A549 and A549-fLuc cells were maintained in high glucose Dulbecco modified
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1 Cell culture and animal model A549 and A549-fLuc cells were maintained in high glucose Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum at 37 C in humidified atmosphere containing 5% CO2. All animal experiments were performed in accordance with the Guidelines of Peking University Animal Care and Use Committee. To establish the A549-fLuc subcutaneous tumor model, tumor cells were inoculated subcutaneously into the right front flank of female BALB/c nude mice (4 5 weeks of age; Department of Laboratory Animal Science, Peking University). Tumor growth was measured using a caliper. Tumor volume was calculated using the following formula: tumor volume = a (b 2 )/2, where a and b were the tumor length and width, respectively. Bioluminescence imaging After anesthetizing with 2% isoflurane in oxygen, nude mice bearing A549-fLuc tumor xenografts received an intraperitoneal injection of 150 mg/kg of D-luciferin. (Gold BioTechnology, St Louis, MO). Bioluminescence imaging (BLI) was then performed using the IVIS spectrum system (Xenogen) 10 min after D-luciferin administration. The BLI signal intensity in the tumor regions was quantified as the sum of all detected photon counts within the region of interest (ROI) after subtraction of background luminescence. Immunofluorescence staining and analyses
2 A549-fLuc tumor tissues were stained for glucose transporter 1 (GLUT-1), Ki67 proliferation marker, human integrin αvβ3, murine integrin β3, and human VEGF. Briefly, frozen tumor sections were incubated using goat anti-glut-1 (Millipore, Billerica, MA), goat anti-ki67 (Millipore, Billerica, MA), mouse anti-human integrin αvβ3 (clone LM609, Millipore, Billerica, MA), or hamster anti-murine integrin β3 antibody (BD Biosciences, San Jose, CA), stained with dye-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), and examined using confocal microscopy. For human VEGF staining, fluorescein isothiocyanate (FITC)-bevacizumab was synthesized by conjugating the VEGF-specific antibody bevacizumab with FITC-succinimidyl ester (Pierce, Rockford, IL, USA) using a previously described method (1). Frozen A549-fLuc tumor sections were incubated with FITC-bevacizumab (2 μg/ml) with or without a blocking dose of bevacizumab (20 μg) for 1 h at room temperature. After washing with phosphate buffered saline the sections were visualized by confocal microscopy. After staining, 20 random views in the tumor slices were selected for the analyses. The GLUT-1, human integrin αvβ3, and VEGF expression levels were calculated by measuring the integrated optical density of images of an equivalent area (mm 2 ) as previously described (2). The tumor cell proliferation index was calculated as the percentage of Ki67-positive nuclei in relation to the total number of nuclei. The murine integrin β3 expression was determined by counting and averaging the number of integrin β3-positive vessels per field of view.
3 Radioligand binding assay The effect of dasatinib on integrin αvβ3 inactivation was determined using cell radioligand binding assays. The integrin αvβ3-specific radioligand 125 I-c(RGDyK) was prepared as previously described (3). Experiments were performed on high integrin αvβ3-expressing U87MG cells with or without 24 h pre-incubation with 30 nm dasatinib, following a previously described procedure (4,5). The best-fit 50% inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression using Graph-Pad Prism 5.0 (GraphPad Software, Inc. San Diego, CA). Experiments were performed twice using quadruple samples. Matrigel plug assay The anti-angiogenic effect of dasatinib was evaluated by Matrigel plug assay as previously described (6), with some modifications. Briefly, growth factor-reduced Matrigel matrix (400 µl; High Concentration, Phenol Red-Free; BD Biosciences, San Jose, CA) was mixed with 100 ng recombinant mouse VEGF120 (ebioscience, San Diego, CA), 100 ng recombinant basic fibroblast growth factor (Invitrogen, Carlsbad, CA), 10 IU of heparin (M&C Gene Tech, Beijing, China), and 1 mg dasatinib (in 10 µl dimethyl sulfoxide [DMSO])) or DMSO (vehicle control). Matrigel mixtures were then inoculated subcutaneously in the right flank of female BALB/c nude mice. Nude mice injected with Matrigel matrix alone were used as the negative controls. Seven days later, the Matrigel plugs were harvested and photographed.
