The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.

Aim . To approve the COrDIS kit (Gordiz, Russia) for the authenticity of cell lines from the Bioresource Collection of the N.N. Blokhin National Medical Research Center of Oncology by the short tandem repeat (STR) profiling. Material and methods . The chosen method proved to be a reliable and reproducible option. With this approach, a number of polymorphic STR loci are amplified using commercially available primer sets. Polymerase chain reaction (PCR) products are analyzed simultaneously with size standards using automated fluorescent detection methods. The results are presented as a simple number code corresponding to the lengths of the PCR products amplified at each locus. By applying this method to cell lines, the laboratory can both authenticate commercial cell lines and build a database of their lines. In the work, we used the COrDIS EXPERT 26 kit (Gordiz, Russia), validated for molecular genetic identification of personality based on multiplex PCR analysis of 26 highly polymorphic loci of human genomic deoxyribonucleic acid. PCR results were analyzed by capillary electrophoresis using an automatic genetic analyzer with laser-induced fluorescence detection (Applied Biosystems 3500xL). Results . When testing the method, profiling of 37 cell lines was carried out, of which 18 were announced in international databases and 19 were unique, obtained at the N. N. Blokhin National Medical Research Center of Oncology, as well as a cell line mixture in order to determine the limits of contamination detection. The obtained results showed the correspondence of commercial cell lines with the data in international databases. Within the framework of this work, profiles of unique lines were obtained and the foundation of own genetic database was laid. Studies to identify the limit of contamination detection by another line have shown that even 4% of the contaminant culture in the total pool can be used to identify its individual alleles. Conclusion . The results obtained indicate the possibility of using the method to identify samples of the collection and detect intraspecific contamination.

Group B streptococci, or Streptococcus agalactiae, are the major cause of severe diseases in newborns and adults. The PAI-A and PAI-A1 pathogenicity islands containing the sspB1 and sspB1a genes, respectively, were found among group B streptococci mobile genetic elements. The presence of sspB genes correlates with urogenital tract infections. The aim of this study was to determine the frequency of group B streptococci strains with the PAI-A and PAI-A1 pathogenicity islands, circulating in Moscow, in comparison with strains from St. Petersburg. The sspB1 gene, and hence the PAI-A pathogenicity island, was not found in the genomes of strains from Moscow. The frequency of the sspB1a gene and the PAI-A1 pathogenicity island in the genomes of clinical strains was three times higher than in the genomes of colonizing strains. Thus, it can be assumed that the genes of the sspB family are more specific of group B streptococci colonizing pregnant women and newborns.

Aim. To study and describe the most common types of artifacts in detection of short tandem repeat (STR) amplicons by capillary electrophoresis and cause difficulties in interpreting the obtained STR profiles.Material and methods. Cell lines were obtained from the bioresource collection of cell lines of the Blokhin National Medical Research Center of Oncology. DNA was isolated according to the manufacturer’s instructions of the DNeasy Blood & Tissue (QIAGEN, Germany) and ExtractDNA Blood & Cells (Evrogen, Russia) kits. DNA concentration was measured using a Qubit 4.0 device (Thermo Fisher Scientific, USA) and a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, USA). Multiplex PCR was performed using a COrDIS EXPERT26 reagent kit (Gordiz, Russia). Capillary electrophoresis of PCR products was performed on a 3500xL Genetic Analyzer (Applied Biosystems, USA). GeneMapper Software v6.0 (Thermo Fisher Scientific, USA) was used to process electrophoresis data.Results. The most well-known artifacts associated with the STR profiling and subsequent capillary electrophoretic separation of amplicons were studied. Cases of detection of these artifacts from personal practice are given. Recommendations for improving the electrophoresis pattern are given.Conclusion. The paper studies the artifacts of analysis in cell line STR profiling by capillary electrophoresis (STR-CE), which researchers encounter in laboratory practice. Common types of analysis artifacts that cause difficulties in interpreting the results obtained during STR profiling, as well as possible reasons for their occurrence, are described in detail and illustrated with examples from our own practice. Recommendations are given for reducing the number of non-specific fluorescent signals and their intensity.

Background. Plasma circulating tumor DNA (ctDNA) is a potential marker for tumor process monitoring. However, the feasibility of using mouse syngeneic subcutaneous melanoma model to assess ctDNA levels remains unclear.Aim. To evaluate the feasibility of ctDNA detection in mouse B16-F10 melanoma model using droplet digital polymerase chain reaction (ddPCR).Materials and methods. We developed and validated a ddPCR assay to quantify plasma ctDNA from B16-F10 cells. To form experimental tumors, C57Bl6 mice were inoculated with B16-F10 cells. On the first, 7th, 14th , 21st days after tumor inoculation blood was collected by retroorbital sinus puncture. ctDNA was extracted from blood plasma. On the 21st day after tumor inoculation mice were sacrificed.Results. We validated a ddPCR assay to quantify plasma ctDNA in B16-F10 melanoma syngeneic model in C57Bl6 mice. The assay linear range was 0.5–32 copies/ul, R2 = 0.997. The empirical limit of detection of ctDNA was 1 copy/ul in 5 ng normal tissue genomic DNA background. The coefficient of variation values ranged from 44.5 % (1 copy/ul) to 16.6 % (16 copies/ul). Plasma ctDNA was detected on 21st day after tumor inoculation in B16-F10 melanoma syngeneic subcutaneous model (p = 0.004). ctDNA concentration positively correlated with tumor volume (ρ = 0.95, p = 0.05) and total circulating DNA concentration (ρ = 1, p = 0.0).Conclusion. B16-F10 melanoma syngeneic subcutaneous model in C57Bl6 mice can be used to monitor cDNA in studies of new approaches for melanoma treatment.

Background. Loss of heterozygosity, loss of the Y chromosome, and other types of genetic alterations are characteristic of tumor cell lines. Standard methods for detecting such changes are effortand time-consuming, and costly. Routine laboratory analysis of the authenticity and absence of intraspecific cell lines contamination using short tandem repeats (STR) profiling also allows for monitoring some genetic changes that cells undergo during the life cycle. The location of some potential STR loci is close to regulatory oncogenic loci, so many researchers note the prognostic and diagnostic utility of using STR profiling at the primary stage of genetic characterization of cell lines.Aim. Control of cell identity and genetic variability during the establishment of a stable cell line.Materials and methods. Profiling was carried out for primary cell cultures, for xenografts samples mel Lap nude, mel Kas nude and mel Pet nude, as well as during cell culture at 8th, 10th, 20th passages of the mel Lap, at 10th, 20th passages of the mel Kas and at 5th, 10th, 20th, 49th passages of the mel Pet, as well as after defrosting archived samples.Results. For the mel Lap culture, by passage 8, a loss of heterozygosity was observed at the SE33 locus, and then the genetic profile remained stable. By passage 10, mel Kas cells lost heterozygosity for two loci SE33 and D6S1043, and in the CSF1PO locus at passage 0, amplification of three alleles was observed – 11, 12, 13. Subsequently, the culture maintained a stable STR profile. By the 5th passage, the mel Pet cell culture lost heterozygosity for almost all the studied loci, but then its STR profile remained unchanged throughout 49 passages and in the xenograft material.Conclusion. The STR profiling method allows not only to monitor genetic stability in a cell line and the absence of intraspecific contamination during cultivation, but also, being fast and cheap, can be used as an additional primary test for significant genetic changes in cells.