The present study was approved by the Ethics Committee of the Krasnoyarsk State Medical University (protocol no. 73/2016; approval date, 16 December, 2016) and the Krasnoyarsk Regional Clinical Oncology Center named after A.I. Kryzhanovskiy (protocol no. 8, 14, June, 2017). All procedures were performed according to the Institutional Safety Instructions, which included biosecurity. All researchers are trained in Institutional Safety/Biosafety and follow-up instructions are provided periodically every 3 months, followed by signature confirmation. The present study was also approved by the Institutional Bioethical Commission (protocol no. 3; approval date, 27 October 2017).

The operative samples were obtained from the Department of General Oncosurgery of Krasnoyarsk Regional Clinical Oncology Center named after A.I. Kryzhanovskiy and primary cell lines were prepared as described previously (18). In the present study, two primary melanoma cell populations were obtained from two drug-naïve melanoma patients. Written informed consent was obtained from each patient before the study. Inclusion criteria were as follows: pathologically confirmed primary malignant melanoma and no know second primary malignancies; age more than 18 years old; no systemic treatment in the four weeks prior to surgical treatment. Tumor tissues predominantly consisted of tumor cells that did not contain an abundance of stromal or vascular cell types. They were processed immediately after surgical excision and melanoma cell suspensions were prepared within 3 h. Single cell suspensions were obtained by mechanical dissociation of tumor tissues. These two melanoma cell populations were named K2303 and B0404 thereafter.

K2303 cells were obtained from a 30-year-old female patient with the melanoma localized on the right leg. It exhibited a superficial type of spreading with a Breslow’s depth of 2.2 mm. B0404 cells were obtained from a 55-year-old male patient, the melanoma was localized on the right shin with a superficial spreading type and Breslow’s depth of 2.0 mm.

Cells were cultured in RPMI-1640 medium with L-glutamine (Gibco, Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) in the presence of antibiotics and antimycotics (10,000 U/ml penicillin G, 10,000 µg/ml streptomycin and 25 µg/ml amphotericin B; HyClone; Cytiva) at 37°C and 5% CO₂ using a CO₂ incubator (Sanyo Electric Co., Ltd.).

To estimate the 50% inhibitory concentration (IC₅₀) of DTIC, cells were treated with a series of DTIC (Sigma-Aldrich Co., USA) dilutions. For this purpose, melanoma cells were placed in a 96-well plate at a density of 1x10⁴ cells per ml and cultured for 48 h at 37°С. In total, four different concentrations of DTIC in DMSO (Panreac Quimica S.A.) were added to the culture medium to achieve the final concentrations of 250, 500, 750 and 1,000 mg/l. Since each dose also contains 1% DMSO, 1% DMSO was used as the negative control. The cells were incubated with DTIC for 72 h at 37°С and 5% CO₂.

To evaluate cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Invitrogen; Thermo Fisher Scientific, Inc.) assay was used. The culture medium was first replaced with that without the drug before 0.5 mg/ml MTT was added. After 4 h at 37°С the cells were lysed with DMSO and the amount of formazan formed was evaluated using an EFOS-9305 spectrophotometer (Shvabe Photosystems, Jsc) at a wavelength of 560 nm to measure light absorbance. The experiment was performed in triplicate. Using the dose-dependency curve of DTIC-mediated inhibition, IC₅₀ was calculated using the GraphPad Software Prism 7.05/e (San Diego, CA, USA).

Melanoma cell lines were seeded into 24-well plates at a density of 1x10⁵ cells per ml. After 24 h the cells were treated with 5.5 mM DTIC or 1% DMSO. In parallel, 1% DMSO was used as the negative control. After 72 h at 37°C and 5% CO₂ incubation the cells were stained using an annexin V-fluorescein isothiocyanate (FITC)/7-aminoactinomycin D (7-AAD) kit (Immunotech; Beckman Coulter, Inc.) according to the manufacturer’s protocol. For this, 10 μl of Annexin V-FITC and 20 μl of 7-AAD were added to each sample containing 100 μl of melanoma cell suspension in binding buffer at a concentration of 5x10⁶, incubated for 15 min in the dark on ice, then 400 μl of binding buffer was added followed by analysis using a Cytomics FC-500 flow cytometer (Beckman Coulter Inc.) with NAVIOS software v.1.3. (Beckman Coulter, Brea, USA).

