The reasons for discordance data on the contribution of a TUBB3 microtubule protein to resistance to antitumor drugs and the metastatic potential of tumors with an assessment of prognostic significance of TUBB3 expression in nonsmall cell lung cancer tissue are analyzed. The need to introduce quantitative analysis methods in molecular diagnostics of tumors (in particular, immunofluorescence assessment associated with flow cytometry) is substantiated.

Objective: The transition to culturing continuous cell lines in media based on serum substitutes of various origins has numerous ethical, financial and methodological reasons. Material and methods: The aim of this study was to evaluate the possibility of replacing fetal bovine serum (FBS) with a domestically produced serum substitute eBS. Results and discussion: It was demonstrated that eBS exhibits the main advantages and disadvantages inherent in serum-free media. For melanoma cells mel Il cultured in media supplemented with 10

Background. The cytoskeletal protein β-tubulin class III (Tubb3) is associated with tumor resistance to taxanes and vinca alkaloids, as well as with the metastatic potential of neoplasm, however, data from immunohistochemical analysis of Tubb3 expression in non-small cell lung cancer (NSCLC) tissue are few and contradictory. Purpose. Characterization of the level and intensity of Tubb3 expression in NSCLC tissue and analysis of the identified parameters correlation with clinically significant characteristics of the disease. Methods. Quantitative assessment of the level and intensity of Tubb3 expression in 120 surgical samples of NSCLC was carried out by immunofluorescence method associated with flow cytometry. Primary rabbit monoclonal antibodies specific to Tubb3 and secondary anti-rabbit antibodies conjugated with fluorescent dye DyLight650 (ab98510, UK) were used. The expression of the marker was assessed by two parameters: the level of expression measured as the percentage of the cells expressing Tubb3 and the intensity of expression in conventional units (CU) represented as the ratio of the geometric mean fluorescence intensity in the experimental and control samples (cells incubated with secondary antibodies only). Results. 1. Tubb3 expression was detected in all NSCLC samples studied. The median level and intensity of Tubb3 expression was 30.5% and 2.0 CU with significant differences (up to 10 times) in the quantitative values of both parameters in different patients. 2. The distribution of the studied tumors in terms of the level and intensity of Tubb3 expression differs from normal (P<0.001), the associative relationship between the assessed parameters is very strong (Spearman's rank correlation coefficient was 0.91; P<0.0001). 3. Statistical analysis did not reveal correlations between the level of Tubb3 expression and the gender and smoking status of the patients, with the degree of tumor differentiation, as well as with the stage of NSCLC. 4. In the group of lung adenocarcinomas, the median level of Tubb3 expression is higher compared with squamous cell lung cancer in male and female patients (P=0.01). Conclusion. High heterogeneity of Tubb3 expression level in NSCLC tissue in the patients and differences in the parameters between the tumors of various histotypes indicate the importance of further correlation analysis of Tubb3 expression level with the patients' life span in order to identify the prognostic value of the marker.

Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells that are capable of vasculogenic mimicry that is inhibited upon the action of an inhibitor of vasculogenic mimicry. Here, we show that the drug induces a strong upregulation of 50 genes that control the cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes that control different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude that one way for coordinated regulation of large groups of genes is regulation simultaneously by multiple transcription factors.

Preventive oncology can solve such problems as preventing the appearance of tumors, relapses after primary treatment, and complications after chemo-radiation therapy. The prospects for use of phytoadaptogens (Panax ginseng, Rhodiola Rosea, Eleutherococcus senticosus etc.), which pleiotropically influence the body along the homeostasis axis (neuro-endocrine-immune systems), are obvious.

