Ultra-Low Dose Amiodarone Reduces Tumor Growth and Angiogenesis

Background: Pre-clinical studies suggest that Amiodarone induces cytotoxicity in several types of cancer cells, thus making it a potential candidate for use as an anti-cancer treatment. In this study, we examined Amiodarone's effects on glioblastoma multiforme (GBM), a highly aggressive and hypervascularized cancer. We hypothesized that Amiodarone would show an anti-angiogenic effect on GBM in addition to its previously suggested anti-cancer activity, and that an ultra-low dose would be both effective and possibly avert the drug’s side effects. Methods: The anti-cancer activity of Amiodarone was assessed by several in vitro assays using GBM cells. This included cytotoxicity, proliferation, transwell migration, Anoikis, colony-formation and three-dimensional (3D) spheroid growth assays. The anti-angiogenic effect of Amiodarone was tested on endothelial cells, using toxicity, proliferation, migration and tube formation in vitro assays. The anti-cancer and anti-angiogenic activity of Amiodarone was examined in vivo on three different murine models. C57BL/6J mice were utilized for the corneal neovascularization model and the Matrigel plug assay. Foxn1 nu mice were inoculated with GBM cells and used for the GBM tumor xenograft model. Results: In this study, we showed that Amiodarone has a signicant anti-cancer and anti-angiogenic activity in vitro. Moreover, ultra-low dose Amiodarone markedly reduced the size of GBM xenograft tumors and displayed a strong anti-angiogenic effect in vivo. Conclusions: Our results strongly suggest that Amiodarone possess dual cancer ghting properties.

Transwell Migration 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% CO 2 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% CO 2 for 18 h to allow cells to migrate toward the underside of the inserts' lter. After 18 h, cells were xed, stained and an inverted microscope (OLYMPUS IX73, Japan) was used to detect and count cells on the lower side of the inserts' lter (5 different elds per well) and the average number of migrated cells was calculated, n=6.

Anoikis Assay
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 Scienti c NuNclon TM Delta Surface, Denmark). The plates were left in a sterile biological safety cabinet until the ethanol evaporated, and the pHEMA solidi ed 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% CO 2 . 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 Technologies TM ) 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% CO 2 . Medium with Amiodarone treatment was refreshed every 5 days. On day 15, colonies were visualized using an inverted microscope (Nikon ECLIPSE TS100). Colonies were xed, stained and counted (5 different elds 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 2methacryloyloxyethyl phosphorylcholine (MPC) and treated with varying concentrations of Amiodarone (0, 1, 10 and 50 µM). The plates were incubated at 37°C and 5% CO 2, 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 g. 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% CO 2 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 [70] in C57BL/6J mice.
Micropockets in the mouse cornea were surgically created and pellets containing 80 ng of carrier-free recombinant human basic-broblast 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 (mm 2 ) = [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. Quanti cation 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 [71]. Immunostaining was performed for FACS analysis in the presence of rat antimouse CD45-APC and CD31+PE (BD Biosciences). Endothelial cells were de ned as CD31+CD45-cells, n=3.

GBM Tumor Xenograft Model
Foxn1 nu mice were inoculated S.C. with 5 × 10 6 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 × width 2 × 0.52). When tumors reached an average volume of ∽200 mm 3 , 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 mm 3 was reached. Tumor weight and volume were measured, followed by embedment in OCT medium and prior to sectioning using a microtome-cryostat.

Replications
The in vitro experiments were repeated independently at least twice, while the in vivo experiments were conducted two times, each with a different dose.

Statistics
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 signi cant 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 signi cant for p < 0.05.

