Compliance with Ethical Standards
Mice (C57BL/6J and Foxn1 nu) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All protocols were approved by the Institutional Animal Care and Use Committee at Children’s Hospital Boston and were conducted in accordance with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research.
Human glioblastoma cell line U-87 MG was purchased from ATCC (VA, USA). Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Switzerland). All cells were characterized before use, mycoplasma free (using an EZ-PCR Mycoplasma Test Kit (Biological Industries) and used for the experiments up to p15. All cells were kept in a humidified incubator at 37°C with 5% CO2. For U-87 MG cells EMEM-conditioned medium (Life Technologies, MA, USA) was used and supplemented with 1% L-glutamine, 1% sodium pyruvate, 10% fetal calf serum (FCS) and penicillin/streptomycin. HUVECs were maintained in a specific medium supplemented with PeproGrow-MacroV kit (ENDO-BM & GS-MacroV, PeproTech) and penicillin/streptomycin, and were seeded in wells that were previously coated with gelatin 0.1% (Sigma-Aldrich).
Cytotoxicity Assay with U-87 MG Cells and HUVECs
Each well of the 96-well culture plates (Thermo Fisher Scientific NuNclonTM Delta Surface, Denmark) was seeded with proper medium containing U-87 MG cells (8 000 cells/well) or HUVEC (6 000 cells/well) and incubated in 37°C and 5% CO2 for 24 h. Amiodarone (INJ 50 mg/ml, Sanofi-Winthrop, France) was added to each well in varying concentrations (0, 1, 10, and 50 μM with U-87 MG cells, and 0, 0.1, 1, 3, 5, 7 and 10 μM with HUVECs). Stock solution of Amiodarone contains 2% Benzyl alcohol (further diluted), therefore, the proper concentration of benzyl alcohol was added to each treatment. The plate was incubated in 37°C and 5% CO2 for another 24 h. After incubation, WST-8 reagent (Sigma-Aldrich, Japan) was added into each well for viability detection and incubated in 37 °C and 5% CO2 for 1 h. Absorbance was measured at 450 nm using a plate reader (Wallac 1420 VICTOR plate-reader, Perkin-Elmer Life Sciences, USA), n=10.
Proliferation Assay with U-87 MG Cells and HUVECs
U-87 MG cells (3 000 cells/well) and HUVEC (3 000 cells/well) were incubated for 24 h in their proper conditioned media. Amiodarone in varying concentrations (0, 1, 10, and 50 μM for U-87 MG cells, and 0, 0.1, 1, 3, 5, 7 and 10 μM for HUVECs) was added into all wells. Plates were incubated in 37°C and 5% CO2 for another 72 h followed by viability detection using WST-8 reagent, n=9.
U-87 MG cells were harvested and centrifuged for 5 minutes at room temperature (RT). Next, cells were suspended in media containing 1% FCS and counted. For this assay a 24-well cultured plate with 8 μm pore size polycarbonate membrane transwell inserts (Corning Incorporated, USA) was used. Proper medium containing 10% FCS was added to the lower compartments. All inserts were adjusted to allow the membrane to be uniformly submerged in the medium. Next, 1% FCS medium containing 70 000 U-87 MG cells was added to each of the inserts' upper compartments and incubated at 37°C and 5% CO2 for 30 minutes. After incubation, 1% FCS medium containing different concentrations of Amiodarone (0, 1, 10 and 50 µM) was added to each of the inserts' upper compartments and then incubated at 37°C and 5% CO2 for 18 h to allow cells to migrate toward the underside of the inserts' filter. After 18 h, cells were fixed, stained and an inverted microscope (OLYMPUS IX73, Japan) was used to detect and count cells on the lower side of the inserts' filter (5 different fields per well) and the average number of migrated cells was calculated, n=6.
