Abstract 4929: Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis

Abstract

Abstract Expression of Claudin-4, a member of the Claudin protein family that is integral in adherens and tight junction complexes, is widely dysregulated in epithelial malignancies and is overexpressed in a number of premalignant precursor lesions, including those of cancers of the breast, pancreas and prostate. Increased Claudin-4 expression in breast cancer is also associated with poor prognosis. Optical imaging of Claudin-4 has been shown previously to be an effective way to detect precancerous lesions of the pancreas, using a fluorescently labelled form of a non-cytotoxic c-terminal fragment of Clostridium perfringens enterotoxin (cCPE, aa184-319), the natural ligand for Claudin-4. Here, we demonstrate whole-body quantitative SPECT imaging of Claudin-4 in preneoplastic breast cancer tissue using 111In-labelled cCPE. Our aim is to improve overall outcome by detection of lesions at an earlier stage. cCPE was produced as a GST-fusion protein from a pGEX plasmid. cCPE.GST or GST was conjugated to the metal ion chelator, benzylDTPA, to allow radiolabelling with 111In. Radiolabeling yield was >95%. Affinity of radiolabelled cCPE-GST for Claudin-4 was confirmed by binding to Claudin-4 expressing MDA-MB-468 and SQ20b cells, compared to Claudin-4 negative HT1080 cells. Radioactivity associated with MDA-MB-468 cells was 24.1±0.9 times higher for 111In-cCPE.GST vs. 111In-GST; P<0.001. Association of 111In-cCPE.GST was 4.7±0.2 times higher with MDA-MB-468 vs. HT1080 cells; P<0.001. 111In-cCPE.GST, but not 111In-GST, was internalised in MDA-MB-468 and SQ20b cells, but not in HT1080 cells. Athymic balb/c mice carrying subcutaneous xenograft tumors of MDA-MB-468 or HT1080 cells were injected intravenously with 5 µg 111In-labelled cCPE.GST or GST (5 MBq). After 24 h, SPECT/CT images were acquired, and the amount of radioactivity in selected organs was determined after dissection. Uptake of 111In-cCPE.GST in Claudin-4 positive MDA-MD-468 tumors was significantly higher compared to 111In-GST or HT1080 tumors (26.14±12.31 vs. 4.84±1.29 vs. 2.36±1.25 percent of the injected dose per gram of tumor tissue (%ID/g); p=0.021). No other significant differences were observed in any of the examined organs. Balb/neuT mice, expressing rat neuT under mmtv promotor control, spontaneously obtain tumorous lesions within their mammary fat pads over the course of 130 days. Balb/neuT mice were imaged monthly using 111In-cCPE.GST or 111In-GST. Mammary fat pads in mice aged around 90 days, bore aplastic lesions which were positive for Claudin-4, as determined by immunohistochemistry, and attracted 111In-cCPE.GST (3-5 %ID/g), but not 111In-GST (<1 %ID/g). In summary, 111In-cCPE.GST targets Claudin-4 expression in frank tumors and preneoplastic tissue, and cCPE imaging may be used as an early detection tool for breast, prostate, pancreas. Citation Format: Michael Mosley, James Knight, Albrecht Neesse, Patrick Michl, Veerle Kersemans, Bart Cornelissen. Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4929. doi:10.1158/1538-7445.AM2014-4929