Amiodarone Affects Viability, Proliferation and Mobility of GBM Cells
Viability assays were carried out on U-87 MG cells, human primary GBM cells, after either 24 or 72 h of incubation with Amiodarone, to investigate cytotoxic and proliferation-rate effects, respectively. Amiodarone induced a mild cytotoxic effect with 50 μM, the highest treatment concentration used in our in vitro experiments, reducing the viability of the cells by 28% after 24 h. However, a reduction in the proliferation rate after 72 h already occurred with 10 μM, where 10 and 50 µM treatments led to 18% and 56% reduction, respectively (Fig. 1a). A transwell migration assay was conducted to explore Amiodarone's effect on cell mobility, a pivotal step in cancer progression. In this, cells were seeded in the upper compartment containing 1% FCS and were allowed to migrate to the underside of this higher compartment which contained 10% FCS. After 18 h, migrated cells were counted as presented in Fig. 1b.

Amiodarone Increases Susceptibility of GBM Cancer Cells to Anoikis
We evaluated Amiodarone's effect on non-adherent cell survival, since GBM is known to be highly metastatic, and non-adherent cell survival in the blood circulation is a crucial factor in metastatic development [17,18]. In this assay, we examined the ability of detached U-87 MG cells treated with Amiodarone to withstand apoptosis for 72 h on a non-adherent surface generated by pHEMA. Fig. 2a shows that control cells had higher survival rates than treated cells: 1 µM and 10 µM signi cantly reduced cell viability by 34% and 38%, respectively, compared with untreated cells. A marked decrease of 48% in cell viability was detected with 50 µM compared with untreated cells.

Amiodarone Reduces Colony Formation of GBM Cells
There is a correlation between the malignant behavior of tumors and their ability to form colonies in different types of matrixes [19]. In order to investigate whether Amiodarone affects GBM cells' ability to form colonies, we performed a soft agar colony formation assay over 15 days. Fig. 2b, c show Amiodarone's dose-dependent effect on the GBM cells' ability to establish colonies. Cells treated with 5, 7 and 10 µM Amiodarone presented a signi cant reduction in the number of colonies formed, 23%, 36% and 45%, respectively. Notably, 50 µM of Amiodarone exhibited almost a complete reduction of 92% in the number of colonies formed.

Amiodarone Inhibits GBM Spheroid Growth
Spatial cell-cell interaction and proliferation of U-87 MG cells can be monitored using a 3D spheroid assay which consists of an assembly step and cell growth. Relative spheroid size grown in roundedbottom 96-well plates under varying concentrations of Amiodarone (0, 1, 10 and 50 µM) was calculated using MATLAB code analysis, as previously shown [20], after 1, 5 and 7 days, as mentioned in the Materials and Methods section (Supplementary g. 1). Cells under all conditions assembled into spheroids within 24 h of cell seeding (Fig. 2d, e). All control and 1 µM Amiodarone-treated spheroids proliferated and increased in size over time. The control spheroids more than doubled their size after 5-7 days, whereas spheroids treated with 1 µM increased by only ∽40% up to day 7. Spheroids treated with 10 µM Amiodarone preserved their size and did not show further growth. However, 50 µM Amiodarone reduced spheroid size on days 5-7 by ∽30-40%.

Amiodarone Exhibits Anti-Angiogenic Activity In Vitro in HUVECs
Since angiogenesis is a prominent feature of GBM with a strong impact on tumor growth and progression [15,21,22], the functionallity of endothelial cells was investigated. HUVEC viability, mobility and functionality were assessed under Amiodarone treatment. HUVEC cytotoxicity and proliferation assays were measured after 24 and 72 h of drug incubation, respectively. Amiodarone induced cytotoxic effects in all the concentrations used (0.1 -10 μM) while 10 μM led to a 32% reduction in cell viability. Even the lowest concentration used, 0.1 µM Amiodarone, signi cantly reduced HUVEC proliferation (Fig. 3a). 0.1, 1 and 3 µM of the drug reduced cell proliferation by 10-18%. A signi cant dose-dependent effect was observed with higher concentrations of Amiodarone: 5, 7 and 10 µM inducing a reduction of 42%, 57% and 67%, respectively. These results imply that HUVECs are more susceptible to Amiodarone's inhibiting action compared with U-87 MG cells.
The drug's effect on HUVEC mobility was measured in the scratch assay. After 36 h of exposure to the drug, 1 µM Amiodarone-treated cells nearly populated the entire scratch wound area (relative wound density ∽90%), much like the untreated cells shown in Fig. 3b, c. The cells' migration decreased by ∽15% with 5 µM and by ∽30% with 7 and 10 μM.
The tube formation assay predicts the ability of endothelial cells to form stable tubes/branches by mimicking in vivo angiogenesis [23]. In this assay, HUVECs treated with 0.1 µM Amiodarone showed no signi cant effect; however, cells treated with drug concentrations of 1 and 5 µM reduced by ∽40% the number of meshes and branches formed (Fig. 4a, b) compared with untreated cells.

