Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention

Abstract

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Claudin-4: A New Molecular Target for Epithelial Cancer Therapy

“…Furthermore, by conjugating toxins and anticancer drugs to C-CPE, it becomes a carrier that delivers these to cancer cells expressing CLDN4 [ 63 ]. For example, Pseudomonas aeruginosa exotoxin A [ 132 , 133 ], diphtheria toxin fragment A [ 134 ], doxorubicin-loaded polysialic acid nanoparticles [ 135 ], 111 In [ 136 ], TNF [ 137 ], and nanomaterials such as gold nanoparticles [ 138 ] bound to C-CPE induces cell death in CLDN4-expressing cancer cells. However, the immunogenicity and potential toxicity of CPE may limit its clinical application [ 139 ].…”

cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids

“…Another advantage of cCPE fusion proteins is that they can be expanded in a modular manner: (i) with other toxin domains, e.g., for cytotoxic targeting of claudin-overexpressing tumors (full-length CPE variants or cCPE-PSIF fusions) [ 41 , 48 , 76 ], (ii) to generate xenon biosensors for magnetic resonance imaging [ 55 ] or radiolabeled probe-based SPECT imaging allowing detection of claudin-overexpressing cells and tumors [ 77 ], (iii) to engineer functionalized nanoparticles to target prostate adenocarcinoma in in vitro and in vivo models [ 78 , 79 ], or (iv) fluorophore conjugation [ 80 , 81 ] or—as in this study—fluorescent protein fusion for fluorescence-based detection of claudin-expressing cancer cells in vitro, ex vivo and in vivo. Thus, cCPE fusion proteins are flexible and useful additions to the diverse toolbox of claudin binders.…”

Diseases of the Canine Prostate Gland

“…Modern studies investigate molecular targets like tight junction proteins. A recent in vitro approach used prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell lines to investigate whether it is possible to destroy tumor cells by gold-nanoparticle-mediated laser perforation (GNOME-LP [143]), a noninvasive thermotherapy. The gold-nanoparticles (AuNPs) were conjugated to Clostridium perfringens enterotoxin (C-CPE); the latter are known to bind to claudins, which are tight junction proteins frequently expressed in tumors.…”