A Strained Cyclooctyne Scaffold Based Multimodality Imaging Probe for Prostate Cancer Diagnosis and Imaging Guided Surgery

Objectives In this investigation, we designed an urokinase plasminogen activator receptor (uPar) targeted PET/optical imaging probe based on strained cyclooctyne platform and demonstrated it for in vivo prostate cancer diagnosis and imaging guided surgery.

Methods The uPar targeted probe CHS1 was synthesized based on strained cyclooctyne scaffold strategy that renders the molecules to be easily conjugated with fluorescent dye and radiolabel such as Cy5.5 and ⁶⁸Ga. The radiolabeling yield, purity, and stability were tested and analyzed using RP-HPLC. For cell imaging studies, PC-3 cells were cultured withCHS1 (10 nM) for 30 minutes and then were imaged with fluorescent microscope. To test the cell uptake, PC-3 cells were cultured in a 24-well plate (1×10⁵ cell per well, quadruplicate) and incubated with ⁶⁸Ga-CHS1 (1 μCi /well) with or without blocking agent AE105 for 0.5h, 1h, and 2h. For in vivo studies, PC-3 xenograft mice (n=4) were injected with 100 μCi ⁶⁸Ga-CHS1 and then the mice were performed for PET/CT imaging, Cerenkov imaging, and fluorescence imaging with 680 nm emission filter at multi-time points. After last imaging study, the tumor was resected under optical imaging guidance. Finally, the tumor and other organs were weighted and measured for radioactivity to calculate the biodistribution of the probe.

Results CHS1 was efficiently labelled with ⁶⁸Ga in 20 minutes at 37 ^oC and showed high yield (>90%) and purity (>99%). The 2 h mouse serum stability test indicated that the ⁶⁸Ga-CHS1 was highly stable (>95% at 2h) for in vivo application. Strong fluorescent signal was observed on PC-3 cells after incubation with CHS1 but weak signal was observed in the presence of excess AE105 (1 mM) for blocking. ⁶⁸Ga-CHS1 exhibited good uptake in PC-3 cells as 1.53±0.24, 1.92±0.37, and 2.63±0.34 % at 0.5h, 1h, and 2h, respectively. While significantly reduced uptake in control group as 0.36±0.12, 0.47±0.22, and 0.49±0.31 %. The in vivo PET imaging showed that the ⁶⁸Ga-CHS1 was gradually increased after injection, with 2.98±0.24, 3.38±0.29, and 3.75±0.36 %ID/g at 0.5h, 1h, and 2h. And significant Cerenkov luminescence and fluorescence signal from the tumor at 680 nm were also observed. However, the tumor uptake in blocking group are significantly reduced by excess AE105, with with 0.74±0.15, 1.21±0.26, and 1.55±0.42 %ID/g at 0.5h, 1h, and 2h. Under the guidance of optical imaging, the PC-3 tumor of the unblocked group was fully resected with minimal lesion of normal tissue.

Conclusions We have developed an uPar targeted multimodality imaging probe based on the strained cyclooctyne platform. The dual modality imaging probe demonstrates favorable imaging characteristics in vivo and can be used for image-guided surgery. Research Support: This work was supported, in part, by the Office of Science (BER), U.S. Department of Energy (DE-SC0008397).