Systemic Amiodarone Treatment Has an Anti-Angiogenic Effect in the Mouse Corneal Neovascularization Model
The mouse corneal micropocket assay using bFGF-induced neovascularization was utilized to evaluate the direct anti-angiogenic activity of Amiodarone following systemic administration in C57BL/6 mice. Amiodarone was administered I.P., 0.05 mg/kg or 0.1 mg/kg q.d., over a course of 5 days. These treatments resulted in a reduction of 22% or 41%, respectively, in the corneal vessel area compared with the control (Fig. 5a, b) with no evidence of side effects. These results indicate a signi cant inhibition of vessel formation after 5 days of drug treatment.

Amiodarone Decreases Angiogenesis in the Matrigel Plug Assay In Vivo
Amiodarone was locally administered in a quantitative mouse Matrigel angiogenesis assay to investigate its ability to inhibit angiogenesis. Matrigel containing VEGF and bFGF mixed with Amiodarone 0.05 mg/plug was injected S.C. into C57BL/6 mice. On day 8, immunohistochemical staining of blood vessels was performed using anti-CD31 antibodies, enabling visual evaluation of the vasculature in the Matrigel plugs (Fig. 6a). The Matrigel plugs mixed with Amiodarone showed signi cantly reduced blood vessel formation, as can be visually assessed in Fig. 6a-c. Untreated mice presented bloody plugs surrounded by massive blood vessels with open lumen structures, suggesting functional vessels, compared with Amiodarone-treated mice that showed poor vasculature. Quanti cation of the in ltrated endothelial cells in Matrigel plugs was accomplished in a single-cell suspension by Fluorescence-Activated Cell Sorting (FACS) (Fig. 6d). In ltrating endothelial cells were substantially reduced by 60% in the Amiodaronetreated mice group compared with the control group.

Amiodarone Suppresses Tumor Growth and Vasculature of GBM Xenografts
For the in vivo anti-tumor activity of Amiodarone, U-87 MG cells were evaluated in S.C. injected Foxn1 nu mice. When the tumors reached an average volume of ∽200 mm 3 , the mice were divided into two groups of treated (Amiodarone 0.1 mg/kg q.d. I.P. injection) and untreated mice. After the experiment was terminated, when an average volume of ∽1 000 mm 3 was reached (on day 14), the tumors were surgically resected and collected for histological examination. Signi cant differences in tumor size and weight between the treated and untreated groups were observed, with average tumor weights of 0.75 gr or 2.36 gr (∽3-fold reduction) and average tumor volumes of 308 mm 3 or 1106.25 mm 3 (>72% reduction), respectively ( Fig. 7a-d). Histological H&E staining showed that in addition to reduction in volume, the treated tissues exhibited a more porous and defective structure compared with untreated tumors, possibly due to increased necrosis (Fig. 7e). Immunohistochemical staining of microvessels with anti-CD31 antibodies presented notably lower vascularization compared with untreated tumors (Fig. 7f). Gross pathology revealed that spleens retrieved from the control group displayed signi cant splenomegaly compared with the Amiodarone-treated mice (Supplementary g. 2). With the 0.1 mg/kg q.d. I.P. injection treatment, there were some indications of toxicity in the form of stomach swelling, abnormal liver, and weight loss of up to 14%. Two out of the ve mice died on day 10 and 14. One mouse from the control group died on day 14. Reducing the dose to a 0.05 mg/kg q.d. I.P. showed good safety without any signi cant weight loss while still maintaining effective activity, reducing tumor volume by 40% compared with the control mice on day 24 (Supplementary g. 3).