Aim of the study: to compare the antitumor efficacy and immunogenicity of vaccines with the same antigens but different adjuvants: Ridostin Pro or Poly(I:C); to evaluate the effect of Ridostin Pro and Poly(I:C) on the cytokine profile of serum and the immunophenotype of mouse spleen cells. Material and Methods. To evaluate the antitumor efficacy of vaccines with different adjuvants, two transplantable tumor lines were used: melanoma B16-F10 and EG 7-OVA lymphoma (expressing ovalbumin) for C57BL/6 mice. Against melanoma B16-F10, vaccination with the peptide TRP2 180–188 with the studied adjuvants was performed in a mixed (preventive/therapeutic) and therapeutic regimens. Ovalbumin with adjuvants was vaccinated against EG 7 lymphoma in a therapeutic mode. The immunogenicity of vaccines with different adjuvants in mice without tumors was evaluated by the ELISPOT method. In this case, the peptide TRP2 180–188 and the protein ovalbumin also served as antigens. The cytokine profile of blood serum and changes in the immunophenotype of mouse spleen cells after administration of Ridostin Pro or Poly(I:C) were studied using flow cytometry. Results. In the B16-F10 model, vaccination in a mixed mode protected mice from tumor formation, and the mice lived for more than 100 days. For B16-F10 and EG 7, vaccination in the therapeutic mode led only to inhibition of tumor growth. Ridostin Pro and Poly(I:C) showed a similar ability to develop specific immunity to the peptide TRP2 and ovalbumin. Ridostin Pro increased cytokine levels in the blood serum of mice more strongly than Poly(I:C). Both drugs caused similar changes in the immunophenotype of spleen cells, but Ridostin Pro increased the number of CD 69+ T cells more strongly than Poly(I:C). Conclusion. The comparison of two drugs as adjuvants for antitumor vaccines showed that the domestic drug Ridostin Pro did not inferior in effectiveness to Poly(I:C) on mouse models. In this regard, Ridostin Pro can be considered as a promising adjuvant for antitumor vaccines and deserves further study.

Background. Progesterone receptors (PR) are regulators of cell proliferation and therefore can be considered as an aim for targeted medications in the treatment of oncological diseases. At the same time, a quantitative assessment of PR expression in the tissue of non-small cell lung cancer (NSCLC), which has not yet been carried out in other studies, will determine the possibility of using PR modulators for the treatment of this disease and identify the potential category of patients most susceptible to these drugs.Purpose. To characterize NSCLC by quantitative indicators of PR expression and to determine the correlation of clinically significant characteristics of patients and clinical and morphological parameters of a NSCLC tumor with the PR expression to assess the possibility of using PR modulators in the treatment of this disease.Methods. The PR expression in 130 surgical samples of NSCLC was quantified using an immunofluorescence method associated with flow cytometry. Primary antibodies to PR (NBP2-46388) and secondary antibodies conjugated with DyLight650 (ab98729) were used.Results. The expression of progesterone receptors was detected in all the studied tumors; an abnormal distribution of the marker expression level was noted (P=0.01). The mean expression level was 55.3±16.2%, and the median was 57% with a range of 70%, which indicates heterogeneity of PR expression in tumors of different patients. There were no statistically significant differences in the level of PR expression depending on the histotype and stage of NSCLC, as well as on the sex of patients. At the same time, the level of expression and the frequency of overexpression of PR (>67%) in tumors in non-smoking patients are higher than in smokers (P⩽0.02).Conclusion. The high frequency of occurrence and level of PR expression in NSCLC indicate the possible effectiveness of the use of their modulators in the treatment of this disease, especially in non-smoking patients.

Quantitative assessment of estrogen receptors alpha (ERα) expression was carried out in 115 samples of non-small cell lung cancer (NSCLC) by an immuno uorescent assay and ow cytometry. It has been shown that high level of ERα ≥20% predicts higher aggressiveness of NSCLC than at low level <20%: median survival at 78 mos of follow-up is increased by 1.5 times; the risk of death is reduced by almost 2.0 times (p = 0.04). Time to death was increased by an average of 18 mos in about 20% of patients with low ERα expression. The results validate informative value of immuno uorescence analysis and owcytometry for quantifying the ERα expression and substantiate the prospects of antiestrogen therapy as a new option for NSCLC treatment, in particular, by analogy with breast cancer - in a long-term adjuvant therapy in ERα+ NSCLC patients.