A pHEMA (Sigma, USA) culture was used to generate Anoikis. A solution containing 20 mg/ml poly(2-hydroxyethyl methacrylate) (pHEMA) in 95% ethanol was prepared and left on a stirrer at RT to dissolve. Once dissolved, the solution was pipetted into 6-well culture plates (Thermo Fisher Scientific NuNclonTM Delta Surface, Denmark). The plates were left in a sterile biological safety cabinet until the ethanol evaporated, and the pHEMA solidified and coated the wells evenly. Proper growth medium containing different concentrations of Amiodarone (0, 1, 10 and 50 µM) was placed in each plate. All plates were seeded with 50 000 U-87 MG cells/well and incubated for 72 h at 37°C and 5% CO2. After incubation, cell viability was measured using WST-8. Absorbance was measured as previously mentioned, n=6.
Soft Agar Assay
Soft agar assay was performed with U-87 MG cells. Six-well culture plates were coated with a bottom layer of 2% agar (Invitrogen by Life TechnologiesTM) and left to solidify in RT. 0.6% agar containing 30 000 U-87 MG cells/well was added to all wells left to solidify in RT. Proper medium containing different concentrations of Amiodarone (0, 1, 5, 7, 10, and 50 μM) was added to all wells. Plates were incubated at 37°C and 5% CO2. Medium with Amiodarone treatment was refreshed every 5 days. On day 15, colonies were visualized using an inverted microscope (Nikon ECLIPSE TS100). Colonies were fixed, stained and counted (5 different fields per well). Images were processed, and the average number of colonies per well was analyzed and calculated using ImageJ, n=3.
Spheroid Growth Assay
15 000 U-87 MG cells/well were seeded in round-bottom 96-well plates (Lipidure®, NY) coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) and treated with varying concentrations of Amiodarone (0, 1, 10 and 50 µM). The plates were incubated at 37°C and 5% CO2, and images were taken 24 h, 5 and 7 days later using an inverted microscope, as mentioned before. MATLAB code was used to generate the calculated area of the different spheroids, as detailed in the spheroids' image analysis section below, n=3.
Spheroid images were cropped to the exact same dimension so that unnecessary background (including pixels of the scale bar) were excluded. Using a MATLAB code, binary B&W images were then formed, in which live spheroids were distinguished from the background (Supplementary fig. 1). The number of pixels was counted in each spheroid. Since individual pictures may be of different resolutions, the total number of pixels in the picture was counted as well and the scaled size of the spheroid was calculated as the ratio between spheroid pixels and the total number of pixels. In some cases, where many dead cells surrounded the live spheroid, the background was cleaned using Photoshop before the B&W image was constructed. In all cases, the scaled spheroids' size was calculated by the ratio between the number of spheroid pixels and the total number of pixels in the cropped image. A further normalization was done in the plots — the spheroid growth was calculated as the ratio between the current size and the initial size, scaling the initial size values of day 1 as 100%.
Scratch Wound Migration Assay
7 000 HUVECs/well were seeded in an IncuCyte® ImageLock 96-well Plate (Sartorius, USA) and incubated in 37°C and 5% CO2 for 24 h. Varying concentrations of Amiodarone (0, 0.1, 1, 3, 5 and 10 μM) were added to each well and a scratch wound was made in each well using the IncuCyte® 96-well WoundMaker Tool (ESSEN, BioScience). The plate was placed in an incubator containing the IncuCyte® S3 Live-Cell Analysis System (ESSEN, BioScience) for 36 h. Real time automated images were taken every 2 h and cell migration analysis was preformed using the IncuCyte® Scratch Wound Cell Migration Software Module. Final images were processed using ImageJ, n=9.
Tube Formation Assay
An endothelial tube formation assay was performed with HUVECs. The wells' bottoms were coated with Matrigel® Matrix (Corning®) 50 μl/well and left to polymerize for 1 h at RT. This was followed by adding 200 μl of Amiodarone in varying concentrations (0, 0.1, 1 and 5 μM) combined with 20 000 cells/well to the Matrigel-coated 96-well plate. The plate was placed in an incubator containing the IncuCyte® S3 Live-Cell Analysis System for 6 h. Real time automated images were taken each hour. Tube formation analysis (calculation of the number of meshes formed) and image processing were done using ImageJ, n=4.
