Radiolabelled Bacillus anthracis toxin-based probes for molecular imaging of MMP activity in tumour models

Abstract

Increased activity of matrix metalloproteinases (MMPs) is associated with poor prognosis and metastasis in different cancer types. To exploit this feature and target cancer cells, the protective antigen (PAWT) of the binary anthrax lethal toxin (LT) was modified to form pores in cell membranes only when cleaved by MMPs (PAL1). Anthrax lethal factor (LF) is then able to translocate through these pores into the cytosol of tumour cells. The aim of this work was to develop a novel non-invasive imaging agent for the detection of matrix metalloproteinase (MMP) activity based on engineered variants of LT. Here, 111In- radiolabelled form of LF was used with the PAL1/LF system to allow non-invasive in vivo imaging and quantification of MMP activity in tumour tissue by SPECT. Initially, PAL1 MMP-activation was tested in a cell free assay. Subsequently, the activation of PAL1 was evaluated by a cytotoxic assay in a range of cancer cell lines, with or without a broad spectrum MMP inhibitor. MMP2 and MMP9 activity in the different cancer cells was assessed by gelatin zymography. Expression levels of MMP14, and LT native receptors (TEM8 and CMG2) were evaluated by western blot analysis. LT components were radiolabelled with 111In. Cell abundance of TEM8 and CMG2 was determined by saturation binding assays using 111In-radiolabelled PAWT. After 111In-radiolabelling non-toxic variants of LF, their cell uptake was evaluated in the presence or not of PAL1. Evaluation of the in vivo pharmacokinetics and biodistribution of 111In-radiolabelled LT components was performed by SPECT/CT in naive mice or MMP-expressing MDA-MB-231 tumour-bearing mice. PAL1 MMP-cleavage was confirmed in a cell free assay. Notably, PAL1 capacity to cause cytotoxicity in a panel of cancer cells correlated with anthrax toxin receptor and MMP14 expression. Additionally, in the presence of MMP inhibitors PAL1 mediated cytotoxicity was prevented. Selective delivery of 111In-radiolabelled LF variants to MMP-expressing cells by PAL1 was demonstrated in vitro, and corroborated using confocal microscopy with fluorescently labelled variants of LF. Dynamic SPECT imaging demonstrated superior and selective uptake of radiolabelled PAL1 in tumour tissue when compared to radiolabelled PAWT. Finally, a radiolabelled LF variant was selectively delivered to MMP-positive MDA-MB-231 tumour bearing mice by PAL1, presenting excellent target-to-background contrast. Taken together, these results indicate that radiolabelled forms of mutated anthrax lethal toxin hold promise for non-invasive imaging of MMP activity in tumour tissue.</p