Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe

<div><p>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 <i>in vivo</i> 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.</p></div

<i>In vitro</i> MMP expression in SKOV3 and WS1 cell cocultures.

<p>(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. *<i>p</i><0.05, significantly different compared with SKOV3 cell cultures. ELISA, enzyme-linked immunosorbent assay; MMP, matrix metalloproteinase.</p

NIRF imaging of SKOV3 tumor masses <i>in vivo</i>.

<p>(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), *<i>p</i><0.001, significantly different compared with the control tissue. NIRF, near-infrared fluorescence; SI, signal intensity.</p

Histological and NIRF analysis of SKOV3 tumor tissues.

<p>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.</p

Characterization of the novel stromelysine-1 peptide substrate.

<p>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) <i>In vitro</i> 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) <i>In vitro</i> 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.</p

<i>Ex vivo</i> NIRF imaging of SKOV3 tumors.

<p>NIRF images revealed signals in the dissected tumor masses (right) as compared with in the adjacent control tissue (left). NIRF, near-infrared fluorescence.</p