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.

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.

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.

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. 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.

Breast cancer (BC) is the leading cause of morbidity and mortality of malignant neoplasms in women worldwide. In spite of a large number of studies devoted to the etiopathogenesis of this group of malignant tumors, many questions concerning specific mechanisms of their origin and development are still unresearched. Based on numerous studies in recent years, in addition to traditional risk factors (such as hereditary factors, hormonal disorders, environmental factors, etc.), foreign and Russian authors have identified a new risk factor for BC development - the sphere of molecular disorders affecting circadian rhythms and circadian genes, as well as metabolic and regulatory intracellular pathways regulated by them. It has been shown that the balance between oncogenes and suppressor genes and processes such as neoangiogenesis, metastasis and antitumor immune response depend on the functioning of the circadian rhythm system. One of the key metabolites in the circadian rhythm regulation system is melatonin, which physiological effects extend not only to the central nervous system, but also to peripheral tissue cells. The study of the relationship between circadian rhythm disturbances, melatonin secretion and malignant tumors is one of the important tasks of modern oncology. In the present review the following aspects of this problem are considered in detail: relationships between circadian rhythms, melatonin and BC; molecular mechanisms of melatonin antitumor action in BC; therapeutic potential of melatonin in prevention and treatment of malignant breast tumors as a direct antitumor agent, a component of accompanying treatment, and as a radiosensitizer and radioprotector for radiotherapy. The purpose of this review is to investigate the problem of the relationship between the circadian rhythm system, melatonin and molecular mechanisms of development and progression of breast malignancies, as well as the therapeutic potential of melatonin in their prevention and treatment. The review is based on the study of relevant and reliable scientific articles, reviews, meta-analyses, systematic reviews, and clinical trials published in Scopus, PubMed, Web of Science and in peer-reviewed Russian journals mainly during the last 5 years.

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.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.

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.

Introduction. Melanoma belongs to the group of the most malignant tumors characterized by aggressive growth and active metastasis. At the same time, the effectiveness of therapy, primarily targeted therapy, is largely limited by the rapid development of drug resistance. Aim. To study the effect of chronic ultraviolet (UV) irradiation on the formation of a population of radiation-resistant melanoma cells; to study the features of cell signaling and the sensitivity of UV-resistant melanoma cells to the antitumor drugs. Materials and methods. The experiments were carried out on in vitro cultured A375 melanoma cells. Cells were cultured in a standard DMEM + 10 % FBS medium; cell growth rate was analyzed using the MTT assay; cell survival after irradiation was analyzed using a colony-forming test. Determination of the transcriptional activity of the estrogen receptor (ER) was performed by reporter analysis upon transfection into cells of a plasmid containing the luciferase reporter gene controlled by estrogen responsive element. The immunoblotting method was used to analyze the expression of cellular proteins; comparative analysis of ERα and ERβ expression was performed by immunofluorescent method. Results. Long-term UV irradiation leads to the formation of a UV-resistant subpopulation of A375 melanoma cells, which is characterized by decreased sensitivity to targeted (vemurafenib) and hormonal (tamoxifen) drugs, increased expression of Snail, an activator of the epithelial-mesenchymal transition, and in the absence of noticeable changes in the expression of PI3K / mTOR signaling. Metformin reduces Snail expression in both parental and UV-resistant A375 cells and enhances the cytostatic effect in combination with vemurafenib or tamoxifen. Conclusion . The data obtained demonstrate a decrease in the sensitivity of melanoma cells to targeted drugs under the long-term exposure to UV. The ability of metformin to potentiate the action of targeted drugs and inhibit Snail allows us to consider metformin not only as an antitumor agent, but also as a potential inhibitor of the epithelial-mesenchymal transition.

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.

Immunofluorescent method by flow cytometry was used to quantify the expression of the tumor-associated protein βIII-tubulin (TUBB3) in the tissue of urothelial bladder cancer and visually normal mucosa (56 samples in total). The expression of the marker was detected in 100% of cases, and heterogeneity of the TUBB3 expression level both in tumor tissue and in "normal" mucosa was revealed. The level of TUBB3 in the "normal" mucosa did not depend on the distance from the tumor (1 cm or more than 3 cm) and, on average, it was lower than in the tumor tissue (21.8 ± 10.8% and 24.9 ± 13.2% vs 35.2 ± 12.4%; p = 0.04 and 0.005, respectively). An increase of the TUBB3 expression in the tumor and in the "normal" mucosa was revealed in muscle invasive bladder cancer compared to non-muscle invasive bladder cancer. Therefore, in urothelial bladder cancer, the tumor-associated protein TUBB3 is a molecular marker of bladder mucosa involvement in the malignancy process and predicts the risk of tumor muscle invasion, which may influence indications for early cystectomy.

The detection of genes related to the lifetime of patients with clear cell renal cancer provides information on the mechanisms of the tumor development and can be the basis for creating approaches to predict patient survival. In this paper, the expression of genes regulated by the HIF2α transcriptional factor was studied. Based on the results obtained here and previously identified genes regulated by the transcriptional factor HIF1α, a new panel of 6 genes, including the BAP1 gene, was proposed. Expression of genes of this panel allows predicting the survival of patients with clear cell renal cancer with high sensitivity (93%), specificity (96%), and relative risk (21.5). After verification, the application of this panel can be useful for personalized treatment of patients with clear cell renal cancer, which will increase the effectiveness of therapy.

