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. 2018 Feb 1;13(2):e0192047.
doi: 10.1371/journal.pone.0192047. eCollection 2018.

Optical Imaging of Ovarian Cancer Using a Matrix metalloproteinase-3-sensitive Near-Infrared Fluorescent Probe

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Free PMC article

Optical Imaging of Ovarian Cancer Using a Matrix metalloproteinase-3-sensitive Near-Infrared Fluorescent Probe

Kuo-Hwa Wang et al. PLoS One. .
Free PMC article

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Abstract

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 MMP-3 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
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 orange-yellow 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 fluorescence.
Fig 2
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.
Fig 3
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.
Fig 4
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.
Fig 5
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.
Fig 6
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 MMP-3-sensitive probe. MMP, matrix metalloproteinase; NIRF, near-infrared fluorescence.

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Grant support

This study was funded by the Ministry of Science and Technology, Taiwan (MOST-103-2314-B-038-053 and 104-2917-I-564-006).
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