Claudin-4 SPECT Imaging Allows Detection of Aplastic Lesions in a Mouse Model of Breast Cancer

Abstract

The expression of claudin-4, a protein involved in tight junction complexes, is widely dysregulated in epithelial malignancies. Claudin-4 is overexpressed in several premalignant precursor lesions, including those of cancers of the breast, pancreas, and prostate, and is associated with poor survival. A noncytotoxic C-terminal fragment of Clostridium perfringens enterotoxin (cCPE) is a natural ligand for claudin-4. Here, we demonstrate wholebody quantitative SPECT imaging of preneoplastic breast cancer tissue using 111 In-labeled cCPE. Methods: cCPE.GST or GST (GST is glutathione S-transferase) was conjugated to the metal ion chelator benzyl-diethylenetriaminepentaacetic acid to allow 111 In radiolabeling. The affinity of radiolabeled cCPE.GST for claudin-4 was confirmed using claudin-4-expressing MDA-MB-468 and SQ20b cells, compared with claudin-4-negative HT1080 cells. In vivo SPECT imaging was performed using athymic mice bearing MDA-MB-468 or HT1080 xenografts and using genetically modified BALB/neuT mice, which spontaneously develop claudin-4-expressing breast cancer lesions. Results: The uptake of 111 In-cCPE.GST in claudin-4-positive MDA-MB-468 xenograft tumors in athymic mice was significantly higher than in 111 In-GST or claudin-4-negative HT1080 tumors (6.72 ± 0.18 vs. 3.88 ± 1.00 vs. 2.36 ± 1.25 percentage injected dose per gram [%ID/g]; P , 0.0001). No other significant differences were observed in any of the examined organs. BALB/neuT mice, expressing rat neuT under mmtv promotor control, spontaneously developed tumorous lesions within their mammary fat pads over the course of 130 d. Overt mammary tumors were claudin-4-positive, and 111 In-cCPE.GST uptake was 3.2 ± 0.70 %ID/g, significantly higher than 111 In-GST (1.00 ± 0.60 %ID/g; P , 0.05). Mammary fat pads in mice aged 80 d bore claudin-4-positive aplastic lesions and accumulated 111 In-cCPE.GST (3.17 ± 0.51 %ID/g) but not 111 In-GST (0.99 ± 0.39 %ID/g; P , 0.001). Conclusion: Taken together, 111 In-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, and pancreatic cancer.

A cCPE‐based xenon biosensor for magnetic resonance imaging of claudin‐expressing cells

“…However, for noninvasive diagnostic detection, fluorescence‐based imaging is limited by penetration depth and autofluorescence. As an alternative, 111 In radiolabeled cCPE and single‐photon emission computed tomography were used to detect Cldn4 + MDA‐MB‐468 xenograft and other tumors in mice . Here, the radiation exposure has to be considered as a drawback.…”

“…As an alternative, 111 In radiolabeled cCPE and single-photon emission computed tomography were used to detect Cldn4 + MDA-MB-468 xenograft and other tumors in mice. 52 Here, the radiation exposure has to be considered as a drawback.…”

Claudin‐targeted drug development using anti‐claudin monoclonal antibodies to treat hepatitis and cancer

“…Among the 27 family members, CLDN‐3 and ‐4 are the CLDNs most frequently overexpressed in malignant tumors, and their expression levels are well correlated with tumor malignancy . Fluorescence‐conjugated C‐CPE has been used to visualize small, early‐stage lesions that are difficult to identify through conventional diagnostic methods . Therefore, a method for detecting CLDNs in biopsy and cytology samples has received attention as a potential diagnostic tool .…”

“…13,14 Fluorescence-conjugated C-CPE has been used to visualize small, early-stage lesions that are difficult to identify through conventional diagnostic methods. [66][67][68] Therefore, a method for detecting CLDNs in biopsy and cytology samples has received attention as a potential diagnostic tool. 69,70 In this context, antibody 5A5 (which recognizes CLDN-3 and -4, but not CLDNs-1, -2, -5, -6, -7, -9, and -10) preferentially accumulated more than control IgG in tumor tissue (Fig.…”

Dual-Targeting Nanoparticles for In Vivo Delivery of Suicide Genes to Chemotherapy-Resistant Ovarian Cancer Cells

“…Interestingly, although the full-length CPE is highly toxic when injected intravenously in animals, the carboxi-terminal fragment (i.e., the C-terminal 30aa) of CPE, is devoid of any toxicity while sufficient for binding to its receptors(11). Accordingly, several strategies have been developed that used the c-CPE as a tumor-specific carrier for diagnostic and therapeutic agents(12, 13). Importantly, recent data from our research group showed that c-CPE conjugated to the NearInfraRed-Dye CW800 is highly effective in identifying microscopic/metastatic ovarian tumor in the abdomen of mice harboring ovarian cancer xenografts(12, 14).…”