4 Cell migration, proliferation, and colony formation assays A wound-healing assay was performed to determine the effect of dasatinib on cell migration. Briefly, A549-fLuc cells ( /well) were cultured in 12-well plates overnight. Monolayer wounds were generated using a pipette tip scratched through the center of the well. The wells were gently washed with PBS. Cell culture medium containing DMSO (vehicle control), 3, 10, or 30 nm dasatinib (in DMSO) was added to each well. Twenty-four hours later, cell migration was examined using a microscope. The distance of migration in each well was measured as pixel units (7). Data are expressed as the average of six samples plus the standard deviation. Next, to determine the effect of dasatinib, DTX, or dasatinib plus DTX on cell viability, cell proliferation and colony formation assays were performed. For the cell proliferation assay, A549-fLuc cells ( /well) grown in 96-well plates were treated with dasatinib or docetaxel (DTX; with or without the combination of 30 nm dasatinib) at various concentrations. After 72 h of incubation, the cell viability was determined using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). For the colony formation assays, A549-fLuc cells ( /well) grown in 12-well plates were treated with cell culture medium containing vehicle control, dasatinib (10 nm), DTX (10 nm), or both dasatinib (10 nm) and DTX (10 nm) for 48 h. Afterwards, the culture medium was replaced and the cells were grown for another 120 h until colonies were visible. The cells were then directly examined using light microscopy or stained with 0.1% Coomassie Brilliant Blue for 20 min and subsequently photographed.
5 SUPPLEMENTAL REFERENCES 1. Liu Z, Sun X, Liu H, et al. Early assessment of tumor response to gefitinib treatment by noninvasive optical imaging of tumor vascular endothelial growth factor expression in animal models. J Nucl Med. 2014;55: Yang M, Gao H, Yan Y, et al. PET imaging of early response to the tyrosine kinase inhibitor ZD4190. Eur J Nucl Med Mol Imaging. 2011;38: Liu Z, Li ZB, Cao Q, Liu S, Wang F, Chen X. Small-animal PET of tumors with 64 Cu-labeled RGD-bombesin heterodimer. J Nucl Med. 2009;50: Liu Z, Liu H, Ma T, et al. Integrin αvβ6-targeted SPECT imaging for pancreatic cancer detection. J Nucl Med. 2014;55: Liu Z, Huang J, Dong C, et al. 99m Tc-labeled RGD-BBN peptide for small-animal SPECT/CT of lung carcinoma. Mol Pharm. 2012;9: Lee E, Koskimaki JE, Pandey NB, Popel AS. Inhibition of lymphangiogenesis and angiogenesis in breast tumor xenografts and lymph nodes by a peptide derived from transmembrane protein 45A. Neoplasia. 2013;15: Shor AC, Keschman EA, Lee FY, et al. Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival. Cancer Res. 2007;67:
6 Supplemental Figure 1. (A-B) Bioluminescence imaging of varying numbers of A549-fLuc cells plated on 96-well plates showed a linear correlation between the bioluminescence signal and tumor cell number (R 2 = ; P <0.0001). Data are expressed as means ± SD, n = 5. (C) Cell proliferation assay showed that firefly luciferase transfection had no significant effect on A549 cell proliferation at various time points. Data are expressed as means ± SD, n = 6.
7 Supplemental Figure 2. Immunofluorescence staining of glucose transporter 1 (GLUT-1) (A) and Ki67 (B) in A549-fLuc tumor tissues with or without dasatinib treatment.
8 Supplemental Figure 3. Immunofluorescence staining of human integrin αvβ3 (A) and murine integrin β3 (B) in A549-fLuc tumor tissues with or without dasatinib treatment.
9 Supplemental Figure 4. Immunofluorescence staining for human vascular endothelial growth factor (VEGF) (with or without blocking with bevacizumab) in dasatinibtreated or control A549-fLuc tumor tissues.
10 Supplemental Figure 5. Dasatinib inhibited A549-fLuc tumor cell migration but had limited cytotoxicity compared to docetaxel (DTX). (A) Wound healing assay of dasatinib on A549-fLuc cell migration after incubation with various concentrations of dasatinib (Das) for 24 h. (B) Pixel values of the denuded area of the A549-fLuc cells treated with various concentrations of dasatinib for 24 h. (C) Cytotoxicity of dasatinib (Das), DTX, and dasatinib plus DTX (Das + DTX) on A549-fLuc cell growth. For the dasatinib plus DTX group, increasing concentrations of DTX and a constant concentration of dasatinib (30 nm) were used. Data are expressed as a percentage relative to the control (100%). (D-E) Photography of the 12-well-plate (D) and microscopy examination (E) of colony formation of A549-fLuc cells treated with dimethyl sulfoxide (DMSO; control), dasatinib (Das), DTX, or dasatinib plus DTX (Das + DTX). **, P <0.01; ***, P <0.001.
11 Supplemental Figure 6. Representative 18 F-FDG PET (A), 99m Tc-3PRGD2 SPECT/CT (B), and Dye755-Ran NIRF (C) images in A549-fLuc tumor-bearing nude mice with or without dasatinib plus docetaxel (Das + DTX) treatment for 6 days. Arrows indicate the location of tumors.
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