We have adopted stringent criteria for establishing the identity of the cell lines used in this study. New lines are purchased directly from trusted repositories (e.g., ATCC), while legacy cell lines (e.g., those cell lines which are gifts from collaborators) are stratified into 3 levels of confidence after sequencing. ‘CONFIRMED' cell lines are either new lines from ATCC or those which have demonstrated an unequivocal match between our designation and the STR database after STR genotyping. For those with ‘no hits' in the STR database, we performed a manual search of key melanoma mutations [e.g., BRAF(V600E), NRAS(Q61), etc.] reported for each line in either the Catalogue Of Somatic Mutations In Cancer (COSMIC), the Cancer Cell Lines Encyclopedia (CCLE), or in individual publications in the literature. We subsequently compared Sanger or whole exome sequence information generated in our laboratory for these lines, and if there is a direct match between our sequence information and the public domain data, these cell lines are designated as ‘CONSISTENT'. For cell lines without public domain information, we analyzed the levels of MITF (M-isoform) to determine whether the cells are compatible with melanocytic cells. Those that express significant M-isoform MITF are designated as ‘COMPATIBLE'.

When selecting cell lines for experimentation, the order of preferential selection is ‘CONFIRMED' > ‘CONSISTENT' > ‘COMPATIBLE'. Furthermore, all cell lines have been tested for common pathogens, such as mycoplasma, and are pathogen-free. The following are accession numbers for cell lines from Cellosaurus (web.expasy.org/cgi-bin/cellosaurus/search): MP41 (CVCL_4D12), Mel-270 (CVCL_C302), Mel-202 (CVCL_C301), OMM2.3 (CVCL_C306), CHL-1 (CVCL_1122), MeWo (CVCL_0445), SK-MEL-2 (CVCL_0069), SK-MEL-119 (CVCL_6077), Mel JuSo (CVCL_1403), IPC-298 (CVCL_1307), UACC-903 (CVCL_4052), SK-MEL-28 (CVCL_0526) and A375 (CVCL_0132).

To initially identify UMM-active compounds, the UMM cell line, OMM2.3, was subjected to viability screening using an FDA-approved Drug Library (Selleckchem) that included 1,018 drugs (Table SI). Cells were seeded at 2,000 cells/well using an automated plate filler in a 384-well plate and incubated for 24 h at 37°C. Subsequently, a 3 µM concentration of each drug was added to the plate via pin-transfer and the plates were incubated for an additional 72 h at 37°C. Drug efficacy was measured via a proliferation ratio using CellTiter-Glo^® Luminescence assay (Promega) and an EnVision plate reader (PerkinElmer).

Based on the results of this initial drug screen, 4 UMM (MP41, Mel-270, Mel-202 and OMM2.3) and 10 CMM (CHL-1, MeWo, SK-MEL-2, SK-MEL-119, Mel JuSo, IPC-298, UACC-903, SK-MEL-28, MGH-CH-1 and A375) cell lines were selected for additional drug screening analysis with the dihydropyridine calcium channel blocker (CCB), amlodipine (Selleck Chemicals), and the non-dihydropyridine CCBs, verapamil and diltiazem (Sigma-Aldrich). The cells were seeded at 2,000 cells/well in 96-well plates, incubated at 37°C for 24 h, and subjected to 9 doses of each drug in triplicate. Cell viability was measured after 72 h of incubation at 37°C in the presence of drug using CellTiter-Glo^® Luminescence assay (Promega). An equivalent volume of DMSO was used as a control, and the results were normalized to this control value. IC₅₀ values were calculated using GraphPad Prism software (Version 7) and compared between the UMM and CMM lines using two-sided t-tests. Intracellular calcium depletion after 48 h of treatment with 0, 2, 4 and 8 µM amlodipine was additionally visualized using 1 µM calcium-sensitive Rhod 2 fluorescent dye (Thermo Fisher Scientific) on one UMM line (OMM2.3) and one CMM line (A375).

All CMM lines were cultured in Dulbecco's modification of Eagle's medium (DMEM; Thermo Fisher Scientific) with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin/streptomycin (Thermo Fisher Scientific). UMM lines were cultured in RPMI-1640 with 10% FBS, 1% each of HEPES, L-glutamine, penicillin/streptomycin, and 0.1% of β-mercaptoethanol (all from Thermo Fisher Scientific).

We then performed drug screening analyses on a smaller cohort of melanoma cell lines in a 3D culture system to evaluate the ability of the drug to penetrate a tumor-like spheroid. For spheroid generation in 3D culture, 100 µl of Matrigel^® were added to 96-well plates followed by incubation at 37°C for 1 h to allow for matrix solidification. Cell lines suspended in 70 µl of their respective growth media were then seeded on top of the matrix base at an empirically-determined density to allow for proper growth over the experimental duration (A375 cells, 800 cells/well; CHL-1 cells, 1,000 cells/well; IPC-298 cells, 2,000 cells/well; MP41 cells, 2,500 cells/well; OMM2.3 cells, 2,500 cells/well). Following an additional incubation at 37°C for 30 min, a 10 µl mixture of Matrigel^® and DMEM (at a ratio of 1:10, respectively) was carefully transferred to each well and allowed to settle to the bottom of the cell culture medium. Thus, the cell layer was secured in place between 2 layers of Matrigel^® extracellular matrix.

