Abstract
Overexpression of tight-junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for claudin-4 and holds potential as a targeting vector for molecular imaging of claudin-4 overexpression. A glutathione S-transferases (GST)-tagged version of the C terminus of CPE (cCPE) was previously used to delineate claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194-319 mutants with putatively improved binding affinity for claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radiolabeling with 111In. The binding affinity of all radioconjugates was evaluated in claudin-4-expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to claudin-4 was assessed in HT1080 cells stably transfected with claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the claudin-4-targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5, 10 ± 0.9, and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194-319, as measured by SPECT, was considerably faster than that of [111In]In-cCPE.GST (half-life, <1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([111In]In-cCPE194-319, 2.7 ± 0.8 vs. 0.4 ± 0.1 percentage injected dose per gram [%ID/g], P < 0.001; [111In]In-S313A, 2.3 ± 0.9 vs. 0.5 ± 0.1 %ID/g, P < 0.01; [111In]In-S307A + N309A + S313A, 2 ± 0.4 vs. 0.3 ± 0.1 %ID/g, P < 0.01; [111In]In-D284A, 2 ± 0.2 vs. 0.7 ± 0.1 %ID/g, P < 0.05; [111In]In-L254F + K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P < 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.
Highlights
L254F 1 K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P, 0.001)
The latter served as a negative control in this study. Because of their fast growth rate and ability to establish robust and reproducible subcutaneous tumor xenografts in mice, PSN-1 cells were selected among all claudin-4– expressing Pancreatic ductal adenocarcinoma (PDAC) cell lines for further experiments [28]
Claudin-4 protein was found to be localized at the cell–cell contacts and associated with intracellular vesicles in confluent PSN-1 cells but not in HT1080 cells
Summary
L254F 1 K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P , 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4–targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second-leading cause of cancer-related death within the decade, with current 5-y survival rates reaching less than 5% This poor prognosis is partly due to late diagnosis of this mostly nonsymptomatic disease at the metastatic stage, and mostly in the emergency medicine setting [1,2]. Radionuclide-based imaging agents for SPECT and PET are exquisitely sensitive tools, capable of detecting molecular markers associated with malignant tissue in vivo. The use of these targeted molecular probes has the potential to greatly improve the diagnosis of pancreatic cancer by enabling the detection of neoplastic transformation and providing functional and prognostic information about the disease. We generated a series of smaller mutant cCPE-based radiotracers with the aim of improving the pharmacokinetics and claudin-4–targeting ability of wild-type cCPE and evaluated these using in vitro and in vivo mouse models of PDAC
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