After detection, with the distribution of cells displayed in their respective regions of two-parameter diagrams, living cells were defined as unstained. By contrast, cells were defined to be in early apoptosis if they stained positive for phosphatidylserine by annexin V-FITC but negative for 7-AAD due to preserved membrane integrity. Cells were defined to be at late-stage apoptosis and necrotic if there is evidence of violation in the integrity of the membranes and were stained simultaneously with both fluorescent dyes. Cells that stained positive for annexin V alone were defined as necrotic cells. The experiment was performed in triplicate.

Melanoma cells were plated into six-well plates at a density of 1x10⁵ cells per ml. After 24 h at 37°C and 5% CO₂, the cells were treated with 5.5 mM DTIC or 1% DMSO. After 72 h, the cells were transferred to serum-free RPMI-1640 medium with 5.5 mM DTIC and diluted to a final density of 1x10⁴ cells per ml. Cell migration and invasion assays were performed using CytoSelect^™ 24-Well Cell Migration and Invasion assays (8 μm, colorimetric format; Cell Biolabs, Inc.) according to the manufacturer’s protocols. Briefly, a 300 μl suspension of melanoma cells in RPMI-1640 medium without serum was placed into the upper part of special chambers for migration and invasion. The bottom layer of the chambers for migration assays was made of a polycarbonate membrane with pores, whilst for measuring invasion it was lined additionally with a layer of Matrigel. The chambers containing the cell suspension were incubated in a CO₂ incubator at 37°C and 5% CO₂ for 24 h in the wells of a 24-well plate filled with RPMI-1640 medium containing 10% FBS, which constitutes the lower part of the chamber. After incubation, the chambers were removed and the Matrigel was mechanically removed. Cells that did not migrate or invade located on the membrane inside the chamber and the invasive and migratory cells located on the outer side of the membrane were fixed and, stained by Cell Stain Solution, washed in dH20, dried at room temperature for 10 min and then lysed by Extraction Solution. The color intensity levels of lysates were measured on an EFOS-9305 spectrophotometer at a wavelength 560 nm, which were used to calculate the levels of cell migration and invasion. The experiment was performed in triplicate.

Total RNA was extracted using RecoverAll™ Total Nucleic Acid Isolation kit (Invitrogen™; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol, using on-column DNase digestion. Extracted RNA was examined using a Qubit^® 2.0 fluorimeter (Invitrogen; Thermo Fisher Scientific, Inc.) with the use of a Qubit^® RNA HS Assay kit (Invitrogen by Thermo Fisher Scientific, Inc.). In total, 10 ng total RNA was amplified, purified, reverse transcribed and labeled with biotin using the GeneChip™ WT Plus kit (cat # 902280, Affymetrix; Thermo Fisher Scientific, Inc.) following the manufacturer’s protocols. The samples were then hybridized to GeneChip™ HuGene 2.1 ST Array Strips (cat # 902114, Applied Biosystems; Thermo Fisher Scientific, Inc.). Post-hybridization staining and washing were processed according to manufacturer’s protocol using GeneAtlas™ Hybridization, Wash, and Stain kit for WT array strips (cat # 900720-C, Affymetrix; Thermo Fisher Scientific, Inc.). The strips were scanned in a GeneAtlas™ Imaging Station (Affymetrix; Thermo Fisher Scientific, Inc.). Data were collected using the Transcriptome Analysis Console software version 4.0.0 and subsequent releases (Thermo Fisher Scientific, Inc.).

