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PET Imaging of Platelet-Derived Growth Factor Receptor # in Colorectal Tumor Xenograft using Zirconium-89 Labeled Dimeric Affibody Molecule Huawei Cai, Qiuxiao Shi, Yu Tang, Lihong Chen, Yue Chen, Ze Tao, Hao Yang, Fang Xie, Xiao-Ai Wu, Nan Liu, Yuanyou Yang, Haoxing Wu, Rong Tian, Xiaofeng Lu, and Lin Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01317 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019
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Molecular Pharmaceutics
PET Imaging of Platelet-Derived Growth Factor Receptor β in Colorectal Tumor Xenograft using Zirconium-89 Labeled Dimeric Affibody Molecule
Short Running Title: 89Zr-DFO-ZPDGFRβ affibody PET imaging All the authors declare there is no conflict of interest. The contribution of each author has been substantiated in cover letter. Author list: Huawei Cai1, Qiuxiao Shi2, Yu Tang3, Lihong Chen4, Yue Chen5, Ze Tao2, Hao Yang2, Fang Xie6, Xiaoai Wu1, Nan Liu5, Yuanyou Yang4, Haoxing Wu7, Rong Tian1, Xiaofeng Lu*2, Lin Li*1 1. Laboratory of Clinical Nuclear Medicine, Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu, 610041, China. 2. Key Lab of Transplant Engineering and Immunology, Regenerative Medical Research Center, West China Hospital, Sichuan University, Chengdu, 610041, China. 3. Key Lab of Radiation Physics and Technology, Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu, 610064, China. 4. Department of Biochemistry & Molecular Biology, West China School of Basic Sciences & Forensic Medicine, Sichuan University, Chengdu, 610041, China. 5. Departments of Nuclear Medicine, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China. 6. PET Center, Huashan Hospital, Fudan University, Shanghai 200040, China. 7. Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital and West China School of Medicine, Sichuan University, Chengdu, 610041, China. *For correspondence or reprints contact: Xiaofeng Lu, PhD., Key Lab of Transplant Engineering and Immunology, Regenerative Medical Research Center, West China Hospital, Sichuan University, No.1 Ke Yuan Road, Chengdu, 610041, China. Tel: +86-28-85164031, Fax: +86-28-85164031, Email:
[email protected] Lin Li, MD., Department of Nuclear Medicine, Laboratory of Clinical Nuclear Medicine, West China Hospital, Sichuan University, No.37 Guo Xue Alley, Chengdu, 610041, China. Tel: +86-28-85422155, Fax: +86-28-85422155, Email:
[email protected] 1
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First-author Information Huawei Cai, Assistant Professor, Department of Nuclear Medicine, Laboratory of Clinical Nuclear Medicine, West China Hospital, Sichuan University, No.37 Guo Xue Alley, Chengdu, 610041, China. Tel: +86-28-85422155, Fax: +86-28-85422155, Email:
[email protected] Word Count: 3564 words
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Molecular Pharmaceutics
Abstract Platelet-derived growth factor receptor β (PDGFRβ) is overexpressed in a variety of malignant cancers and plays critical role in tumor angiogenesis, and has been proven as a valuable target for cancer treatment. In this pilot study, a dimeric affibody molecule ZPDGFRβ, was prepared and radiolabeled with positron emission radionuclide zirconium89 for PET imaging of colorectal tumors by targeting PDGFRβ expression in vivo. The PDGFRβ-binding capability of dimeric affibody was evaluated by flow cytometry, immunofluorescent staining and whole-body optical imaging. Then, ZPDGFRβ was conjugated with DFO-Bn-NCS and radiolabeled with 89Zr. Targeted binding capability of 89Zr-DFO-ZPDGFRβ to PDGFRβ expressing cells was investigated by cellular assay in vitro and microPET/CT imaging in vivo. Dimeric ZPDGFRβ affibody had specifically higher binding capability with PDGFRβ expressing pericytes rather than LS-174T cancer cells, and well co-localized with tumor neovasculature by flow cytometry and immunofluorescent assay. ZPDGFRβ was successfully labeled with 89Zr by DFO chelating with yield of 94.1 ± 3.53 %.
