Patient-derived metastatic melanoma cells treated with vitamin D and anticancer drugs

1. Immunofluorescence staining

For immunofluorescence analyses cells isolated from malignant melanoma tumour were seeded in 8-well chamber slides, fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized for 5 min in 0.2% Triton X100 in PBS. Slides were washed three times with PBS and incubated with 1% BSA for 30 min at RT. Following blocking appropriate primary antibody in 0.1% BSA (Thermo Fisher Scientific: PMEL (HMB45) MA5-13232, 1:40; Melan-A MA5-15237, 1:100 or MA5-32217, 1:200; Cell Signaling Technology: FAP Cat. No. 66562, 1:100) was applied and the slides were incubated in humidified chamber at 4 °C overnight. Subsequently slides were washed three times in PBS, and incubated with secondary antibody (ThermoFisher Scientific either Alexa Fluor® 488 conjugate goat anti rabbit IgG A11008 or Alexa Fluor® 594 conjugate donkey anti mouse IgG A21203) solution in PBS for 1 h at RT in dark. Following three washing steps in PBS the slides were counterstained with 4′,6′-diamidinio-2-phenylindole (DAPI). Images were collected with a fluorescence microscope Olympus cell Vivo IX 83. For a-SMA (alpha-smooth muscle actin) immunofluorescence analyses the procedure of staining with Melan A was proceeded to the appropriate secondary antibodies as described above. Subsequently antibody against a-SMA PE‑conjugated (R&D Systems: IC1420P, 1:50) was applied to the specimen for an additional 30 min in humidified chamber at 4 °C. Following three washing steps in PBS the slides were counterstained with DAPI. Images were collected with a fluorescence microscope Olympus cell Vivo IX 83 as Z‑stacks.

1. SRB viability assay

The viability of cells isolated from metastatic malignant melanoma tissues treated with 1,25(OH)₂D₃, vemurafenib or both was assessed with sulforhodamine B (SRB). The cells were seeded in 96-well plates at 2,000 of cells per well density and left overnight. Subsequently the cells were treated simultaneously with serial dilutions of vemurafenib (3,15-200 nM) and 1,25(OH)₂D₃ at 100 nM or 1 µM concentration for 72 h. Cells were fixed with 10% trichloroacetic acid for 1 h at 4°C, washed with distilled water and stained for 15 min with 0.4% SRB solution (Sigma–Aldrich; Merck KGaA) in 1 % acetic acid. SRB dye was solubilized using a solution of 10 mM buffered Tris Base (pH 10.5) and absorbance measured at 570 nm using an Epoch spectrophotometer (BioTek, Winooski, USA). The relative IC50 value was calculated as the mid‑point between no inhibition and the maximum observed decrease in proliferation (n=6).

1. Proliferation rate assay

Proliferation rate analysis of the cells isolated from malignant melanoma tissues treated with 1,25(OH)₂D₃ at 100 nM concentration and cediranib at 1000 nM concentration was carried out as a live imaging with Olympus cell Vivo IX 83, equipped with CO₂ incubator and enables for stable cell cultivation at 37°C, controlled humidity and 5% CO₂ during the whole 72 h experiment. Results were calculated with the Olympus cell Vivo IX 83 software with use of TruAI technology, normalized with 1.0 at the beginning of the experiment (n = 4).

1. Wound closure rate

The experiment was carried out as a live imaging with Olympus cell Vivo IX 83 and cell migration process was observed during 72 h incubation. The cells isolated from malignant melanoma tissues were seeded on a 8-well chamber slide (1.0 × 10⁵ cells per well). A mechanical wound was created by scraping the confluent monolayer of cells with a 200 µL pipette tip. Cediranib at 1000 nM concentration was added to the cells pretreated with 1,25(OH)₂D₃ at 100 nM concentration for 24 h. The cell free area was calculated as a percentage closure relative to original size [(wound area in mm²)*100/(original wound area in mm²)] with use of TruAI technology of the Olympus cellSens software (n = 4).

1. Mitochondrial membrane potential

Patients-derived cells were seeded on an 8-well chamber slide (2.0 × 10⁴ cells per well). After 24 h, cells were stained with MitoTracker™ Red CM-H₂Xros (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 70 nM concentration for 20 min at 37°C, and washed two times with PBS. Cells were pretreated with 1,25(OH)₂D₃ at 100 nM concentration for 24h. Subsequently cediranib at 1000 nM concentration was added to the cells in a fresh medium and mitochondria were observed for additional 24 h. The experiment was carried out as a live imaging with Olympus cell Vivo IX 83. Results were calculated as an area fraction ROI (“the mitochondria”) normalized with 1.0 at the beginning of the experiment with the Olympus cell Vivo IX 83 software, (n = 3 - 4).

1. Measurement of melanoma bioenergetics

Patients-derived cells were seeded at a density of 20,000 cells per well in an Agilent Seahorse microplate and cultured at 37°C in 5% CO₂ until reaching 80% confluency in 80 μL culture medium per well. Subsequently cells were incubated for 24 h with vemurafenib at 200 nM concentration or cediranib at 1000 nM concentration with or without 1,25(OH)₂D₃ at 100 nM concentration. Additionally, selected cells were also pretreated with 1,25(OH)₂D₃ at 100 nM concentration for 24 h and then incubated with the chosen anticancer drug at appropriate concentration for 24h. The Cell Mito Stress Test was performed as described by Franczak M. et al. (Franczak, M.; Kutryb-Zajac, B.; El Hassouni, B.; Giovannetti, E.; Granchi, C.; Minutolo, F.; Smolenski, R.T.; Peters, G.J. The effect of lactate dehydrogenase-A inhibition on intracellular nucleotides and mitochondrial respiration in pancreatic cancer cells. Nucleosides Nucleotides Nucleic Acids 2022, 1-11, doi:10.1080/15257770.2022.2031215.). The medium was changed before analysis for the XFp assay medium with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate (Agilent, US). During the assay 1.5 μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and 0.5 μM rotenone with antimycin were added sequentially for the Cell Mito Stress Test. The XFp sensor cartridge, with compounds used in the test, was incubated at 37 °C in the non-CO₂ incubator for 15 minutes before the assay and plate with cells for 45 minutes. Each analysis was performed by the Agilent Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). The oxygen consumption rate (OCR) and the mitochondrial respiration parameters were calculated with the software provided with the Agilent Seahorse XFp Analyzer (n = 2-5).