Abstract
Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1–mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A. Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.
Highlights
Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that together can degrade most components of the extracellular matrix
We show that this novel pretargeted imaging system is selective for matrix metalloproteinases (MMPs)-expressing cancer cell lines in vitro, and we show selective, noninvasive, in vivo imaging in MMP-expressing tumor xenografts grown in mice
Cleavage of protective antigen (PA)-L1 by MMP2 and MMP14 at the intended site was further confirmed by mass spectrometry (Supplemental Fig. 2)
Summary
Protein Production and Synthesis of Labeled Compounds All components of B. anthracis LT were expressed and purified as previously described [12]. In vitro assays were performed to evaluate the capacity of 111In-LFE687A or 111In-LFn to interact with PA pores and to be delivered into cells after radiolabeling. Dynamic SPECT/CT imaging of tumor-naıve SCID mice was performed after intravenous administration of 111In-LFn (3 mg, 3 MBq; 100 mL) or 111In-LFE687E (10 mg, 10 MBq; 100 mL) through a cannula inserted in the tail vein. To determine the pharmacokinetics of PA-L1 in vivo, dynamic SPECT images were acquired in MDA-MB-231 tumor–bearing mice over 3 h after intravenous injection of 111In-PA-L1 or 111In-PAWTK563C (20 mg, 10 MBq; 100 mL). To determine the PA-L1 selectivity of tumor uptake, and of MMP-mediated uptake, groups of control animals were administered 111In-LFE687A alone or in combination with PA-WT (20 mg; 50 mL), PA-U7 (20 mg; 50 mL), or PA-L1 and an excess of unlabeled LFE687A (1 mg; 100 mL). All results are reported as mean 6 SD of at least 3 independent replicates, unless otherwise indicated
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