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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. (Cancer Sci 2011; 102: 1897–1903)
Cancer is a major cause of mortality and morbidity in the world and its incidence continues to increase. Although conventional treatment options have advanced significantly, they remain far from optimal due to ineffective diagnosis. Accordingly, effective methods for detecting malignant tumors in their early stages are needed for successful therapy.(1,2)
Matrix metalloproteinases (MMP) are a family of zinc-binding, calcium-dependent proteolytic enzymes that can remodel the extracellular matrix (ECM) as well as cleave a number of cell surface modulators associated with several pathological conditions.(3–6) In humans, the MMP family includes 16 secreted MMP and seven membrane-associated MMP (MT-MMP).(7) Among the MMP, MT1-MMP (MMP-14) not only promotes tumor growth and ECM proteolysis, but also activates pro-MMP-2 and pro-MMP-13 on the cell surface, especially at lamellipodia, the migration front of cells.(8) Therefore, MT1-MMP has a close relationship with tumor malignancy and holds great promise as an early biomarker for malignant tumors.(9,10)
Currently, optical imaging techniques using target-specific probes are of great interest.(11–16) As such, we aimed to develop a MT1-MMP-specific activatable probe that couples the fluorescent label rhodamine X (ROX) to an anti-MT1-MMP monoclonal antibody (anti-MT1-MMP mAb-ROX). This probe can be obtained in a self-quenched form, and on specific interaction with MT1-MMP expressed on the surface of malignant cells the probe is internalized by the cell and is subsequently degraded by the lysosome,(17) which dequenches the probe fluorescence allowing optical signal emission.(17) This sequence follows a mechanism similar to that in a report describing the development of ROX-labeled avidin for use in optical imaging of a wide range of tumors.(11) Thus, anti-MT1-MMP mAb-ROX might permit estimation of tumor malignancy.
In the present study, we prepared anti-MT1-MMP mAb-ROX and evaluated its effectiveness as a specific imaging probe for MT1-MMP-expressing tumors by characterizing cellular uptake and probe biodistribution in C6-implanted mice.
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In the present study, we developed an activatable-type fluorescence probe, anti-MT1-MMP mAb-ROX. Anti-MT1-MMP mAb-ROX was dramatically activated under antibody-degeneration conditions (Fig. 2), and in stained C6 cells (Fig. 4). Activation of anti-MT1-MMP mAb-ROX was not seen in the negative control MCF-7 cells (Fig. 5). Changes in fluorescence could be blocked by pretreating C6 cells with an endocytosis inhibitor (Fig. 6). These results serve as proof of concept for the development of fluorescence probes having a monoclonal antibody core structure and having the specificity, selectivity and high sensitivity necessary to target molecules. In addition, high T/M and T/B ratios were observed in tumor-bearing mice expressing MT1-MMP (Table 1), suggesting that our probe would be clinically useful for in vivo imaging of MT1-MMP expressed in malignant tumors.
We used ROX as the fluorophore for the MT1-MMP specifically activatable fluorescence probe. Rhodamine derivatives bound to proteins have been reported to form H-dimers easily at low concentrations,(21) which might cause self-quenching, and fluorescence activation can be triggered on unfolding and degeneration of the rhodamine-conjugated protein. Because MT1-MMP expressed at the cell surface is known to be internalized and delivered to the lysosome for degradation,(17) this strategy would be suitable for MT1-MMP-specific activatable fluorescence imaging.
A variety of malignant tumors such as breast cancer, glioma and fibrosarcoma are reported to express MT1-MMP.(22–24) In the present study, we used C6 glioma cells that express MT1-MMP at high levels(25) and MCF-7 human breast adenocarcinoma cells that show low MT1-MMP expression as model cell lines for evaluating the efficacy of our probe. Preliminary in vitro studies using the cell lines MDA-MB-231 and HT1080 derived from human blastoma and human fibrosarcoma, respectively, indicate that this probe will also be suitable for these types of tumors (data not shown). The fluorescence in C6 cells treated with anti-MT1-MMP mAb-ROX gradually increased with time and was significantly observed from 3 h post-incubation. This time-course seems rather long compared with radiolabeled anti-MT1-MMP mAb in our previous study.(26) However, considering that MT1-MMP expressed on the cell surface is reportedly internalized to the endosome and eventually recycled to the cell surface within 1 h,(27) it would be expected that anti-MT1-MMP mAb-ROX would require additional time to emit a fluorescent signal following its degeneration in cells.
To further verify the fluorescence emission mechanism, we next planned an in vitro inhibition study using Col I, which has been shown to be an inhibitor of dynamin-dependent MT1-MMP internalization.(20,28,29) Pretreatment of cells with Col I significantly depressed the fluorescence intensity of the probe as expected. The moderate fluorescent signal observed in cells pretreated with Col I might be due to other internalization processes, such as clathrin-independent mechanisms involving caveolae, which also process MT1-MMP in addition to dynamin-dependent internalization in tumor cells.(20,29,30) Considering these aspects, MT1-MMP imaging in the present study was indeed achieved by activation after MT1-MMP-dependent internalization.