Background. Adjuvant is necessary for enhancing the efficacy of cancer peptide vaccines. Our previous work has demonstrated the efficacy of TRL-3 agonists, which include Poly(I:C) and Ridostin Pro, as part of peptide neoantigen vaccines against murine melanoma B16-F10. Aim. To evaluate the antitumor efficacy of Ridostin Pro or Poly(I:C) against murine lymphoma E.G7-OVA. Materials and methods. The study was performed on C57Bl / 6 mice with subcutaneously transplanted E.G7-OVA lymphoma containing the complete chicken ovalbumin sequence. The antitumor effects of Ridostin Pro and Poly(I:C) were evaluated in monotherapy as well as in vaccines containing chicken ovalbumin in addition to the adjuvant. The antitumor effect of Ridostin Pro and Poly(I:C) was evaluated when used in different vaccination regimens: in one case treatment was started after tumour transplantation and in the other case before tumour transplantation. The criteria of antitumor response were inhibition of tumour growth, increased survival of mice and cure. Results. Ridostin Pro and Poly(I:C) both as part of the vaccine and when administered without ovalbumin increased the percentage of tumour growth inhibition and survival of mice with E.G7-OVA lymphoma. In a regime where vaccination with ovalbumin and Ridostin Pro or Poly(I:C) was started before tumour transfection, a complete cure of the mice was shown. Conclusion. Ridostin Pro and Poly(I:C) enhance the antitumor effect of a peptide vaccine against E.G7-OVA lymphoma.

Background. The search for effective combinations of immune checkpoint inhibitors with common cytostatics, targeted cancer drugs and other treatments is a modern trend to improve the effectiveness of immunotherapy.Purpose. Development of a panel of lung cancer cell cultures and cells of normal lung tissue with a characterized molecular phenotype by expression of one of the targets of immunotherapy — programmed cell death ligand 1 (PD-L1).Methods. PD-L1 expression was quantitatively analyzed by immunofluorescence method associated with flow cytometry.Results. A panel of lung cancer cell cultures of different histotypes and cells of normal lung tissue with characterized molecular phenotype was formed according to the expression of the immunotherapy target PD-L1. In terms of PD-L1 expression intensity, cell cultures can be arranged in a series: Calu-1 HFL-1 Calu-6 Wi-26 A-549 H-596 H-211, with a 7-fold difference in the index between Calu-1 and H-211.Conclusion. The panel of cultures of cancer and normal lung cells is recommended for the search and development of effective modifiers of the immunotherapy target PD-L1.

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.

Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that yet are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells capable to vasculogenic mimicry upon action of inhibitor of vasculogenic mimicry. Here, we describe that the drug induces a strong upregulation of 50 genes controlling cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes controlling different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude, that one way for coordinated regulation of big groups of genes is the regulation simultaneously by multiple transcription factors.

An herbal formulation of a multiphytoadaptogen (MPA) developed at the N. N. Blokhin Cancer Center and containing phytocomponents from Oplopanax elatus Nakai has been studied in preclinical and clinical trials for preventive oncology. Secondary metabolites were identified in the extract of rhizomes and roots of O. elatus and in the MPA pharmaceutical composition using high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Compounds identical to ginsenosides Rd and Re (tetracyclic triterpene saponins from Panax ginseng) and araloside C and stipuleanoside R2 (pentacyclic triterpene saponins, oleanolic acid derivatives, from Aralia mandshurica) were identified in the MPA and in the extract of rhizomes and roots of O. elatus. The spectra of biological activity of ginsenosides Rd and Re, araloside C, and stipuleanoside R2 were analyzed in silico using the PASS computer program. According to published data and in silico analysis, the activities of ginsenosides Rd and Re, araloside C, and stipuleanoside R2 correspond to properties previously confirmed in the MPA studies for preventive oncology.