A total of three replicates of each cell line before and after DTIC treatment at 5.5 mM for 72 h were prepared. Quality Control of the experiment was automatically estimated at the imaging stage and all arrays passed the quality controls. The data generated in the present study were deposited at the Array Express repository (accession no. E-MTAB-10359; https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10359/).

The expression data were used to perform principal components analysis using all probe sets by Transcriptome Analysis Console Software v.4.0.1. (Thermo Fisher Scientific, Inc.). The expression data were also used to cluster the samples using a hierarchical clustering method (19). All P-values were false discovery rate-corrected for multiple hypothesis testing. Differentially expressed probe sets were defined using the threshold of absolute fold change ≥2 and the Q-value ≤0.05. PANTHER™ v.10.0 classification system (URL: PanterDB.org) was used to interpret the biological function of the differentially expressed genes.

The cells were seeded at a density of 2x10⁵ cells per ml and cultured for 24 h at 37°C and 5% CO₂. The culture medium was then replaced and the cells were treated with DTIC at a final concentration of 5.5 mM, with 1% DMSO used as the negative control. The cells were then incubated for 72 h, detached with 0.25% trypsin-ethylenediaminetetraacetic acid and washed twice with cold 0.01 M phosphate buffered saline (Amresco LLC). Isolation of total RNA was performed using the RecoverAll™ Total Nucleic Acid Isolation kit (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Total RNA was then subjected to reverse transcription with random primers using the MMLV RT kit (cat # SK021, Evrogen, Russia). The reaction was performed with incubation at 40°C for 50 min, and the enzyme was subsequently inactivated by incubation at 70°C for 10 min.

Amplification of the obtained cDNA in an amount of 2 μl was performed in a StepOne ™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in 20 μl reaction mixture containing 1 μl probe and primers for the detection of integrin β8 (ITGB8), plexin-A2 (PLXNA2) and matrix metalloproteinase-1 (MMP-1) from the TaqMan™ Gene Expression Assay (Assay names, CYP1A1 Hs01054796_g1, CYP1A2 Hs00167927_ml, CYP2E1 Hs00559367_ml, ITGB8 Hs00174456_ml, PLXNA2 Hs00300697_ml and MMP1 Hs00899658_ml; cat. no. 4331182; Applied Biosystems, Thermo Fisher Scientific Inc.) and 8 μl 2.5X reaction mixture for RT-PCR (Syntol, Russia) in the presence of the reference ROX dye (Syntol, Russia), using the following thermocycling protocol: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of denaturation at 95°С for 15 sec and annealing and elongation at 60°С for 1 min. Endogenous normalizing controls were β-actin and hypoxanthine phosphoribosyltransferase-1 (Assay names, ACTB Hs01060665_g1 and HPRT1 Hs01003267_m1; TaqMan™ Gene Expression Assay; cat. no. 4331182; Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression levels were calculated using the 2^-ΔΔCq method as previously described (20).

Statistical analysis was performed using the non-parametric Mann-Whitney U-test for comparisons between two independent groups (DTIC-treated group vs. control untreated group) using the Statistica 6.1 software (StatSoft, Inc.). P<0.05 was considered to indicate a statistically significant difference.

According to MTT analysis, the IC₅₀ value of DTIC for the K2303 melanoma cells was calculated to 4.2 mM, whilst the IC50 of DTIC for B0404 melanoma cells equated to 10.3 mM ( Figure 1A ). The final concentration of DTIC was chosen to be 5.5 mM. Overall, K2303 melanoma cells were found to be more sensitive to DTIC treatment compared with B0404 cells. As DTIC metabolized extrahepatically by cytochrome P450 1A1 (CYP1A1), it’s mRNA levels were estimated in K2303 and B0404 cells by qRT-PCR as well as CYP1A2 and CYP2E1 levels which can metabolize DTIC also (21). We determined CYP1A1 expression in both cell lines studied, CYP1A2 expression in B0404 and CYP2E1 expression in K2303 cells ( Figure 1 В ). Relative expression level was.