Discussion
Amiodarone, a widely administered anti-arrhythmic drug [1], was shown by several pre-clinical studies to reverse multidrug resistance and sensitize several types of cancers to chemotherapy. Pre-clinical studies demonstrated its anti-cancer effects as a single therapeutic or in combination with other anti-cancer drugs on different cancers, including prostate, hepatocellular and breast carcinomas [6][7][8][9][24][25][26]. Based on this, we opted to investigate its anti-cancer effects on GBM, the most common and aggressive primary malignant brain tumor with a dismal prognosis outcome [13]. GBM is a highly vascularized tumor, but in many cases, it shows the potency to invade adjacent and distant areas of the brain in an angiogenesisindependent manner [18,27]. Therefore, nding drugs with dual effects that can target both cancer cells and angiogenesis is of high value. We hypothesized that, in addition to Amiodarone's previously demonstrated anti-cancer effects, it also exhibits prominent anti-angiogenic activity, consequently offering a possible new approach to treating highly vascularized tumors.
The direct effects of Amiodarone on GBM cells were demonstrated in a wide variety of in vitro assays using U-87 MG cells. Its most prominent effect was shown to be on cell mobility as demonstrated in the migration assay, where 10 µM of Amiodarone led to a more than 80% inhibition in the number of migrated cells. Proliferation was also reduced with exposure to Amiodarone, but in higher doses (~60% inhibition was obtained with 50 µM).
The observed effect of the drug on cell viability may be related to the induction of cell death as previously shown. Kim et al. demonstrated that Amiodarone induced apoptosis in U-87 MG cells in a dosedependent manner, and this effect was synergistically enhanced on various malignant glioma cells when combined with tumor necrosis factor (TNF)-related apoptosis-inducing ligand. The associated effect was shown to be mediated by induction of the unfolded protein response (UPR) and an increase of intracellular Ca 2+ levels, which induced cell death [26]. More recent work demonstrated that the UPR is involved in affecting tumorigenic hallmarks such as cellular metastatic potential and angiogenesis [28,29] and plays an important role in GBM growth and progression [30].
To further investigate Amiodarone's ability to impede the metastatic potential of cancer cells, we measured its effect on the survival of cells in circulation. Anoikis is a programmed cell death activated when cells lose their contact with the extracellular matrix (ECM) occurring during their invasion into the blood circulation. Cancer cells that succeed in evading Anoikis may then colonize in distant organs and begin the metastatic process [17]. We show that Amiodarone in the range of 1-50 µM, reduced the survival of U-87 MG cells on a non-adherent surface, by ~30-50% (Fig. 2a).
The colonization potential of cancer cells involves an anchorage-independent process requiring cell-cell interactions and proliferation [31]. In our experiments, Amiodarone reduced colony formation and signi cantly inhibited U-87 MG cells' ability to form aggregates in soft agar in a dose-dependent manner, >90% inhibition. Recently published data by Chang et al. showed a similar trend of Amiodarone's effect on cervical carcinoma cell colonization, suggesting an associated increase in truncated serine and arginine-rich splicing factor 3 (SRSF3), and reduction of RNA (miR)-224. Amiodarone's suppressive effects on the SRSF3 promoter activity were further veri ed in GBM8401 and U118MG glioma cell lines [6]. Numerous studies demonstrated that SRSF3 and miR-224 are frequently upregulated in glioma cells and are associated with tumor progression and a poor prognosis in glioma patients [32,33].
Similar to colony formation that requires 3D cell interactions, the multicellular spheroid model provides an excellent physiologically relevant model in an ECM-free condition. Unlike monolayer culture, spheroids enable spatial cell-cell interactions and the generation of tissue-like features [20,34]. In the spheroid model, Amiodarone was found to signi cantly suppress spheroid growth, while 50 µM also induced a notable shrinkage in size. Similar results were reported for patient-derived glioblastoma stem-like cultures showing induction of apoptosis and decreased invasion of tumor neurospheres in association with change in cell cycle and survival gene expression [35]. This is in agreement with other reports demonstrating that Amiodarone inhibited cancer cell migration in vitro, and inhibited invasion in a Matrigel assay of human breast cancer cells MDA-MB-231, as well as other murine tumor cells, B16OVA melanoma, JC and 4T-1 breast cancer [24]. Our results point to Amiodarone's potential for reducing tumorigenesis and invasiveness, as demonstrated by the con rmed reduction in motility, invasiveness, proliferation, adherence-free survival and colonization -factors indicative of a tumor's metastatic potential [36].
