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.

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.

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.

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.

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.

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.

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.

The expression level of estrogen receptors (ERα and ERβ) in surgical tumor biopsy specimens from 167 patients with non-small cell lung cancer (NSCLC) was quantified by flow cytometry-based immunofluorescence analysis. ERα and ERβ expression was revealed in all the tumor samples investigated. The level of ERα expression in the tumors varied from 10 to 58% and ERβ — 12% to 80%, indicating significant heterogeneity of estrogen receptor expression in tumors of different patients. The mean ERβ expression level was approximately 2-times higher compared to ERα (42.1±15.3% vs 21.4±11.3%; p<0.001). It allows to consider ER>β as a major estrogen target in NSCLC tissue. The level of ERα and ERβ expression in NSCLC tissue is independent of gender, smoking status, and the histological type of the tumor. Increased ERβ expression was detected only in male patients with lung adenocarcinoma in comparison with squamous cell carcinoma (p=0.02). The assessment of the correlation between ERα and ERβ in both the whole cohort of patients and subgroups with clinically relevant disease parameters revealed that the level of one marker does not predict the other one’s expression. The coefficient of determination, which characterizes how differences in one variable can be explained by the difference in a second variable, was less than 25% in any comparison group. The authors consider that the high level of ERβ expression and ERα coexpression in NSCLC tissue substantiates the clinical perceptiveness of a new treatment option for the disease, namely, adjuvant hormone (antiestrogen) therapy.

In the study of 125 patients after radical surgery for non-small cell lung cancer (NSCLC), distinctive clinicopathological parameters of this disease were found. Thus, the majority of patients with NSCLC were smokers (73%), men had significantly higher rate of the disease than women (80% vs 20%). Patients of different sex varied by the tumor histological type: squamous cell carcinoma was characteristic of male patients (70%), while adenocarcinoma predominated in women (80%). Authors conclude that the described incidence patterns and the association of NSCLC with significant clinical features are comparable with the literature data, which indicates the absence of significant changes over the past 8 years. Survival rates of the patients after radical surgery for NSCLC also coincided with results of the previous studies. Overall survival rate varied by the stage of the disease, smoking status, and the tumor histological type: patients with earlier stage at diagnosis, never-smokers and patients with adenocarcinoma rather than squamous cell carcinoma were associated with a more favorable prognosis. The heterogeneity of the patients who survived 5 years after surgical treatment was shown: the group included patients not only with stage I of the disease, but also with more advanced II and even III stages of the disease. Finally, the absence of gender differences in the overall survival of the patients with NSCLC was demonstrated; this fact differs from the literature data on the better survival of female patients compared to men. The authors suggest that the result may indicate the emergence of factors in the Russian population that improve the course of disease in men or adversely affect the disease in women.

The results of epithelial-mesenchymal transition analysis in serous ovarian cancer by the developed method of double immunofluorescent staining and flow cytometry while surgical tumor specimens and paraffin embedded tissue blocks are compared. When estimating de novo expression of the mesenchymal protein vimentin in epithelial tumor cells expressing cytokeratins, the results turn out to be close not only in quantitative parameters but also visually in the nature of cell distribution on the dotted plots. The results obtained indicate that the introduction of the developed new technology for the molecular diagnostics of the epithelial-mesenchymal transition of human tumors into routine laboratory practice is promising.

In several drug repurposing trials, metformin has shown promising effects against various cancers. Metformin, a complex I inhibitor, actively regulates the metabolism of tumor cells through LKB/AMPK, Akt and mTOR pathways. This work aims to investigate the antiproliferative effects of metformin on A431 cells and to evaluate its activities as a possible anti-PD-L1 adjuvant. Epidermoid carcinoma cell culture A-431 (ATCC, USA) was incubated with metformin (15 mM) in a high (4.5 g/L) and low (1.0 g/L) glucose growth medium. The antiproliferative activity of metformin was assessed by the MTT test. Expression of PD-L1 was evaluated quantitatively by immunofluorescent assay and flow cytometry. The primary anti-PD-L1 (ARG65862) and secondary (DyLight650, ab98729) antibodies were used. Two indexes of PD-L1 expression were calculated: level is the ratio (%) of specifically fluorescent cells to the control (incubation with the secondary antibody only) and intensity is the ratio of mean fluorescence intensity in the experimental sample to the control. Metformin has shown significant antiproliferative effects on A431 cells. A high level of the phosphorylated form of AMPK (p-AMPK), one of the metformin targets, was found in A431 cells. Metformin treatment did not increase p-AMPK but significantly reduced the expression of cyclin D1, a key regulator of the cell cycle, and also blocked the expression of glucose transporter GLUT1. Low glucose significantly enhanced metformin effects on GLUT1 expression. In untreated A431 cells, there was no difference in both the level and intensity of PD-L1 expression between high vs low glucose growth medium. In the high glucose growth medium, 24h or 72h metformin treatment did not alter PD-L1 expression. In the low glucose growth medium, metformin treatment of A431 cells for 24h and 72h decreased the PD-L1 expression: the level – in 1.4 and 1.6 times; intensity of the marker expression – in 1.3 and 1.7 times, respectively. Metformin exhibits pronounced antiproliferative effects against skin cancer cells, including through blocking the cell cycle and glucose transporter GLUT1. Low glucose enhances the action of metformin as a potential adjuvant to anti-PD-L1 therapy.

