Tumor Imaging Using Radiolabeled Matrix Metalloproteinase-Activated Anthrax Proteins.

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. <b>Methods:</b> 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-WT^K563C, or ¹¹¹In-LF^E687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. <b>Results:</b> 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-LF^E687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LF^E687A 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-LF^E687A 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). <b>Conclusion:</b> Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.