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Published OnlineFirst June 6, 2017; DOI: 10.1158/1541-7786.MCR-17-0164 administered to PBS-washed cells 1 hour after NIR-PIT which were analyzed on a BLI system (Photon Imager; Biospace Lab). Regions of interest (ROI) were placed on each entire well, and the lucif- erase activity (photons/minute) was then calculated using M3 Vision Software (Biospace Lab). Animal and tumor models All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to 8-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick). During the procedure, mice were anesthe- tized with inhaled 3%–5% isoflurane and/or via intraperitoneal injection of 1 mg of sodium pentobarbital (Nembutal Sodium Solution, Ovation Pharmaceuticals Inc.). To determine tumor volume, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were measured with an external caliper. Tumor volumes were based on caliper measure- ments and were calculated using the following formula; tumor volume 1⁄4 length width2 0.5. Body weight was also measured. Mice were monitored daily for their general health. The presence of skin necrosis or toxicity attributable to the anti-PSMA-IR700 was evaluated with the observation of skin color and general health, including weight loss and appetite loss. Tumor volumes were measured three times a week until the tumor volume reached 2,000 mm3, whereupon the mice were euthanized with inhala- tion of carbon dioxide gas. In vivo fluorescence imaging studies PC3pip-luc cells (2 106) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. Serial ventral and dorsal fluorescence images of IR700 were obtained with a Pearl Imager using a 700-nm fluorescence channel before and 0, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 hours after intravenous injection of 100 mg of anti-PSMA-IR700 via the tail vein. Pearl Cam Software (LI-COR Biosciences) was used for analyzing fluorescence intensities. ROIs were placed on the tumor and liver. ROIs were also placed in the adjacent nontumor region as background (left dorsum and lower abdomen). Average fluo- rescence intensity of each ROI was calculated. TBRs (fluorescence intensities of target/fluorescence intensities of background) were also calculated (n 1⁄4 10). In vivo NIR-PIT PC3pip-luc cells (2 106) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. To examine the therapeutic effect of in vivo NIR-PIT on PC3pip-luc tumors, tumor-bearing mice were randomized into 4 groups of at least 10 animals per group for the following treatments: (i) no treatment (control); (ii) 100 mg of anti-PSMA-IR700 i.v., no NIR light exposure (anti- PSMA-IR700 i.v.); (iii) NIR light exposure only, NIR light expo- sure was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 (NIR light exposure); (iv) 100 mg of anti-PSMA-IR700 i.v., NIR light exposure was administered at 50 J/cm2 on day 1 and 100 J/ cm2 on day 2 after injection (NIR-PIT). These therapies were performed every week for up to 3 weeks. Serial fluorescence images, as well as white light images, were obtained using a Pearl Imager with a 700-nm fluorescence channel. www.aacrjournals.org Near-Infrared Photoimmunotherapy Targeting PSMA For in vivo BLI, D-luciferin (15 mg/mL, 200 mL) was injected intraperitoneally and the mice were analyzed on a BLI system (Photon Imager) for luciferase activity (photons/minute). ROIs were set on the entire tumors to quantify the luciferase activities. ROIs were also placed in the adjacent nontumor region as back- ground. Average luciferase activity of each ROI was calculated. To clarify therapeutic effect luciferase activity of the tumor before NIR-PIT set to 100%. Histologic analysis To detect the antigen-specific microdistribution in the tumor, fluorescence microscopy was performed. Tumor xenografts were excised from mice without treatment, 24 hours after injection of anti-PSMA-IR700 (anti-PSMA-IR700 i.v.) and 24 hours after NIR-PIT. Fluorescence images, as well as white light images, of extracted tumors were obtained using a Pearl Imager with a 700-nm fluorescence channel. Then, extracted tumors were frozen with optimal cutting temperature (OCT) com- pound (SAKURA Finetek Japan Co.) and frozen sections (10- mm thick) were prepared. Fluorescence microscopy was per- formed using the BX61 microscope with the following filters: excitation wavelength 590 to 650 nm, emission wavelength 665 to 740 nm long-pass for IR700 fluorescence. DIC images were also acquired. To evaluate histologic changes, light microscopy study was also performed using Olympus BX61. Extracted tumors were also placed in 10% formalin and serial 10-mm slice sections were fixed on glass slide with hematoxylin and eosin (H&E) staining. Statistical analysis Data are expressed as means SEM from a minimum of five experiments, unless otherwise indicated. Statistical analyses were carried out using GraphPad Prism version 7 (GraphPad Software). For multiple comparisons, a one-way ANOVA fol- lowed by the Tukey correction was used. The cumulative probability of survival based on volume (2,000 mm3) were estimated in each group with a Kaplan–Meier survival curve analysis, and the results were compared with use of the log-rank test. Student t test was used to compare the treatment effects with that of control. A P of <0.05 was considered statistically significant. Results In vitro characterization of PC3flu and PC3pip-luc cell lines As defined by SDS-PAGE, anti-PSMA-IR700 and nonconjugat- ed control anti-PSMA mAb showed an identical molecular weight, around 150 kDa, and fluorescence intensity was confirmed in the band of anti-PSMA-IR700 (Fig. 1A). After 6-hour incubation with anti-PSMA-IR700, PC3pip-luc cells showed high fluorescence signal, which was confirmed with flow cytometry (Fig. 1B) and fluorescence microscopy (Fig. 1C). On the other hand, PC3ful cells showed no fluorescence signal. Fluorescence signal in PC3pip-luc cells was completely blocked by adding excess anti- PSMA mAb (Fig. 1C), indicating that anti-PSMA-IR700 specifi- cally binds to PSMA on the cells. In vitro NIR-PIT Immediately after exposure, NIR light exposure induced cellular swelling, bleb formation, and rupture of vesicles Mol Cancer Res; 15(9) September 2017 1155 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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