It is well established that the tumor microenvironment and, more speci cally, angiogenesis, have an immense impact on tumor growth [21,22]. Amiodarone's direct effect on endothelial cell function was measured in many in vitro assays using HUVECs. We found that HUVECs were more sensitive to Amiodarone compared with U-87 MG cells with respect to viability. Our observations are in agreement with previous studies indicating that Amiodarone may have selective activity, showing relatively nontoxic effects with normal astrocytes while inducing apoptosis in GBM cells [26,35]. In concentrations ranging from 0.1 to 10 µM, Amiodarone signi cantly reduced cell proliferation (up to ~70%), cell mobility (∽30%) and formation of tubes/branches (∽40%) (Fig. 3, 4). Considering that angiogenesis involves a series of events, including endothelial cell migration, proliferation and tubular formation [37], our in vitro results imply that Amiodarone has an anti-angiogenic effect that may be clinically relevant. Moreover, these results, showing tumor angiogenesis suppression, are in line with our results of the in vivo angiogenesis model -the Matrigel plug assay [38]. In this animal study, Amiodarone that was mixed into Matrigel plugs (0.05 mg/plug), as a mode of local treatment, extensively reduced the vascularization of the plugs over the course of 8 days. This model provides a direct assessment for endothelial cell recruitment and their morphogenesis into the blood vessel network. A similar trend was con rmed in the mouse corneal micropocket angiogenesis assay, where Amiodarone, administered systemically over a course of 5 days, induced up to a 40% reduction of vessel formation.
Although angiogenesis is considered to be a hallmark of GBM progression [15,29,30,52], currently the only FDA-approved drug targeting angiogenesis in GBM is the anti-VEGF antibody Bevacizumab [53,54], prescribed for recurrent GBM or as a second-line treatment after standard treatment failure [55]. Several meta-analyses demonstrated that the addition of Bevacizumab to standard therapy improved progression-free survival but failed to improve the overall survival rate. Moreover, there are associated side effects of neurocognitive decline and in health-related quality of life [56][57][58]. The lack of signi cant responses to this treatment along with its serious adverse effects emphasizes the advantage of agents with selective and wider action directed against cancer and endothelial cells.
GBM tumor xenografts provide an overall assessment of tumor progression, including both direct processes of cancer cells and indirect anti-cancer tumor microenvironment processes. Amiodarone at the dose of 0.1 mg/kg q.d. I.P., over a course of 14 days, showed over a 70% reduction in tumor volume and weight. Immunohistological staining con rmed an associated reduction in angiogenesis, where untreated tumors showed rich microvessel networks compared with treated mice. The drug's underlying mechanism of action is yet unclear and involves multiple possible target sites, but taken together, our results suggest that Amiodarone possesses anti-angiogenic attributes along with its anti-tumor activity -a dual effect.
One of the key issues with Amiodarone is its adverse side effects when used to treat cardiac dysrhythmias. These include: dangerous polymorphic ventricular arrhythmia due to its blocking potassium channels [39]; dermatologic changes, including photosensitivity and blue-gray skin discoloration [40]; and thyrotoxicosis due to its similarity to thyroxine [10,41]. But the drug's most serious side effect is pulmonary toxicity that can lead to pulmonary brosis and death [42]. These clinical symptoms may last months after cessation due to a long biological half-life [12] and may explain the cohort study by Boursi et al., who found that among patients with GBM, active users of Amiodarone for the treatment of their cardiac conditions had a worse survival rate than those who previously had never received the drug [43].
Given this, our study highlights the fact that the anti-cancer activity in the GBM xenograft model can be obtained with an ultra-low dose of Amiodarone compared with the human equivalent dose that is currently prescribed for cardiovascular conditions. The dose of 0.1 mg/kg q.d. I.P. in mice showed some signs of toxicity. However, half of this dose was well tolerated over a course of 24 days, while still inducing a signi cant reduction in tumor growth. When the same doses of 0.1 mg/kg or 0.05 mg/kg q.d.
I.P. were administered for 5 days in the mouse corneal micropocket assays, no side effects were evident, suggesting the possibility of ne tuning the dosage for chronic treatment. Since the pharmacokinetics of substances administered I.P. are similar to those of oral administration [44], it can be concluded that our highest dose administered to mice is equivalent to 0.48 mg/day in a human adult, based on the FDA's guidelines and Reagan-Shaw's conversion formula [45,46]. This is a signi cantly lower dose than the one used for treating antiarrhythmic diseases (typically between 100 to 400 mg/day), which is between 200 and 800-fold lower [47].
The positive outcomes using an ultra-low dose, combined with the drug's dual effect, emphasizes Amiodarone's great potential for treating highly vascularized tumors, either systemically or locally.