The cells were cultured for 11 days to allow for spheroid formation, after which time a 20 µM concentration of amlo-dipine was added on day 0. Following a 48-h incubation at 37°C in the presence of drug or the DMSO control, the diameter of randomly-sampled spheroids in each colony was measured on day 2 using light microscopy (Zeiss Axiovert 100 TV) and ImageJ software. Mean spheroid diameters for each colony were calculated from these measurements and normalized to the mean diameter of the corresponding cell line's colonies on day 0. The normalized mean diameters on day 2 were compared between conditions using two-sided t-tests.

To characterize the mechanism responsible for the observed inhibitory effects of calcium channel blockade, we performed several functional assays on UMM and CMM cell lines. Cell cycle analysis was first performed using propidium iodide (PI) staining. The cell lines were plated on 6-well plates at 50% confluence and cultured for 24 h. They were then treated with 5, 10 or 20 µM of amlodipine and incubated at 37°C for an additional 24 h. The cells were then trypsinized, centrifuged at room temperature (1,500 rpm, 5 min), and washed with PBS. Following re-suspension in 100 µl of 1X PBS, 250 µl of 70% cold ethanol was added drop-wise while vortexing. The samples were frozen at -20°C overnight for fixation. The following day, the cells were thawed, and the ethanol was removed. The cells were then washed with 1X PBS and resuspended in 0.5 ml PI staining solution [1 ml of PI (100 µg/ml), 80 µl of RNAse (100 µg/ml), and 20 µl of NP-40 (0.1%); Molecular Probes] prior to incubation at room temperature in the dark for 30 min. All analyses were performed on FACSCalibur Flow Cytometer (BD Biosciences).

After observing significant growth inhibition in our cell viability analyses, we hypothesized that amlodipine may be initiating its effects on cell growth through the induction of apoptosis. We used Annexin V expression as an early apoptotic marker. An Alexa Fluor 488 Annexin V/PI staining kit was used following the manufacturer's protocol (Life Technologies; Thermo Fisher Scientific). Briefly, the cells were collected after 24 h of 5, 10 or 20 µM amlodipine treatment, washed twice with PBS, and stained with Alexa Fluor Annexin V for 20 min followed by PI for 1 min both at room temperature. Flow cytometry was performed on a BD FACSAria (BD Biosciences), and all data were analyzed using FlowJo 10.0.8 software.

To detect for the association of a senescent-associated effect of amlodipine treatment, 2 UMM (MP41 and OMM2.3) and 2 CMM (A375, CHL-1 and IPC-298) cell lines were plated on a 12-well plate and cultured for 24 h. The cells were then treated with 3 different concentrations of (2, 4 and 8 µM) of amlodipine and incubated at 37°C for an additional 48 h. The medium was then removed, and the cells were washed with 1X PBS followed by the addition of 0.5 ml of fixing solution for 15 min. The plate was again washed with 1X PBS prior to the addition of 0.5 ml of staining solution mix detection kit (470 µl staining solution, 5 µl staining supplement, 25 µl of 20 mg/ml SA-β-gal in DMF; BioVision). The cells were incubated overnight at 37°C and senescence was visualized at 24 h using light microscopy (Zeiss Axiovert 100 TV).

Western blot analysis was also preformed to validate the apoptotic effects. Uveal lines (MP41 and OMM2.3) were grown on 60 mm plates, treated with either 10 µM of amlodipine or an equivalent volume of DMSO for the controls, and incubated at 37°C for 24 h. Protein extraction was then performed using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS; Boston Bioproducts). A Lowry assay was performed to determine protein quantification, and sample concentrations were adjusted accordingly based on the standard curve produced. A total of 10 µg of each sample were loaded per well and run through a 4-12% SDS-PAGE gel prior to transfer to a nitrocellulose membrane (Bio-Rad Laboratories). Following incubation with 5% non-fat milk in TBST for 1 h at room temperature, the membrane was washed with TBST and probed with rabbit antibodies against cleaved PARP (cat. no. 9541s; Cell Signaling Technology) and mouse antibodies against GAPDH as a loading control (cat. no. ab8245; Abcam) at a 1:3,000 dilution for 16 h at room temperature. The blots were washed again with TBST and then incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated anti-rabbit (cat. no. 7074s; Cell Signaling Technology) or anti-mouse antibodies (cat. no. 7076s; Cell Signaling Technology) for 16 h at 4°C. The blots were washed again with TBST and developed with Clarity western ECL substrate (Bio-Rad Laboratories).

All viability and functional assays were performed in triplicate and with biological replicates of each class of melanoma cell lines (UMM and CMM) where appropriate. The Student's t-test was used to perform both comparisons of mean IC₅₀ values following two-dimensional CCB treatment in CMM vs. UMM and comparisons of mean spheroid growth after three-dimensional CCB treatment in treated vs. control groups of each individual cell line. The comparison of apoptosis induction between treated and control groups was performed using one-way ANOVA and Dunnett's multiple comparisons test (with control group as reference). Significant differences for all tests were assumed at P-values <0.05. All statistical analyses were performed using GraphPad Prism 8 software.