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Nagaya et al. Published OnlineFirst June 6, 2017; DOI: 10.1158/1541-7786.MCR-17-0164 Figure 1. Confirmation of PSMA expression as a target for NIR-PIT in PC3 cells, and evaluation of in vitro NIR-PIT. A, Validation of anti-PSMA-IR700 by SDS-PAGE (left, colloidal blue staining; right, fluorescence). Diluted anti-PSMA mAb was used as a control. B, Expression of PSMA in PC3flu and PC3pip-luc cells was evaluated by FACS. After 6 hours of anti-PSMA-IR700 incubation, PC3pip-luc cells showed high fluorescence signal. Fluorescence in PC3pip-luc cells was completely blocked by adding excess anti-PSMA. On the other hand, PC3flu cells showed no fluorescence signal. C, DIC and fluorescence microscopy images of PC3flu and PC3pip-luc cells after incubation with anti-PSMA-IR700 for 6 hours. High fluorescence intensities were shown in only PC3pip-luc cells. Necrotic cell death was observed upon excitation with NIR light (after 15 minutes) in PC3pip-luc cells. Scale bars 1⁄4 20 mm. D, Bioluminescence imaging (BLI) of a 10-cm dish demonstrated that luciferase activity in PC3pip-luc cells decreased in a NIR-light dose-dependent manner. E, Luciferase activity in PC3pip-luc cells was measured, which also decreased in a NIR-light dose-dependent manner (n 1⁄4 5; P < 0.05 vs. untreated control; P < 0.01 vs. untreated control, by Student t test). F, Membrane damage of PC3flu cells induced by NIR light exposure was measured with the dead cell count using propidium iodide (PI) staining. No membrane damage was observed in PC3flu cells after NIR light exposure. G, Membrane damage of PC3pip-luc cells induced by NIR-PIT was measured with the dead cell count using PI staining, which increased in a light dose-dependent manner (n 1⁄4 5; , P < 0.01, vs. untreated control, by Student t test). There was no significant cytotoxicity associated with NIR light exposure alone in the absence of anti-PSMA-IR700 and with anti-PSMA-IR700 alone without NIR light exposure. 1156 Mol Cancer Res; 15(9) September 2017 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on December 1, 2020. © 2017 American Association for Cancer Research.PDF Image | Near-Infrared Photoimmunotherapy Targeting Prostate Cancer
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