The expression of PD-L1 protein was quanti ed in 56 samples of urothelial bladder cancer (BC). PD-L1 expression was detected in all BC samples, while in non-muscle-invasive bladder cancer (NMIBC) the median value is 1,5 times higher than in muscle-invasive bladder cancer (MIBC) (p = 0,001). High PD-L1 expression level (≥30%) was observed in 20% of MIBC samples and among NMIBC tumors - in 68% of cases, that is 3,4 times more often (p = 0,0004). The last fact justi es the prospect of starting therapy with immune checkpoint inhibitors already at the onset of the disease - at NMIBC.

Many human diseases including cancer, degenerative and autoimmune disorders, diabetes and others are multifactorial. Pharmaceutical agents acting on a single target do not provide their efficient curation. Multitargeted drugs exhibiting pleiotropic pharmacological effects have certain advantages due to the normalization of the complex pathological processes of different etiology. Extracts of medicinal plants (EMP) containing multiple phytocomponents are widely used in traditional medicines for multifactorial disorders' treatment. Experimental studies of pharmacological potential for multicomponent compositions are quite expensive and time-consuming. In silico evaluation of EMP the pharmacological potential may provide the basis for selecting the most promising directions of testing and for identifying potential additive/synergistic effects. Multiphytoadaptogen (MPhA) containing 70 major phytocomponents of different chemical classes from 40 medicinal plant extracts has been studied in vitro, in vivo and in clinical researches. Antiproliferative and anti-tumor activities have been shown against some tumors as well as evidence-based therapeutic effects against age-related pathologies. In addition, the neuroprotective, antioxidant, antimutagenic, radioprotective, and immunomodulatory effects of MPhA were confirmed. Analysis of the PASS profiles of the biological activity of MPhA phytocomponents showed that most of the predicted anti-tumor and anti-metastatic effects were consistent with the results of laboratory and clinical studies. Antimutagenic, immunomodulatory, radioprotective, neuroprotective and anti-Parkinsonian effects were also predicted for most of the phytocomponents. Effects associated with positive effects on the male and female reproductive systems have been identified too. Thus, PASS and PharmaExpert can be used to evaluate the pharmacological potential of complex pharmaceutical compositions containing natural products

Background. Estrogen receptors beta (ERβ) are an important biological regulator and target of antiestrogens, however, unlike estrogen receptors alpha (ERɑ), their significance in the prognosis and treatment of breast cancer remains unclear. Purpose . Evaluation of the ERβ prognostic value in the comparative assessment of frequency and level of the marker expression in groups with good and poor prognosis by Ki-67 proliferative index score in breast cancer. Methods . ERβ expression level (% of cells expressing the marker) in 68 breast tissue samples was quantified by immunofluorescence and flow cytometry. Primary antibodies to ERβ (clone 14C8, ab288) and secondary antibodies conjugated with DyLight650 (ab98729) were used. In the same samples, the Ki-67 expression level was assessed by the immunohistochemical method. Results . The ERβ and Ki-67 were detected in 100% breast tissue samples with high heterogeneity of the markers’ expression in different patients. Statistical analysis of good and poor prognosis in accordance with the Ki-67 proliferative index score (Ki-67≤20% and Ki-67>20%) showed the prognostic value of the ERβ expression level of 50%. There was no association between the Ki-67 and ERβ expression levels in the same tumor sample (Spearman's rank correlation coefficient R=–0,16; P>0,05). At the same time, high expression of ERβ≥50% was 2,3 times more frequently detected in the good vs poor prognostic group by Ki-67 — 41% vs 18%, P=0,02. Conclusion . The ERβ expression level ≥50% in the tumor can be considered as a factor of good prognosis of breast cancer.