To evaluate the sensitivity of the primary melanoma cells originated from the two patients to DTIC in vitro, the proportions of live and apoptotic cells were measured using flow cytometry before and after DTIC treatment. Both cell lines demonstrated notable responses to treatment, since DTIC increased the percentage of cells in early apoptosis by two-fold ( Figure 2 ). The proportion of late apoptotic and necrotic cells was not altered in K2303 and reduced in B0404.

The rates of migration and invasion were altered following DTIC treatment. We observed evident reductions in the migratory and invasive capacities of K2303 cells, whilst B0404 cells exhibited increased cell migration and invasion ( Figure 3 ). Therefore, melanoma cells appeared to respond differentially in terms of the ability to disseminate in response to DTIC treatment in the present study.

To reveal the phenotype of primary melanoma cells obtained from the two different patients, we performed a transcriptomic study ( Figures 4A–C ). We identified a total of 5,042 genes with altered expression in K2303 cells and 8906 in B0404 cells after DTIC treatment. Upregulated genes in K2303 were generally associated with apoptosis (TNFRS 10D), DNA repair (TDP2), extracellular matrix remodeling (TFPI2 and RPTN) and cell proliferation (EPGN, GPRC5A, VASN, FOSB, SCUBE3 and SULF1). Among the list of downregulated genes, we observed them to be associated with cell proliferation (FRAS1 and ACAN) and extracellular matrix remodeling (ACAN). In B0404 cells, it was found that the genes that were upregulated following DTIC treatment were typically associated with T-cell mediated immune response (GAGE1), apoptosis and DNA repair (RHOB). By contrast, genes that were associated with cell proliferation (AREG and DKK1) and extracellular matrix remodeling (MXRA5, MMP-1, EDIL3, CCDC80, TNC, ITGA8, FN1 and CCBE1) were downregulated.

We next focused our attention on signaling pathways where the differentially expressed genes were also components. We observed the top downregulated genes in B0404 to be components of the vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor R2 (VEGFR2), PI3K/AKT, focal adhesion, interleukin-18, mitogen-activated protein kinase (MAPK), RAS and transforming growth factor (TGF)-β signaling pathways. Genes as components of PI3K/AKT, VEGFA/VEGFR2, focal adhesion, endothelin pathway, transforming growth factor (TGF)-β and hippo-Merlin signaling were downregulated in K2303 cells.

Since we found a clear dysregulation in the expression of genes associated with cell adhesion and migration, the expression levels of a number for these genes, components of up-regulated pathways were subsequently validated by RT-qPCR. Pathway Enrichment analysis was performed using the PANTHER database (http://pantherdb.org/). Gene Ontology (GO) terms associated with cancer cell biology (‘biological adhesion’, ‘biological regulation’, ‘cellular process’, ‘developmental process’, ‘localization’, ‘locomotion’, ‘multicellular organismal process’, ‘response to stimulus’ and ‘signaling’) were used to identify six genes matched to selected criteria, namely ITGBL1, NOV, PLXNC1, ITGB3, ITGB8 and PLXNA2. Subsequently, ITGB8 and PLXNA2 were chosen as those most likely to be involved in the regulation of melanoma cell migration and invasion, as indicated in (22–24). MMP1 was also selected as a key dysregulated gene in both cells following DTIC treatment, using the GO term ‘cellular process’. Thus, the mRNA levels of ITGB8, plexin-A2 (PLNXA2) and MMP1 were measured in both cell lines before and after DTIC treatment. ITGB8 and MMP1 downregulation were found in both cell lines. However, PLNXA2 mRNA expression was increased in K2303 cells, whereas PLNXA2 expression was reduced in B0404 cells after DTIC treatment ( Figure 4D ). The expression levels of all three of these genes corresponded accurately with the microarray data in the present study, although the MMP1 levels showed a tendency to decrease according to the microarray result but a significant decrease was observed in the RT-qPCR data. Thus, PLNXA2 down-regulation was associated with migrative and invasive phenotype of primary melanoma cells.