Conclusions
Taken together, our in vitro and in vivo results suggest that Amiodarone possesses both signi cant anticancer activity -demonstrated by reduction of tumorigenic processes directly related to tumor progression, such as invasion and colonization -and signi cant anti-angiogenic activity. We found that ultra-low dose Amiodarone induced tumor shrinkage in GBM xenografts and profoundly reduced blood vessel formation in all mouse models. Repurposing Amiodarone for ultra-low dose systemic or local treatment, in light of its suggested multi-targeting mechanisms, may bypass the drug's negative side effects and become a new standard for cancer treatment. Further study of Amiodarone's underlying cellular mechanisms may also reveal its ability to combat other solid tumors, particularly ones that are largely dependent on angiogenesis.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests.  Amiodarone inhibits corneal neovascularization after I.P. treatment. Amiodarone's anti-angiogenic activity evaluated following systemic administration in the mouse corneal micropocket assay using bFGFinduced neovascularization. C57BL/6 mice were injected I.P. with Amiodarone 0.05 mg/kg or 0.1 mg/kg q.d. for 5 days. a Representative images taken from treated (0.1 mg/kg q.d.) and untreated mice on day 5. bFGF pellets detected as a white spot in the center of the cornea. Blood vessels growing from limbal periphery are reduced in the Amiodarone-treated group compared with the untreated group. b

Funding
Normalization of vessel area (%) for untreated and Amiodarone treated mice (0.05 mg/kg and 0.1 mg/kg q.d.). Vessel area was calculated using the following formula: [Clock hours × Vessel length (mm) × π × 0.2 mm]. Graphs indicate signi cant inhibition of vessel formation after 5 days of treatment. Objective lens 400x, (in each group n=10). *p<0.05, ***p<0.001, compared with non-treated control mice. Results are presented as mean ± SEM.

Figure 4
Amiodarone affects HUVEC function in 3D. a, b Angiogenesis tube formation assay of HUVECs seeded on Matrigel® after 6 hours of incubation, using IncuCyte® S3 Live-Cell Analysis System with ranging concentrations of Amiodarone (0, 0.1, 1 and 5 μM). Statistical analysis of tube formation hallmark (number of meshes formed) and images processed using ImageJ software; representative images are presented, n=4. Scale bar: 1.9 mm. *p<0.05, compared with non-treated control cells. Results are presented as mean ± SEM.

Supplementary Files
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