Tumor Imaging Using Radiolabeled Matrix Metalloproteinase-Activated Anthrax Proteins

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-WT^K563C, or ¹¹¹In-LF^E687A (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-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). 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.

Keywords: MMP; SPECT; anthrax lethal toxin; cancer; pretargeting.

FIGURE 1.

Schematic overview of MMP-activated pretargeting of cancer cells using PA-L1/LF system: binding of PA-L1 to anthrax receptors (1), cleavage and activation of PA-L1 by MMPs (2), formation of prepore (3), binding of ¹¹¹In-LF^E687A to PA prepore and formation of PA pore (4), and endocytosis and delivery of ¹¹¹In-LF^E687A to cytoplasm (5).

FIGURE 2.

In vitro characterization of panel of cancer cell lines for protease and anthrax toxin receptor expression. (A) Western blots show differential MMP14, CMG2, and furin expression in whole-cell lysates. (B) MMP2 activity was measured in HT1080 cell lysates, using gelatin zymography. Broad-spectrum inhibitor GM6001 inhibited MMP2 activity. (C and D) Furin-cleavable PA-WT and MMP-cleavable PA-L1 delivered cytotoxic fusion toxin FP59 to panel of cancer cells, with variable efficiency. Controls are defined as MTT signal derived from vehicle-treated cells. *P < 0.05. ***P < 0.001.****P < 0.0001. (E) Comparison of results obtained in panels A–D.

FIGURE 3.

Cy3-LF^E687E was delivered to HT1080, but not MCF7, by PA-L1. Images were acquired after fixation and mounted with 4′,6-diamidino-2-phenylindole (DAPI) to highlight nucleus.

FIGURE 4.

In vitro cell uptake of ¹¹¹In-PA-L1, ¹¹¹In-PA-WT^K563C, and ¹¹¹In-LF^E687A. (A) Saturation binding assays using ¹¹¹In-PA-WT^K563C determined affinity for and amount of PA binding sites on cells. (B) Binding of ¹¹¹In-PA-WT^K563C to cells was not significantly different from ¹¹¹In-PA-WT. (C) Uptake of ¹¹¹In-PA-WT^K563C and ¹¹¹In-PA-L1 in MDA-MB-231 cells could be blocked by 100-fold excess of cold unlabeled PA-WT or PA-L1. (D) PA-L1–mediated uptake of ¹¹¹In-LF^E687A in MDA-MB-231 cells could be significantly reduced by exposure of cells to broad-spectrum MMP inhibitor GM6001. (E) PA-L1 allowed uptake of ¹¹¹In-LF^E687A in MDA-MB-231 cells but not in MCF7 cells. **P < 0.01. ***P < 0.001. ****P < 0.0001.

FIGURE 5.

In vivo evaluation of ¹¹¹In-LFn and ¹¹¹In-LF^E687A in tumor-naïve SCID mice. (A) Blood clearance of ¹¹¹In-LFn (inset: maximum-intensity projections over time). (B) Ex vivo biodistribution showing uptake of ¹¹¹In-LFn in selected tissues and blood, 1 h after intravenous administration. (C) Representative coronal and sagittal sections of SPECT images acquired 1 h after intravenous administration of ¹¹¹In-LFn. (D) Blood clearance of ¹¹¹In-LF^E687A (inset: maximum-intensity projections over time). (E) Ex vivo biodistribution showing uptake of ¹¹¹In-LF^E687A in selected tissues and blood, 3 h after intravenous administration. (F) Representative coronal and sagittal sections of SPECT images acquired 3 h after intravenous administration of ¹¹¹In-LF^E687A. b = bladder; h = heart; k = kidneys; l = liver. Full-sized versions of panels A and D are available in the supplemental materials.

FIGURE 6.

In vivo evaluation of ¹¹¹In-PA-L1 and ¹¹¹In-PA-WT^K563C in MDA-MB-231 tumor-bearing SCID mice. (A) Blood clearance of ¹¹¹In-PA-L1 and ¹¹¹In-PA-WT^K563C. (B) Ex vivo biodistribution showing uptake of ¹¹¹In-PA-L1 or ¹¹¹In-PA-WT^K563C in selected tissues and blood, 3 h after intravenous administration. (C) Tumor-to-blood ratios determined from B. (D and E) Representative coronal and sagittal maximum-intensity projections of SPECT/CT images, 3 h after administration of ¹¹¹In-PA-L1 or ¹¹¹In-PA-WT^K563C. *P < 0.05. **** P < 0.0001. b = bladder; h = heart; k = kidneys; l = liver; t = tumor.

FIGURE 7.

(A) Representative coronal maximum-intensity projections of SPECT images of MDA-MB-231 tumor xenograft–bearing SCID mice, 24 h after administration of ¹¹¹In-LF^E687A, alone or in combination with (left to right) PA-L1, PA-L1 and excess of unlabeled LF^E687A, PA-U7, PA-WT, or no PA protein. Tumors are encircled (B) Ex vivo biodistribution showing tumor uptake of ¹¹¹In-LF^E687A in different experimental groups. b = bladder; h = heart; l = liver; s = spleen; t = tumor. **P < 0.01. ****P < 0.0001.