tze-Chien Chen, Wen-Fu T. Lai.
Funding acquisition: Wen-Fu T. Lai.
Investigation: Kuo-Hwa Wang, Yung-Ming Wang, Li-Hsuan Chiu, Tze-Chien Chen, Yu-Hui
Tsai, Wen-Fu T. Lai.
Methodology: Kuo-Hwa Wang, Yung-Ming Wang, Li-Hsuan Chiu, Tze-Chien Chen, Yu-Hui
Tsai, Chun S. Zuo, Chun Austin Changou, Wen-Fu T. Lai.
Project administration: Li-Hsuan Chiu, Wen-Fu T. Lai.
14 / 17
Resources: Kuan-Chou Chen, Wen-Fu T. Lai.
Software: Chun S. Zuo, Chun Austin Changou, Wen-Fu T. Lai.
Supervision: Li-Hsuan Chiu, Yu-Hui Tsai, Chun S. Zuo, Kuan-Chou Chen, Chun Austin
Changou, Wen-Fu T. Lai.
Validation: Kuo-Hwa Wang, Yung-Ming Wang, Li-Hsuan Chiu, Tze-Chien Chen, Yu-Hui
Tsai, Chun S. Zuo, Chun Austin Changou, Wen-Fu T. Lai.
Visualization: Chun S. Zuo, Kuan-Chou Chen, Wen-Fu T. Lai.
Writing ± original draft: Kuo-Hwa Wang.
Writing ± review & editing: Wen-Fu T. Lai.
15 / 17
progression from benign to advanced ovarian cancer. Clin Cancer Res. 2001; 7(8):2396±404. PMID:
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