Supplementary Figures for Phage display peptide probes for imaging early response to Bevacizumab treatment
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Phage display peptide probes for imaging early response to Bevacizumab treatment Qizhen Cao1, Shuanglong Liu1, Gang Niu1, Kai Chen2, Yongjun Yan2, Zhaofei Liu1, Xiaoyuan Chen1,2 1 Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University School of Medicine, 1201 Welch Road, Stanford, CA 94305, USA 2 Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), 31 Center Drive, Bethesda, MD 20892, USA Corresponding author Xiaoyuan Chen, PhD Laboratory of Molecular Imaging and Nanomedicine (LOMIN) National Institute of Biomedical Imaging and Bioengineering (NIBIB) National Institutes of Health (NIH) 31 Center Drive, Suite 1C14 Bethesda, MD 20892-2281 E-mail: shawn.chen@nih.gov Telephone: 301-451-4246 Fax: 301-480-1613 Key words: Phage display; Angiogenesis; Therapy response; Bevacizumab; Molecular imaging Running title: Imaging Bevacizumab Therapy Response Supporting Figure Legends Fig. S1. Schematic illustration of the discovery of bevacizumab responsive peptide (BRP). LS174T tumor-bearing mice were treated with 3 doses of bevacizumab (intraperitoneal injection at 20 mg/kg every other day). Ph.D.-12TM phage display peptide library expressing random 12mer peptides on M13 phages were injected intracardiacally into the mice 1 day after the third dose of bevacizumab treatment. Mice were sacrificed 10 min after phage injection, and the tumors were harvested. The particles were collected and amplified for the next round of biopanning. After six rounds of biopanning, single plaques from soft agar were isolated. The peptide sequences were deduced from the decoded DNA information. The BRP peptide was then appropriately labeled for optical and positron emission tomography (PET) imaging. Fig. S2. Histological detection of phage particles in tumor tissues with Cy5.5 labeled LLADTTHHRPWT-phage. Saline vehicle (A) or bevacizumab (B) treated LS174T tumor mice were injected with 1 nmol of Cy5.5 conjugate. After 24 h circulation of the phage particles, FITC-conjugated tomato lectin was then injected for in vivo dual staining of tumor vasculature (100×). Cy5.5-BRP phage showed more tumor accumulation in bevacizumab treated than in saline control mice. Cy5.5 and FITC overlay pictures showed that BRP phages bind specifically to bevacizumab-treated microvascular tumor endothelium cells. Note that the relatively weak Cy5.5 signal is likely due to the short circulation half-life of the phage particles. Fig. S3. Metabolic stability of BRP peptide. (A) Analytical HPLC chromatogram of 18F-FP-BRP standard. Pure 18F-FP-BRP had a retention time of about 22 min following the conditions described in methods. (B) HPLC of urine sample at 1 h after injection of 18F-FP-BRP showed about 90% intact tracer, with several minor peaks with different retention times, suggesting good metabolic stability of this linear 12-mer peptide in vivo. Fig. S4. BRP does not bind to VEGF or bevacizumab. Cy5.5-BRP was incubated with VEGF, bevacizumab or VEGF-bevacizumab mixture separately for 0.5 h at 37 ºC. The samples were then centrifuged with Bio-Spin®6 chromatography columns (BIO-RAD) (MW cutoff: 6000 Da). The UV-Vis spectra of the eluents showed no association between Cy5.5-BRP with VEGF, bevacizumab, or VEGF/bevacizumab complex. Fig. S5. HUVEC cell uptake of Cy5.5-BRP. HUVEC only or HUVEC co-cultured with LS174T cancer cells (Transwell culture system) were treated with phosphate buffered saline (PBS) vehicle or bevacizumab (625 μg/ml) for 24h, and then incubated with Cy5.5-BRP (10 nM). Shown are the microscopic photographs of (A) HUVEC only, PBS; (B) HUVEC only, bevacizumab treatment; (C) HUVEC/LS174T co-culture, PBS; (D) HUVEC/LS174T co-culture, bevacizumab treatment. Only HUVEC cells co-cultured with LS174T and pre-treated with bevacizumab had Cy5.5-BRP uptake. 
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