Tumor Imaging Using Radiolabeled Matrix Metalloproteinase-Activated Anthrax Proteins.
Elvina Xavier M-A., Liu S., Bugge TH., Torres JB., Mosley M., Hopkins SL., Allen PD., Berridge G., Vendrell I., Fischer R., Kersemans V., Smart S., Leppla SH., Cornelissen B.
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 <sup>111</sup>In-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 <sup>111</sup>In-radiolabeled PA-L1, <sup>111</sup>In-PA-WT<sup>K563C</sup>, or <sup>111</sup>In-LF<sup>E687A</sup> (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 <sup>111</sup>In-LF<sup>E687A</sup> was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LF<sup>E687A</sup> Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of <sup>111</sup>In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. <sup>111</sup>In-LF<sup>E687A</sup> 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.