This review describes the available works on four main trends of prospective application and study of melatonin in oncology: the main properties and mechanisms of the antitumor effect of melatonin, melatonin as a direct antitumor agent, melatonin as a drug for the accompanying therapy of psychoemotional disorders and sleep disorders in cancer patients, and melatonin as a drug for the accompanying therapy of chronic pain syndrome in oncology. Currently, researchers focus on fundamental and clinical relationships between circadian rhythms and carcinogenesis, and one of the key problems is the potential of melatonin as an agent capable of interfering with the molecular processes of formation and maintenance of the viability of malignant tumors. An equally important problem is the potential of melatonin in terms of using its physiological properties for the prevention and accompanying therapy of undesirable effects of the main treatment in oncological practice, which is emphasized in this review. The purpose of this review is an unbiased study of the prospects for the multidirectional use of melatonin both as a direct antitumor agent and as a component of complex therapy of concomitant adverse events associated with tumor and the treatment. Based on a detailed analysis of original scientific articles and scientific reviews in the PubMed search system, as well as peer-reviewed Russian journals, authors have made an attempt to characterize the current state of melatonin in oncology based on principles of evidence-based medicine. Authors also suggests and justify key prospects for use of melatonin in oncology practice and the range of studies necessary to clarify its properties and place in the combined therapy of malignant neoplasms.Received 9 July 2021. Revised 18 August 2021. Accepted 19 August 2021.Funding: The study did not have sponsorship.Conflict of interest: Authors declare no conflict of interest.Contribution of the authors Conception and study design: I.I. Eremenko, V.E. Ponomarev Drafting the article: I.I. Eremenko, V.E. Ponomarev, S.B. Polikarpova, E.A. Bogush, V.Yu. Kirsanov, B.A. Zisman, M.M. Davydov Critical revision of the article: I.I. Eremenko, V.E. Ponomarev Final approval of the version to be published: I.I. Eremenko, V.E. Ponomarev, S.B. Polikarpova, E.A. Bogush, V.Yu. Kirsanov, B.A. Zisman, M.M. Davydov

Per the majority of authors, melanoma is the most common tumor diagnosed during pregnancy (31 % of all malignant neoplasms). In approximately 1/3 of women melanoma developed in child-bearing age is diagnosed during pregnancy or in the postpartum period. However, only some retrospective studies analyzed the effect of pregnancy on melanoma development, and conclusive data on development, progression and treatment of BRAF-mutant melanoma is lacking. In this subpopulation of patients, BRAF status supposedly can negatively affect disease outcome irrespective of treatment methods.The article presents a clinical case of recurrence of melanoma with the BRAF V600E mutation during pregnancy. The patient underwent lymph node dissection during pregnancy prolongation, after labor she received antitumor drug therapy with МЕК and ВRAF inhibitors. Melanoma recurrence during pregnancy did not worsen treatment outcomes for the mother and embryo.

Estrogens play an extremely important role in regulating the proliferation of ovarian cancer. The estrogen receptor alpha (ERα) stimulates cell growth, whereas ERβ can be attributed to tumor suppressors. The study aims to assess the relationship between the expression of estrogen receptors in tumors and the efficacy of front-line platinum plus taxane chemotherapy in ovarian cancer patients. ERα and ERβ tumor expression was evaluated quantitatively by flow cytometry in a narrowly defined group (31 patients): stage III high-grade serous ovarian carcinoma (HGSOC), suboptimal surgical cytoreduction, front-line platinum plus taxane chemotherapy (front-line, six cycles). The median of progression-free survival (PFS) was 2 times greater (18 vs 8 months, p = 0.04) and the recurrence risk (HR) was 2.2 times (95 % CI: 1.1–6.2, p = 0.04) lower in the group with high (in more than 40% of the cells) vs low level of ERβ tumor expression. The statistically significant difference between PFS in the groups with high vs low tumor ERα expression was not revealed. A high level of ERβ and not ERα expression can predict the efficacy of front-line platinum plus taxane chemotherapy in stage III HGSOC patients. The status of estrogen receptor beta can be considered as one of the possible predictors for evaluating the effectiveness of ovarian cancer therapy.

The immunofluorescence analysis of the tumor-associated protein TUBB3 in gastric cancer tissue and morphologically normal tissue adjacent to the tumor was carried out using flow cytometry (in total 48 samples). Expression of TUBB3 was detected in 100% of the tumors and in 79.2% of the normal tissue samples. The marker expression level in 36.8% of the normal tissue samples was approximately equal to that in the tumor; it was significantly (by a factor of 1.8) lower in 63.2% of the cases. Direct correlation was found between the level of TUBB3 expression in the tumor and normal tissue (p = 0.0061). It is concluded that the TUBB3 expression in morphologically normal tissue adjacent to the tumor can be a molecular marker of the local spread of gastric cancer and a guideline in selecting the optimal postoperative patient management.

Expression of tumor-associated protein beta-III tubulin (TUBB3) was evaluated quantitatively by immunofluorescence analysis using flow cytometry in lung tissue of intact animals and on day 11 after the Lewis lung carcinoma transplantation as well in lung metastasis tissue and in visually “normal” surrounding tissue. It was shown that the tumor is characterized by a high TUBB3 expression, while also the expression of the marker was detected in both variants of visually normal lung tissue: it was lower than in tumor tissue but significantly higher than in lung tissue of intact animals. The detection of TUBB3 outside the tumor indicates that the tissue appeared to be normal has already involved in malignancy and this supports the hypothesis that tumor-associated protein TUBB3 can be used as a molecular marker of local tumor spread.