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

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.

Establishment and Characterization of FusionRed Stable Transfected Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cells.

BACKGROUND/AIM: Cancer cell inoculation is routinely used to evaluate novel therapeutic approaches in vivo. However, without reporter genes enabling deep tissue imaging, study of early tumor progression and therapeutic responses is often limited. We describe the establishment and characterization of two canine cancer cell lines stably expressing red fluorescence proteins as tools for later in vivo imaging. MATERIALS AND METHODS: Two red fluorescence cell lines were generated by plasmid transfection. Fluorescence protein expression was confirmed by flow cytometry and microscopy. Deep tissue imaging was demonstrated in mice using a NightOWL LB 983. Gene expression changes after transfection were analyzed by RNAseq. RESULTS: Both cell lines were detectable in vivo by subcutaneous injection of 1×106 cells. RNAseq revealed up to 2005 transfection-induced differentially expressed genes but no significant changes in cellular key pathways. CONCLUSION: The fluorescent cell lines provide a solid basis for future in vivo studies on canine cancer.

Publisher Correction: Functionalization of gold-nanoparticles by the Clostridium perfringens enterotoxin C-terminus for tumor cell ablation using the gold nanoparticle-mediated laser perforation technique.

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Functionalization of gold-nanoparticles by the Clostridium perfringens enterotoxin C-terminus for tumor cell ablation using the gold nanoparticle-mediated laser perforation technique.

A recombinant produced C-terminus of the C. perfringens enterotoxin (C-CPE) was conjugated to gold nanoparticles (AuNPs) to produce a C-CPE-AuNP complex (C-CPE-AuNP). By binding to claudins, the C- CPE should allow to target the AuNPs onto the claudin expressing tumor cells for a subsequent cell killing by application of the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. Using qPCR and immunocytochemistry, we identified the human Caco-2, MCF-7 and OE-33 as well as the canine TiHoDMglCarc1305 as tumor cells expressing claudin-3, -4 and -7. Transepithelial electrical resistance (TEER) measurements of Caco-2 cell monolayer showed that the recombinant C-CPE bound to the claudins. GNOME-LP at a laser fluence of 60 mJ/cm2 and a scanning speed of 0.5 cm/s specifically eliminated more than 75% of claudin expressing human and canine cells treated with C-CPE-AuNP. The same laser fluence did not affect the cells when non-functionalized AuNPs were used. Furthermore, most of the claudin non-expressing cells treated with C-CPE-AuNP were not killed by GNOME-LP. Additionally, application of C-CPE-AuNP to spheroids formed by MCF-7 and OE-33 cells grown in Matrigel reduced spheroid area. The results demonstrate that specific ablation of claudin expressing tumor cells is efficiently increased by activated C-CPE functionalized AuNPs using optical methods.