Corneal Micropocket Assay
The corneal micropocket assay was carried out as previously detailed  in C57BL/6J mice. Micropockets in the mouse cornea were surgically created and pellets containing 80 ng of carrier-free recombinant human basic-fibroblast growth factor (bFGF) (R&D Systems, Minneapolis, MN) were implanted into them. Amiodarone was administered intraperitoneally (I.P.), 0.1 mg/kg/d or 0.05 mg/kg/d q.d. over a course of 5 days. After 5 days, the vascular growth area was measured using a slit lamp and photos of mouse eyes were taken. The area of neovascularization was calculated as vessel area by the product of vessel length measured from the limbus and clock hours around the cornea, using the following equation: vessel area (mm2) = [clock hours × vessel length (mm) × π × 0.2 mm], n=10.
In Vivo Matrigel Plug Angiogenesis Assay
C57BL/6J mice were injected subcutaneously (S.C.) with growth-factor-reduced Matrigel Matrix (BD Biosciences) mixed with recombinant human vascular endothelial growth factor (VEGF) (10 ng/ml) and bFGF (10 ng/ml). Matrigel plugs in the treated groups were additionally mixed with Amiodarone 0.05 mg/plug. After 8 days, Matrigel plugs were removed, embedded in optimal cutting temperature compound (OCT) medium (OCT, Tissue-Teck, USA) and immunohistochemistry was performed using a Vectastain Elite ABC kit (Vector Laboratories) followed by anti-CD31 (BD Biosciences) reaction for micro-vessel staining. Detection was carried out using a 3,3'-Diaminobenzidine chromogen (DAB), resulting in positive brown staining, in addition to using Gill’s Hematoxylin for nuclei staining. Quantification of the endothelial cells in Matrigel plugs was performed by Flow cytometry using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA, USA) following enzymatic digestion of Matrigel plugs, as described elsewhere . Immunostaining was performed for FACS analysis in the presence of rat anti-mouse CD45-APC and CD31+PE (BD Biosciences). Endothelial cells were defined as CD31+CD45- cells, n=3.
GBM Tumor Xenograft Model
Foxn1 nu mice were inoculated S.C. with 5 × 106 U-87 MG cells/mouse. Tumor growth was measured transcutaneously with a digital caliper every other day. Tumor volume was calculated using the standard equation: (length × width2 × 0.52). When tumors reached an average volume of ∽200 mm3, the mice were divided into groups of treated (Amiodarone 0.1 mg/kg or 0.05 mg/kg I.P. injection q.d.) and untreated (given saline) mice over a course of 14 or 24 days, respectively. Tumors were surgically resected and collected when an average volume of ∽1 000 mm3 was reached. Tumor weight and volume were measured, followed by embedment in OCT medium and prior to sectioning using a microtome-cryostat. Histological sections were stained using Hematoxylin/Eosin (H&E) or anti-CD31 (DAB) followed by Gill’s Hematoxylin, as previously mentioned, n=3-5.
The in vitro experiments were repeated independently at least twice, while the in vivo experiments were conducted two times, each with a different dose.
Statistical data was analyzed on GraphPad Prism 8 (www.graphpad.com, San Diego, CA). Studies containing two groups were assessed using the unpaired two-tailed Student’s t-test. Studies containing more than three groups were compared and analyzed using a one-way analysis of variance (ANOVA), and significant differences were detected using Tuckey's multiple comparison post-test. Spheroid radiuses were measured using MATLAB code (as detailed in the Materials and Methods section) and subsequently analyzed by two-way ANOVA. Differences were considered statistically significant for p < 0.05.