Development of membrane type‐1 matrix metalloproteinase‐specific activatable fluorescent probe for malignant tumor detection

Abstract

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease that activates pro-MMP-2 and pro-MMP13, which are related to tumor malignancy. Therefore, probes that specifically image MT1-MMP would be useful for malignant tumor diagnosis. In the present study, we prepared rhodamine X-conjugated anti-MT1-MMP antibody (anti-MT1-MMP mAb-ROX) as an activatable fluorescent probe and evaluated its usefulness for MT1-MMP-specific imaging. Anti-MT1-MMP mAb-ROX was obtained in a quenched form with approximately three ROX molecules per mAb. Its fluorescence intensity increased approximately 14-fold in the presence of detergent, which is suitable for activatable systems. C6 glioma cells and MCF-7 human breast adenocarcinoma cells were used as MT1-MMP-positive and MT1-MMP-negative models, respectively. The fluorescence intensity of C6 cells treated with anti-MT1-MMP mAb-ROX, but not ROX-conjugated isotype control antibody (NC Ab-ROX), increased with time and was significantly higher than that of MCF-7 cells at 6 h (P < 0.001). The fluorescence intensity of cells treated with anti-MT1-MMP mAb-ROX was also suppressed by pre-treatment with a MT1-MMP endocytosis inhibitor (P < 0.05). Furthermore, the probes were intravenously administered to C6 and MCF-7 xenografted mice. The tumor-to-muscle (T ⁄ M) ratio of the anti-MT1-MMP mAb-ROX group was 15.1 ± 3.2 at 48 h and was significantly higher than that of the NC Ab-ROX group (T ⁄ M ratio = 4.6 ± 3.0, P < 0.05) in C6 xenografted mice, while the T ⁄ M ratio of the anti-MT1-MMP mAb-ROX and NC Ab-ROX groups was not different in MCF-7 xenografted mice. These findings suggest that anti-MT1-MMP mAb-ROX is a promising probe for specifically detecting MT1-MMP-expressing tumors.

Radioiodinated Peptidic Imaging Probes for <i>in Vivo</i> Detection of Membrane Type-1 Matrix Metalloproteinase in Cancers

“…24,25) However, these optical probes were inappropriate for quantitative in vivo analysis of MT1-MMP because optical imaging suffers from photon attenuation due to absorption and dispersion as well as autofluorescence. In this regard, nuclear medical imaging would provide more accurate quantitation; therefore, several protein-based radiolabeled probes for MT1-MMP have been developed.…”

Noninvasive Detection and Imaging of Matrix Metalloproteinases for Cancer Diagnosis

“…The fluorescence intensity of the probe was increased approximately 14-fold in the presence of detergent. It was confirmed that the cellular internalization of MT1-MMP activated anti-MT1-MMP mAb-ROX fluorescence, which allowed the visualization of MT1-MMP expression in C6 glioma cells [41]. However, the fluorescence wavelength of ROX (k ex = 575 nm, k em = 602 nm) was too short for in vivo optical imaging.…”

“…Except for the recognition of MMPs with peptides, another approach of the ''turn-on'' fluorescent probes is realized by MMP-antibody interaction [41,42]. The self-quenching of the probe was caused by fluorophore binding to antibody and fluorescence activation was triggered on unfolding and degeneration of the fluorophore-conjugated antibody.…”

Miniaturized antibodies for imaging membrane type‐1 matrix metalloproteinase in cancers

“…In fact, some fluorogenic probes activated by or binding with MT1-MMP have successfully imaged tumors. (20)(21)(22) However, these probes were insufficient for quantitative analysis of MT1-MMP in vivo. In this regard, radiolabeled probes were expected to achieve more accurate quantitation; therefore, several radiolabeled probes for MT1-MMP have been developed including the 99m Tc-MT1-mAb (9) and 123 I-labelled TIMP-2.…”