Background . The original herbal formula of Multiphytoadaptogen (MPhA) for preventive oncology developed by the N. N. Blokhin Center of Oncology containing phytocomponents from Schisandra chinensis (Turcz.) Baill (Schisandraceae), has been investigated in vitro, in vivo. Preliminary efficiency in clinical trials has also been obtained. This was allowed because MPhA in Russia is registered as a parapharmaceutical agent and therefore standardized according to established requirements. However, due to the high efficiency of MPhA, a detailed study of the chemical composition and standardization of it is required, including the Schisandra chinensis active components, which turned out to be translocated into MPhA as a result of the extraction technology developed. The aim of the study was to identify the Schisandra chinensis biologically active substances in MPhA and to evaluate the biological activity profiles of the identified phytocomponents using in silico analysis. Materials and methods. we used high performance liquid chromatography in combination with mass spectrometry (HPLC–MS / MS). Chromatography was performed on an ACQUITY UPLC BEH C18 column in a gradient mode. A TSQ Vantage triple quadrupole mass spectrometer with electrospray ionization was used. we performed in silico analyzes of Schisandrin and Schisantherin A biological activity spectra using computer programs PASS and PharmaExpert. Result . The secondary metabolites lignans Schisandrin and Schisantherin A were identified in the herbal formula MPhA. Schisandrin and Schisantherin A activities, according the scientific literature and in silico analysis, correspond to the properties studied for MPhA which therefore fits into the concept of medication for preventive oncology. Conclusion. The determined secondary metabolites can be used for identification, standardization and quality testing of the herbal formula MPhA.

PD-L1 (Programmed death-ligand 1), a membrane protein of the immunoglobulin superfamily, is one of the targets for cancer immunotherapy. A panel of 14 cancer cell cultures with a different constitutive PD-L1 expression level is formed and characterized. The panel is recommended for preclinical studies of a cytostatic drug effect on PD-L1 expression and for predicting the efficacy of their combination with immune checkpoint inhibitors.

Introduction. Despite advances in the treatment of melanoma, the results of therapy cannot be considered satisfactory, and the search for new drugs and effective combinations of medicine continues. The drugs are being developed aimed at reducing the metastatic tumor potential – migrastatics. The targets of the drugs can be cytoskeletal proteins of tumor cells – cytokeratin (CK) intermediate filaments and microtubule protein beta-III tubulin (TUBB3). Aim. To estimate of the CK and TUBB3 expression in melanoma cell lines to form an informative in vitro cell model for screening and studying migrastatics. Materials and methods. The molecular phenotype of 21 human melanoma cell lines from the collection of N. N. Blokhin National Medical Research Center of Oncology, and 18 of which were isolated from tumor metastases in the lymph nodes, soft tissues or subcutaneously. The level of TUBB3 expression and de novo expression of CKs in vimentin-expressing cells (CK + Vim) were assessed by an immunofluorescent method and flow cytometry. Results. Beta-III tubulin expression was detected in all cultures studied, de novo expression of CKs was found in 20 / 21 lines. The exception was primary uveal melanoma 92-1, that did not express CK + Vim. Both parameters significantly differed between the cells of the studied panel: CK + Vim co-expression – from 0 to 91 %, TUBB3 – from 18 to 86 %. No correlation was found between the expression level of TUBB3 and CK + Vim (Pearson’s correlation coefficient r = 0.11; p = 0.65). Three groups of the cell lines with different ratio of TUBB3 expression and CK + Vim co-expression were identified: 1) similar level of expression of both markers; 2) the level of co-expression of CK + Vim more or less high than the index for TUBB3; 3) the level of TUBB3 expression more or less high than the index for CK + Vim co-expression. Conclusion. A panel of 21 human melanoma cell lines was formed with quantitatively estimated expression of cytoske-letal proteins responsible for the migration activity of tumor cells – CKs and TUBB3. Groups of the lines with different expression ratio of the markers can be used for screening and preclinical evaluation potential migrastatics that reduce the metastatic potential of melanoma and may reduce resistance to taxanes.