Abstract
Background: Claudin (CLDN) proteins have been described to be found and accordingly targeted to evaluate novel therapeutic approaches. C-terminus of Clostridium perfringens enterotoxin (C-CPE) binds efficiently several claudins and thus recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for cancer cell targeting using gold nanoparticle- mediated laser perforation (GNOME-LP). Cancer cells inoculation is routinely used to generate in vivo models to evaluate novel therapeutic approaches in prostate cancer. However, detailed characterization of cancer spreading and early tumor development and therapeutic response is often limited as conventional cell lines do not allow advanced deep tissue imaging.Methods: two canine prostate cancer cell lines were stably transfected with red fluorescent protein (RFP), followed by G418 selection. RFP marker as well as CLDN3, -4 and -7 expression was comparatively confirmed by flow cytometry, qPCR and immunofluorescences. For cancer cells targeting, GNOME-LP at a laser fluence of 72 mJ/cm² and a scanning speed of 0.5 cm/s was used. Statistical analysis was performed using SAS software 7.1, Dunnett´s Multiple Comparison Test and Student´s two-sided t-test. Differences were considered statistically significant for p<0.05.Results: we established two canine prostate carcinoma cell lines, stably expressing RFP allowing perspective deep tissue imaging. Directed C-CPE-AuNP binding to native and RFP transfected cells verified the capability to specifically target CLDN receptors. Cancer cell ablation was demonstrated in vitro setting using a combination of gold nanoparticle mediated laser perforation and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10 % depending on cell line. Conclusion: the results confirm that this therapeutic approach can be used efficiently to target prostate carcinoma cells carrying a marker protein allowing deep tissue imaging. The established cell lines and the verified proof of concept in vitro study provide the basis for perspective Xenograft model in vivo studies. The introduce red fluorescence enables deep tissue imaging in living animals and therefore detailed characterization of tumor growth and subsequently possible tumor ablation through C-CPE-AuNPs treatment.
Ultrabright red AIEgens for two-photon vascular imaging with high resolution and deep penetration
