Angiosense 680

FMT (FMT 4000 MSIM; PerkinElmer, USA) was used to detect the overall iRFP fluorescence intensity in mice weekly for a total duration of 8 weeks. After weighing the mice, they were anesthetized with a mixture of isoflurane/oxygen and placed comfortably in the portable animal imaging cassette. The setting was established for a full-body scan, with excitation at the wavelength of 680 nm, initiating a 3D scan to quantify iRFP intensity. The scan covered the entire murine body with an excitation power set to 80 mW and exposure time between 2 and 5 s. Each mouse was scanned for 5–10 min using source-detector pair excitation measurements (λ_ex = 670 nm/λ_em = 690–740 nm) to quantify fluorescence signal intensity. The collected fluorescence signal data were reconstructed using TrueQuant v3.0 software (PerkinElmer), and the fluorescence data from a known concentration of the imaging agent, AngioSense 680 (PerkinElmer), was used to establish a standard curve to calibrate the iRFP fluorescence picomolar data representing the size of human prostate cancer tumors in mice.

PF-03084014 and sunitinib were synthesized by Pfizer chemists. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). MDA-MB-231Luc and AngioSense 680 EX was purchased from PerkinElmer (Waltham, MA). The antibodies for IHC analyses were anti-BrdU (BD Pharmingen, San Diego, CA), anti-HIF1α (R&D Systems, Minneapolis, MN), anti-phospho-H2AX, anti-HES1, and anti-VEGFR2 (Cell Signaling Technology, Danvers, MA).

K/BxN serum-induced vascular permeability was monitored in vivo by noninvasive near-infrared fluorescence imaging (NIRF) of the mouse whole-paw. Mice were anesthetized by 2% isoflurane (Butler Schein, 1 L/min flow), implanted with a tail vein catheter, and immobilized with the paw secured by surgical tape. For K/BxN experiments depicted in Fig. 6, mice were imaged using the Kodak In-Vivo FX Pro 400 whole-animal NIRF imaging system (Carestream Health). Mice were injected intravenously through the tail vein catheter with 100 μL of the blood pool probe AngioSense 680 (PerkinElmer), imaged at 1-min intervals (650 nm excitation/700 nm emission, 21.4 mm FOV, 10-s exposure, 2× binning) for 5 min to establish baseline fluorescence followed by intravenous injection of 75 μl K/BxN serum and further imaging for another 25 min. For K/BxN experiments depicted in Fig. 6, mice were imaged using the Pearl Imager NIR fluorescence imaging system (Li-COR), with excitation at 700 nm and 85 μm scan resolution. Quantitative image analysis of the mice was performed using Pearl Cam Software (Li-COR). The K/BxN serum used in this study was sourced from KRNxNOD F1 mice that exhibited severe arthritis. The average fluorescence intensities and the fold change in fluorescence intensities from the initial imaging time point within paws were quantified using custom routines in MatLab (MathWorks).