A novel strategy to tag matrix metalloproteinases-positive cells for in vivo imaging of invasive and metastatic activity of tumor cells

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Abstract

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; therefore, a molecular imaging strategy assessing their activity may help to 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. We constructed 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). In vitro experiments 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. Optical imaging experiments in vivo demonstrated that the probe was cleaved and specifically remained in tumor xenografts in a MMP-dependent manner. 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.

Graphic abstract

We established a novel strategy to tag the surface of matrix metalloproteinase-positive cells and developed an optical imaging probe to visualize the invasive/metastatic ability of malignant tumor.

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Introduction

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 [1], [2], [3], [4]. Among MMPs, MMP2 (also known as gelatinase A) and membrane-type 1 MMP (MT1-MMP) have been especially associated with tumor invasion and metastasis [4], [5]. 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 [4], [5], [6]. Therefore, a molecular imaging strategy assessing the proteolytic activity of both MMP2 and MT1-MMP would help to non-invasively predict tumor malignancies.

A series of optical imaging probes, which are specifically activated by intratumor MMPs, have been devised over the last decade [7], [8], [9]. The typical components of such activatable “smart probes” are a signal domain responsible for fluorescent emission, a specific quencher for the fluorescence, and a substrate polypeptide for MMPs. Release of the quencher as the result of proteolytic cleavage of the substrate polypeptide by MMPs is responsible for the fluorescence emitted from the probe. However, this kind of smart probe still has room for improvement. Because the activated probe is free from MMP-positive cancer cells, the fluorescent signal should diffuse to neighboring cells and be rapidly excluded from MMP-positive regions.

Very recently, the MMPs-dependent activatable strategy was applied to the development of a novel type of optical imaging probe with a polymeric nanoparticle platform [10]. The probe consists of strongly quenched MMPs-specific near infrared fluorogenic peptides on the surface of tumor-homing polymeric nanoparticle as a carrier. The probe showed high enough target-to-background ratios and high enough stability in physiological conditions; however, there still remained a concern about its capability for the detection of invasive cancer cells which locate in invasive fronts far from tumor blood vessels, because in vivo delivery of the probe is fully dependent on the enhanced permeability and retention (EPR) effect.

The development of an imaging probe for positron emission tomography using specific inhibitors of MMPs [11], [12], [13], [14], [15], [16] has had some success in terms of detecting the existence of MMPs, however, the activity of MMPs could not be sensed.

To overcome these problems, imaging probes which specifically sense the activity of MMPs and tag MMP-positive cells are required to accurately predict the malignancy of tumors. In the present study, we established a novel strategy, through which the tagging of malignant cancer cells is triggered by MMP2 and MT1-MMP activity. We applied this method to the development of an optical imaging probe for intratumor MMP activity.

Section snippets

Plasmid DNA

To construct the plasmid pEF6/MT1-MMP-FLAG, a DNA fragment encoding MT1-MMP-FLAG-tag was obtained by digesting pSG-MT1-F with both EcoRI and BamHI, and inserted between the corresponding sites of pEF6/myc-His (Invitrogen Corp.). The pSG-MT1-F plasmid was a gift from Dr. Motoharu Seiki (University of Tokyo) [17]. To construct the plasmid expressing the protein-based imaging probe, a DNA fragment encoding DsRed2 was first inserted between the EcoRI and XhoI sites of pGEX-6P-3 (GE Healthcare UK

Construction of an optical imaging probe to tag MMP2- and MT1-MMP-positive cells

We first designed a novel protein-based optical imaging probe not only to sense the MMP2 and MT1-MMP activity of cancer cells but also to specifically tag the surface of the target cells (Fig. 1A). The probe is composed of four domains: 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

Discussion

In the present study, we established a method to specifically tag MMP2- and MT1-MMP-positive cells with a fluorescent probe. Employing the strategy, we successfully developed an optical imaging probe to visualize and quantify MMP activity in solid tumors.

Based on our experiments in vitro and in vivo, it is obvious that the C-domain is responsible for the MMP2 and MT1-MMP-dependent activation of the probe. However, we have not yet clarified why the membrane-penetrating activity of the internal

Conclusions

We established a novel method to specifically tag the surface of MMP2- and MT1-MMP-positive cells with an arbitrary protein in vivo. Applying it, we successfully developed a protein-based optical imaging probe to visualize and quantify the malignant phenotype of solid tumors. This strategy is applicable to the delivery of anti-cancer drugs specifically to malignant tumors.

Acknowledgements

This study was supported by the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO), Japan, and by Grants-in-aid for Scientific Research on Priority Areas (Cancer Research) and for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was part of the program “R&D of Molecular Imaging Equipment for Malignant Tumor Therapy Support” supported by New Energy and

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