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Matrix metalloproteinases (MMPs) are endopeptidases responsible for degrading the extracellular matrix (ECM) and remodeling tissue in both physiological and pathological processes.MMP2 and membrane-type 1 MMP (MT1-MMP) have been associated with tumor invasion, metastasis and angiogenesis.Purpose: Establish a novel molecular imaging strategy assessing the proteolytic activity of both MMP2 and MTI-MMP positive cells to predict the malignancy of tumors.Methods and results: 1.Construction of an optical imaging probe to tag MMP2-and MT1-MMP-positive cells.A protein-based probe composed of a glutathione-S-transferase (GST)-tag (Inhibitory [I]-domain), a polypeptide as a specific substrate for both MMP2 and MT1-MMP (Cleaved [C]-domain), a transmembrane domain of the epidermal growth factor receptor (Transmembrane [TM]-domain), and DsRed2 (Fluorescent [F]-domain) was constructed by gene recombination.2.MMP-dependent tagging of cellular membrane: The human flbrosarcoma cells (HT1080) were treated with the probe in the presence or absence of GM6001 (pharmacological MMP inhibitor) and then observed under a fluorescence microscope.The results revealed intense fluorescence on the surface of the cells, which GM6001 dramatically suppressed.On the other hand, the mutant imaging probe did not emit strong fluorescence regardless of the presence or absence of GM6001.All of the in vitro data clearly indicate that the probe has the ability to specifically tag the cellular membranes of MMP2 and MT1-MMP-positive cells.3.Optical imaging of MMP2 and MT1-MMP activity in tumor xenografts: Subcutaneous HT1080 tumor bearing mice were intravenously injected with the protein-based probe, and subjected to optical imaging.Just after the probes administration, fluorescent signal was detected throughout the body.The fluorescence from the entire body dramatically decreased thereafter; however, it remained in the tumor xenografts 24 h after the probes administration.The western blotting results showed that the full-length probe was predominantly detected in the xenograft just after the probes administration.On the other hand, it was not detected in the xenograft 24 h later; instead, a cleaved product, TM-F-domain, was detected.Moreover, when the mutant probe was administered, the fluorescent signal did not accumulate in the tumor xenograft 24 h after administration.All of these results clearly demonstrate that the probe was activated through proteolytic cleavage by intratumoral MMPs, and only the active form (TM-F-domain) remained and emitted fluorescence in the xenografts.4.Quantitative analysis of intratumor MMP2 and MT 1-MMP activity using the probe.MT1-MMP deficient cells were stably transfected with either the MT1-MMP-expressing plasmid or its empty vector, and transplanted the stable transfectants into the right hind leg of immunodeficient mice to prepare MT1-MMP-positive and-negative xenografts.The tumor-bearing mice were intravenously injected with the protein-based probe, and subjected to optical imaging.The imaging experiment clearly demonstrated that the probe emitted stronger fluorescence in MT1-MMP-positive xenografts 24 h after its administration than did its counterpart, indicated that the probe was activated and emitted fluorescence in response to intratumor MMP2 and MT1-MMP activity.5.Optical imaging of invasive fronts of a tumor xenograft with the probe.The human cervical epithelial adenocarcinoma cells (HeLa) were intradermally transplanted into skin flap of nude mouse, and the tumor-bearing mouse was injected with the protein-based imaging probe.Red fluorescence from the xenograft was observed with OV-100 optical imaging system in real time.The red fluorescence in invasive fronts of the tumor xenograft was detected.This result directly shows that the probe actually sense invasive tumors in vivo.Moreover, the fluorescence was detected near tumor blood vessels, too.This result further confirms the characteristic of the probe to sense MMPs activity, because it is known that tumor angiogenesis accompanies with high MMPs activity.Conclusions: All of these results indicate that the release of the I-C-domain through the proteolytic cleavage of the C-domain by MMP2 and MT1-MMP triggers the tagging of cellular membranes with the TM-F-domain.The present feasibility study opens the door to the development of a novel imaging probe for tumor malignancy using positron emission tomography as well as an optical imaging device.