In C6 glioma xenografted mice, the T/M ratios increased with time after administration of anti-MT1-MMP mAb-ROX and the ratios were significantly higher than those of the NC Ab-ROX-treated group (P < 0.01 estimated by two-way factorial anova), indicating the effectiveness of anti-MT1-MMP mAb-ROX for detecting tumors. Furthermore, in MCF-7 xenografted mice there was no difference between the T/M ratios of the anti-MT1-MMP mAb-ROX-treated group and those of the NC Ab-ROX-treated group. This result would support that anti-MT1-MMP mAb-ROX can specifically visualize MT1-MMP-expressing tumors. In addition, the T/B ratio of mice administered anti-MT1-MMP mAb-ROX was also significantly higher than that of the NC Ab-ROX group at 48 h after administration, although the fluorescent signals in blood would be diminished by endogenous fluorophores such as hemoglobin.(31) The high T/B ratio of anti-MT1-MMP mAb-ROX might again support the strategy of the probe. In contrast, NC Ab-ROX showed moderate accumulation of fluorescence in tumors, which we also observed in our previous study using radiolabelled MT1-MMP mAb.(26) While this result could be due to the enhanced permeability and retention (EPR) effect,(32) the contrast of T/B ratios between anti-MT1-MMP mAb and NC Ab nevertheless was improved for the activatable fluorescence probe compared with the RI probe (approximately 4 at anti-MT1-MMP mAb-ROX/NC Ab-ROX versus approximately 1.5 at 99mTc-anti-MT1-MMP mAb/99mTc-NC Ab(26)). This discrepancy between optical and RI probes could be derived from differences in signal controllability in vivo. In other words, nuclear imaging probes cannot be signal-activatable type for in vivo usage, unlike the optical imaging probes prepared in this paper. Although macromolecular probes without any targeting moieties, such as antibodies, liposomes and micelles, accumulate in tumors by the EPR effect; the accumulation site is at first in interstitial spaces in the tumors, followed by slow non-specific internalization into the tumor cells.(33) As the macromolecule probes with targeting moieties attach to the tumor cells for active entry, the quantity and rate for the accumulation inside the tumor cells might increase naturally. Importantly, in the case of signal-activatable type optical probes like anti-MT1-MMP mAb-ROX, there is a lower signal (ideally negligible signals) in the interstitial space because the probes emit optical signals only after internalization to the tumor cells. In contrast, a relatively higher signal in the interstitial space is inevitable in the case of radioactive antibody probes. Taken together, the improvement of the contrast between anti-MT1-MMP mAb and NC Ab-ROX compared with the RI probe contributes to the signal-activatable ability of our probe. Therefore, anti-MT1-MMP mAb-ROX would be superior for MT1-MMP-expressing tumor imaging applications.
Several MT1-MMP imaging probes have been developed for use with single photon emission computed tomography (SPECT) and fluorescence imaging.(34–37) These probes used MT1-MMP substrate as a basic structure, and showed good imaging in vitro. However, some of these substrates were also recognized by other MMP subtypes such as MMP-2, because the amino acid sequences of these substrates are not completely specific to MT1-MMP, and as such would produce non-specific signals. In contrast, the anti-MT1-MMP monoclonal antibody we selected for the basic structure in the present study possesses both high specificity(38) and affinity (sub-nanomolar Kd value in our preliminary data) for MT1-MMP, providing strict imaging of malignant tumors expressing MT1-MMP.
However, the fluorescence wavelength of our probe was too short for detection from outside the body because light in the near-infrared (NIR) region (700–1000 nm) is appropriate with respect to tissue permeability.(31,39,40) Provided that a NIR fluorophore is introduced to the probe to satisfy the strategy for MT1-MMP-specific activation, this could be a useful alternative for in vivo optical imaging in the future. Fluorescence endoscopy imaging technology has been rapidly advancing and is expected to provide a navigation system for surgery.(41,42) For endoscopy, fluorescence probes emitting light in the visible region are suitable because most targets exist at shallow tissue depths and require visibility rather than permeability of fluorescence emitted from a probe. Like a variety of probes,(9,14,15,21) the anti-MT1-MMP mAb-ROX (fluorescence around 600 nm) that we developed could thus also be an appropriate choice for diagnosing tumor malignancy using fluorescence endoscopic technology.
In conclusion, we synthesized an anti-MT1-MMP mAb-ROX that specifically interacts with MT1-MMP expressed on tumor cells and is internalized by these cells and its fluorescence dequenched after antibody unfolding and degeneration. The results indicate that anti-MT1-MMP mAb-ROX would be a useful probe for detecting MT1-MMP-expressing tumors.