A Novel Strategy To Tag Matrix Metalloproteinases-positive Cells For In Vivo Imaging Of Invasive And Metastatic Activity Of Tumor Cells

Characteristic of malignant tumors is the ability to invade surrounding normal tissue and metastasize to distant sites. These malignant phenotypes are often observed in cancer patients and the main cause of cancer-related deaths. Matrix metalloproteinases (MMPs) have been identified as important factors facilitating these malignant phenotypes by degrading all the main protein components of the extracellular matrix (ECM) and basement membrane. Among MMPs, MMP2 (also known as gelatinase A) and Membrane-type 1 MMP (MT1-MMP) have been especially associated with tumor invasion and metastasis. MMP2 is overexpressed as an inactive zymogen (proMMP2) in various types of cancer, and the proMMP2 is converted to mature MMP2 through proteolytic cleavage by MT1-MMP. Therefore, a molecular imaging strategy assessing the proteolytic activity of both MMP2 and MT1-MMP would help predict the malignancy of tumors.Here, we established a novel method of specifically tagging the surface of MMP2- and MT1-MMP-positive cells, and applied it to the development of an optical imaging probe. The 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).In vitro experiments,HT1080 cells were cultured with the protein-based imaging probe (10μg/mL) in the presence (+) or absence? () of GM6001 (10μg/mL) for 24 h, and observed under a fluorescence microscope and fluorescent intensity was quantified. The results clearly demonstrated that, after the probe was cleaved at the C-domain by the MMPs, the resultant TM-F-domain was inserted into the cellular membrane. The fluorescent intensity was 23.3-fold higher in the absence of GM6001 compared to its counterpart. We additionally confirmed that the probe has little cytotoxicity at the concentration we applied to our in vitro experiment.In vivo experiments,HT1080 tumor-bearing mice were intravenously injected with the protein-based imaging probe and subjected to an in vivo imaging experiment at 0,12,24h after the probe administration. The fluorescence from the entire body dramatically decreased thereafter; however, it remained in the tumor xenografts 24h after the probe's administration. These optical imaging experiments in vivo demonstrated that the probe was cleaved and specifically remained in tumor xenografts in a MMP-dependent manner.MCF7 tumor-bearing mice with the MT1-MMP-positive (+) and negative (?) cells were intravenously injected with 10 nmol of the protein-based imaging probe, and subjected to optical imaging at 24 h after the probe's administration. Fluorescent intensity detected in above experiment was quantified and divided by each tumor volume. 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. The photon counts from the MT1-MMP-positive and negative xenografts were about 83,000±17,000 (photons/mm3) and about 9100±7800 (photons/mm3), respectively. These results strongly indicate that the probe was activated and emitted fluorescence in response to intratumor MMP2 and MT1-MMP activity.To further examine whether the imaging probe can detect an invasive tumor in vivo as we desired. HeLa cells 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. We detected red fluorescence in invasive fronts of the tumor xenograft. 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.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.