Introduction. The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks.Objective. Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue.Materials and methods. Method validation was carried out by quantitative estimation of βIII-tubulin (TUBB3) expression in single-cell suspensions of non-small-cell lung cancer obtained from surgical tumor samples. Primary antibodies to TUBB3 (ab7751) and secondary DyLight 650-conjugated antibodies (ab98729) were used for immunofluorescent staining. The «Navios» flow cytometer (Beckman Coulter) was used to measure the fluorescence. The validation parameters were assessed by the coefficient of variation calculated as the ratio of standard deviation of TUBB3 level to its mean value.Results. Two parameters were analyzed: intra-assay precision and time stability of the results of the TUBB3 expression assessment. It was demonstrated that the mean coefficients of variation of the marker expression level in the tumor tissue did not exceed 20 % for both parameters. According to recommendations on the analytical validation of methods based on flow cytometry, it proves the validity of the method for these parameters.Conclusions. The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors. The practical value of the time stability of immunofluorescence stain during 24 h storage of a stained cells suspension in the dark at 4 °C was highlighted. It shows the possibility of adjusting the time interval between completion of the analytical study part and flow cytometer measurement.

A new method of double immunofluorescent staining for flow cytometry has been created to evaluate quantitative expression of mesenchymal protein vimentin only in epithelial cells of a solid tumor that is a mix of different origin cells. De novo vimentin expression is strongly associated with epithelial-mesenchymal transition and therefore is a metastatic potential marker of epithelial tumor cells. In comparison with semiquantitative available methods, the proposed one has several advantages, such as the accurate measurement of the marker's expression, and minimization of spatial and temporal tumor heterogeneity. Clinical validation of the method has revealed inverse correlation between the quantitative index of epithelial-mesenchymal transition level and progression-free survival using Kaplan-Meier curves and the COX proportional hazards ratio in 32 ovarian cancer patients.

Comparative quantitative evaluation of estrogen receptors β (ERβ) expression is performed by the immunofluorescence method using flow cytometry in the ovarian cancer cell lines COLO-704, OVCAR-3, EFO-21, and SK-OV-3, as well as the breast cancer cell line MCF-7. A high level of ERβ expression (88–91%) is revealed in all the cell cultures investigated, and significant differences were observed in the intensity of the marker’s expression between ovarian cancer cell cultures (in OVCAR-3, EFO-21, and SK-OV-3 they are higher than in COLO-704 cells by factors of 1.6, 2.3, and 3.5, respectively). Moreover, the intensity of ERβ expression was significantly higher in SK-OV-3, EFO-21, and OVCAR-3 than in MCF-7 cells: in 2.7; 1.4 and 1.9 times, respectively. The authors suggest that in contrast to breast cancer patients, ERβ rather than ERα could be the main target for antiestrogens in ovarian cancer patients. The revealed differences in the ERβ expression in a number of widely used ovarian cancer cell cultures lead us to recommend them for further fundamental research on ERβ to study the biological effects of antiestrogens.

Abstract—A new method of double immunofluorescence analysis by flow cytometry is developed to evaluate the quantitative expression of the mesenchymal marker vimentin in epithelium cells of a solid tumor, which in turn is a mixture of different histogenesis cells. The method has a number of evident advantages compared to the clinically used immunohistochemistry technique for marker investigation: the intralaboratory precision of the results, gentle preanalytical preparation, lack of subjectivity in the interpretation of the results, and integral evaluation of the markers expression in a large number of cells from a large volume of the tumor tissue sample (≥5000 cells from a sample of ≥2 cm in diameter).

Abstract—Molecular heterogeneity in seven human metastatic melanoma cells lines is revealed using a double immunofluorescent assay by flow cytometry. It is evaluated that quite a small population of cells (up to 10%) expressed only vimentin, the main marker of melanoma. Only epithelial cytokeratins expression is revealed in a significantly greater number of cells (from 25 to 50%). The dominant molecular phenotype (50–80% cells expressed both markers), the coexpression of vimentin and cytokeratins, is identified for the first time. The authors assume that the latter phenotype of melanoma cells may be a molecular marker of the tumor’s aggressiveness and metastatic potential.