89Zr-DFO-Z
PDGFRβ
indicated preserved specific binding
ability with PDGFRβ expressing cells and effective inhibiting capability to PDGF-β ligands (P 99.9%) using 0.1 M oxalic acid
solution. For radiolabeling of DFO- ZPDGFRβ, the pH of 89Zr-oxalate (~185 MBq) was initially adjusted to 8.0 with 1.0 M Na2CO3 in a microcentrifuge vial and ceased by CO2 evolution. Then, 100 μg of DFO-ZPDGFRβ conjugate was added and the reaction mixture was incubated at room temperature and shake with 300 rpm for one hour. The unlabeled 6
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Molecular Pharmaceutics
89Zr
was removed by ultrafiltration centrifugation as described above and the product
concentration was determined by Bradford assay. Radiochemical purity was determined by radio-instant thin-layer chromatography (radio-ITLC) with FC-3600 radioactive Flow-Count system (Bioscan, Washington, DC, USA), using 0.05 M ethylenediaminetetraacetic acid (EDTA) as mobile phase. 89Zr-DFO-ZPDGFRβ retained at the baseline (Rf = 0.0~0.2), whereas
89Zr
ions moved to the solvent front (Rf =
0.8~1.0). The stability of 89Zr-DFO-ZPDGFRβ with respect to the loss of radioactivity 89Zr from the affibody was determined by incubation in mouse blood plasma (~3.74 MBq in 1 mL plasma) for up to 6 h at 37 °C. Cellular uptake and Functional Assay of Radiolabeled Affibody Conjugates PDGFRβ overexpressed pericytes were used for cellular binding assay as previously described 25, 26. Briefly, cells were seeded in 24-well plates at a density of 1 × 105 cells per well and cultured overnight before use. Then, cells were incubated with 74 kBq (approximate 0.04 μg) of 89Zr-DFO-ZPDGFRβ for 1 and 2 h, respectively. In the blocking group, 10 μg of non-labeled affibody was added 6 h prior before adding
89Zr-DFO-
ZPDGFRβ to block the binding sites of affibody. After incubation at each time point, the medium was removed and the cells were washed three-times with PBS. The cells were lysed by 0.2 mL of NaOH (1 mol/L); all cell lysates were collected and counted in a FJ-2008PS gamma counter (Xi’an Nuclear Instrument Inc, China). Cells added with 74 kBq of 89Zr instead of any conjugate was used as blank control (BLK). Competitive inhibition of conjugates on pericytes migration was quantified using the scratch assay
27, 28.
5 × 104 cells were inoculated in the 24-well plate and starved in 7
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pericyte medium containing 0.5% fetal bovine serum overnight. Vertical scratches were made in the middle of plate using a 1 mL pipette tip. PDGF-BB (100 ng/mL) was added into each group for pericytes migration induction. Then, 5 μM of Imatinib, 200 nΜ of ZPDGFRβ, 200 nΜ of DFO-ZPDGFRβ, and 200 nΜ of non-radioactive 91Zr-DFO-ZPDGFRβ were respectively added into wells followed by PDGF-BB for inhibition at 37 °C. Cells added with PBS, but no PDGF-BB or other reagents, was used as control for migration evaluation. After incubation for 24 h, the fraction of coverage in each scratch was recorded and calculated as percentages by ImageJ software. Non-radioactive 91Zr was used in this assay for eliminating possible interference from radiobiological effect. Biodistribution and microPET/CT Scanning The biodistribution of 89Zr-DFO-ZPDGFRβ at 5 min, 30 min, 1, 2, 4, 8, and 24 h in mice bearing LS-174T xenografts was investigated at an injected dose of 374 kBq in 100 μL of PBS. The injected mice were randomized divided into groups with 4 mice in each, and sacrificed at predetermined time points. Organs were harvested and the radioactivities were counted by a gamma counter. The data were presented as mean ± SD %ID/g. Additional experiment was performed to assess the potential effect of 89ZrDFO-ZPDGFRβ affibody molecules with existence of other PDGFRβ-specific affibody molecules. In the test group, 89Zr-DFO-ZPDGFRβ was coinjected with 100 μg of DFOZPDGFRβ, and the biodistribution was measured at 1, 2, and 4 h after injection as described above. In the PET/CT imaging experiment, LS-174T xenograft-bearing mouse was injected with 3.7 MBq of 89Zr-DFO-ZPDGFRβ via tail vein. Then, animal PET imaging was conducted on an Inveon micro-PET/CT scanner (Siemens Healthcare 8
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Molecular Pharmaceutics
Molecular Imaging, Knoxville, TN, USA) at approximately 15 min, 1, 2, 4, and 24 h post injection. For each scan, three-dimensional (3D) regions of interest (ROIs) were drawn over the whole-body images. The average radioactivity concentration in the ROI was obtained from the mean pixel values within the ROI volume. Statistics SPSS software (version 20) was used for one-way analysis of variance (ANOVA) for multiple comparisons. The significance level was defined as p
