Abstract
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Objectives Claudin-4, a membrane protein involved in epithelial cell tight junction complexes, is overexpressed in various subtypes of ovarian cancers and its expression could serve as a predictor for cancer prognosis. Clostridium perfringens enterotoxin (CPE) is a small protein that binds to claudin-4 with high affinity. Its C-terminus fragment (CCPE) is adequate for high-affinity binding without causing cytolysis, and therefore has the potential to being an ideal targeting molecule for ovarian cancer detection and prognosis prediction. In this research, we demonstrate the synthesis and characterization of 64Cu labelled CCPE PET probes for ovarian cancer detection.
Methods CCPE with cysteine residue was expressed by E. coli and purified for further experiments. Chelators NODAGA-NCS and NODAGA-Maleimide were conjugated onto CCPE (namely CCPE-NODAGA-NCS and CCPE-NODAGA-Mal) and 64CuCl2 was further utilized for radiolabeling. PET probes were characterized by HPLC analysis and subjected to mouse serum stability assay. The cell uptake and binding affinity tests of CCPE PET probes were conducted using OVCAR3 (claudin-4 positive) and SKOV3 (claudin-4 negative) ovarian cancer cell lines. In vivo small animal PET/CT imaging and ex vivo biodistribution studies to specific organs collected after sacrifice were conducted on ovarian tumor xenograft mice models with (OVCAR3) or without (SKOV3) claudin-4 overexpression, respectively. The mice (n=4 per group) were injected with approximately 7.4 MBq of either 64Cu-CCPE-NODAGA-NCS or 64Cu-CCPE-NODAGA-Mal and subjected to imaging at 1 h, 4 h and 24 h post-injection followed by sacrifice immediately after the last image acquisition. For blocking studies, the probes were co-injected with 250 µg of unlabeled CCPE.
Results CCPE with both NODAGA-NCS and NODAGA-Mal chelators were successfully synthesized. Both probes were labeled with 64Cu in high radiochemical purify (>95%) and demonstrated molecular stability in mouse serum for up to 4 h incubation (>97% was intact probe). In cell uptake assays, both probes exhibited significant higher uptake in claudin-4 positive OVCAR3 cells than in claudin-4 negative SKOV3 cells in a time-course manner. For example, the uptake for 64Cu-CCPE-NODAGA-NCS in OVCAR3 cells and SKOV3 cells were 13.9% and 3.2% (P < 0.05) of applied activity after 2 h incubation, respectively. This high uptake could effectively be blocked by co-incubation with unlabeled CCPE (the OVCAR3 cell uptake was reduced from 13.9% to 4.4% at 2 h incubation, P < 0.05). Interestingly, small animal PET/CT imaging showed that 64Cu-CCPE-NODAGA-NCS had higher tumor accumulation than the 64Cu-CCPE-NODAGA-Mal (1.8 %ID/g vs. 0.5 %ID/g at 1 h post injection, P < 0.05). The liver and spleen uptake at 24 h post-injection were 6.1 %ID/g, 2.4 %ID/g vs. 1.0 %ID/g, 1.0 %ID/g for 64Cu-CCPE-NODAGA-NCS vs. 64Cu-CCPE-NODAGA-Mal, respectively. However, the kidneys uptake data were similar, with 4.3 %ID/g for CCPE-NODAGA-NCS and 4.1 %ID/g for CCPE-NODAGA-Mal at 24 h post injection. Furthermore, uptakes of both PET probes were blocked by coinjection of unlabeled CCPE protein. For example, the 64Cu-CCPE-NODAGA-NCS at 24 h post injection time point had a tumor uptake of 3.4 %ID/g without blocking compared with 1.0 %ID/g with blocking (P < 0.05). Ex vivo biodistribution results also indicated that the probes were accumulated in tumor specifically in moderate levels and metabolized through both kidneys and liver.
Conclusions 64Cu labeled CCPE conjugated to NODAGA-NCS and NODAGA-Mal probes were successfully synthesized and both showed specific uptake for claudin-4 overexpressing ovarian cancer cells. Compared with NODAGA-Mal conjugates, the NODAGA-NCS conjugates exhibited better in vivo metabolism character and imaging contrast. Therefore, 64Cu-CCPE-NODAGA-NCS may demonstrate clinical utility as a novel PET probe for claudin-4 positive ovarian cancer detection and cancer prognosis prediction.