In vivo optical imaging of membrane-type matrix metalloproteinase (MT-MMP) activity

Abstract

Herein we demonstrate for the first time that a fluorogenic probe can be used as an in vivo imaging agent for visualizing activities of membrane-tethered, membrane-type matrix metalloproteinases (MT-MMPs). An MT-MMP fluorogenic probe that consisted of an MT1-MMP (MMP-14) substrate and near-infrared (NIR) dye-quencher pair exhibited rapid, efficient boosts in fluorescence upon cleavage by MT1-MMP in tumor-bearing mice. In particular, unlike similar fluorogenic probes designed to target extracellular, soluble-type MMPs (EC-MMPs)--which can be cleared from the bloodstream after activation--the fluorescence signals activated by MT1-MMP enable clear visualization of MT1-MMP-positive tumors in animal models for up to 24 h. The results indicate that a simple form of a fluorogenic probe that is less effective in EC-MMP imaging is an effective probe for imaging MT-MMP activities in vivo. These findings can be widely applied to designing probes and to applications targeting various membrane-anchored proteases in vivo.

Figure 1

(A) Chemical structure of MT-MMP-specific fluorogenic probe, MT-P. The cleavage site is between Phe and Leu. (B) HPLC and UV-VIS (inset) spectra of MT-P. (C) LC/MS spectrum of MT-P.

Figure 2

Recovery of fluorescence of MT-P in the presence of various activated MMPs. (A) Fluorescence emission kinetic spectra of MT-P in the presence of various concentrations of activated MT1-MMP (0, 4, 20 and 40 nM) and MT1-MMP (40 nM) with a broad spectrum MMP inhibitor (MMP-I) following a 60 min incubation at 37 °C. (B) Fluorescence activation of MT-P in a solution containing various activated MMPs following a 60 min incubation at 37 °C. Means ± SD. (n = 3). ^*P < 0.05 for MT-MMPs relative to EC-MMPs.

Figure 3

(A) Representative Western blot analyses of MDA-MB-435 cell lysates for MT1-MMP (64 kDa). (B-D) Fluorescent immunostaining of MT1-MMP expression in an MDA-MB-435 tumor section counterstained with (B) DAPI (blue, marks the nucleus), (C) primary antibody for MT1-MMP and (D) their merged images.

Figure 4

Whole-mouse optical imaging. (A) Representative serial in vivo NIR fluorescence images of MT1-MMP-positive MDA-MB-435 tumor-bearing mice injected intravenously with MT-P under the condition without and with MMP inhibitor (MMP-I). Images were acquired at the indicated time points and were normalized by the maximum average value. The color bar indicates radiant efficiency (low, 0; high, 0.139×10⁶). Arrows indicate tumors. (B) Tumor ROI T/M ratio (the ratio of the signal in the ROI of the tumor compared to the muscle region) analysis of MDA-MB-435 tumor in vivo. Means ± SD. (n = 3–6 per group). ^*P < 0.05.

Figure 5

(A) Representative images of dissected organs and tissues of MDA-MB-435 tumor–bearing mice sacrificed at 24 hours after intravenous injection of MT-P without (left) and with (right) MMPI. The color bar indicates radiant efficiency (low, 0; high, 0.133×10⁶). (B) Biodistribution of MT-P at 24 h post-injection. (C) Tumor ROI T/M analysis of fluorescence intensity in excised tumor. Means ± SD. (n = 3–6 per group). ^*P < 0.05.

Figure 6

NIR fluorescence images of tumor sections injected with MT-P (red) without and with MMP inhibitor. Tumor sections were counterstained with DAPI (blue). Scale bar, 10 μm.