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The study objective is to analyze the prognostic significance of PRAME protein expression in patients with uveal melanoma using immunohistochemical assay.Materials and methods. A total of 30 patients with uveal melanoma were examined and treated. The average age of patients at the time of treatment was 51.3 ± 11.8 years. In all cases, enucleation was performed according to the indications. A routine clinical-morphological, molecular-genetic, and immunohistochemical assay study was performed (n = 29). The immunohistochemical study was performed using antibodies to PRAME (clone 6H8, dilution 1: 50). The median follow-up period was 86.3 ± 2.9 months.Results. Of the 29 samples studied, staining was determined in 16, which was 55.2 %. Weak intensity of 1+ staining (from 10 to 20 % of tumor cells) was detected in 7 uveal melanoma samples, medium intensity of 2+ (from 10 to 20 % of tumor cells) – also in 7, and strong intensity of 3+ (30 % of tumor cells) – in 2 tumor samples. When assessing the seven-year survival, the cumulative survival rate in the group without PRAME expression was 0.857, while in the group with PRAME expression it was significantly lower and amounted to 0.357 (p = 0.0001). The PRAME protein expression was significantly correlated with the epithelioid cell type of the tumor (p = 0.041) and with the total and partial monosomy of chromosome 3 (p = 0.013).Conclusion. This paper presents the world’s first study of the prognostic significance of PRAME protein expression by immunohistochemical analysis in patients with uveal melanoma. A significant association of PRAME-positive patients with an unfavorable vital prognosis was shown.

Purpose: to study the expression of vascular endothelial growth factor (vegf-a) and its receptors (vegfr-1 and vegfr-2) in renal cell carcinoma (rcc) cells and assess the effect of the expression levels of these markers on the tumor characteristics and prognosis of patients with rcc.Material and methods. The study included 65 patients with rcc (pt1a-t4n0/+m0/+). All patients underwent radical surgery. Histological tumor tissue samples obtained during surgery were used for the study. Expression of vegfa, vegfr-1, -2 was studied by immunohistochemical staining using appropriate antibodies to receptors and growth factors.Results. Expression of vegf and vegfr-1 and vegfr-2 receptors was ound in the cytoplasm and on the membrane of primary tumor cells of patients with rcc. There was a significant direct correlation of overexpression of the markers with g 3-4 anaplasia (vegfr-1, -2) and signs of significant tumor extension, including high pt category (vegfr-1, -2), larger size of the primary tumor (vegfr-1 , -2), tumor invasion of paranephria (vegf, vegfr-1), tumor venous thrombosis (vegfr-1, -2), multiple metastases (vegf-2), metastases in the adrenal gland (vegf, vegfr-2) and liver (vegfr-1) (p0.05).Conclusion. Expression of vegfa, as well as vegfr-1 and vegfr-2 receptors, was found on the surface and in the cytoplasm of cells of the primary tumor of patients with rcc (pt1a-t4n0/+m0/+). There was a significant correlation between vegf/vegfr overexpression with a high grade (g3-4) tumor anaplasia and significant tumor extension. In univariate analysis, a significant adverse effect on specific survival of vegfr-2 overexpression was observed. In regression analysis, vegfr-2 overexpression tended to independently affect specific survival. These results show the importance of vegf/vegfr expression as biomarkers in renal cell carcinoma.

Rationale: Riboflavin (vitamin B2) is one of the most promising agents for photodynamic therapy (PDT). However, its use is limited by the excitation in the ultraviolet (UV) and visible spectral ranges and, as a result, by a small penetration into biological tissue not exceeding a few millimeters. This problem could be solved by approaches ensuring excitation of riboflavin molecules within tumor tissues by infrared (IR) light. Upconversion nanoparticles (UCNPs) can be potentially considered as mediators able to effectively convert the exciting radiation of the near IR range, penetrating into biological tissue to a 3 cm depth, into the photoluminescence in the UV and visible spectral ranges.Aim: To evaluate the efficacy of UCNPs for IR-mediated riboflavin activation in the depth of tumor tissue during PDT. Materials and methods: The water-soluble riboflavin flavin mononucleotide (FMN, Pharmstandard-UfaVITA, Russia) was used as a photosensitizer in in vitro and in vivo experiments. The in vitro experiments were performed on human breast adenocarcinoma SK-BR-3, human glioblastoma U-87 MG, and rat glioma C6 cell lines. Lewis lung carcinoma (LLC) inoculated to hybrid BDF1 mice was used as a model to demonstrate the delivery of FMN to the tumor. UCNPs with a core/shell structure [NaYF4:Yb3+, Tm3+/NaYF4] were used for photoactivation of FMN in vivo. PDT based on FMN, UCNPs and laser radiation 975 nm (IR) was performed on mouse xenografts of human breast adenocarcinoma SKBR-3.Results: We were able to show that FMN could act as an effective in vitro photosensitizer for SK-BR-3, U-87 MG, and C6 cell lines. FMN IC50 values for glioma cells were ~30 μM, and for SK-BR-3 cell line ~50 μM (24 h incubation, irradiation 4.2 J/cm2). In the LLC model, the appropriate concentration of FMN (30 μM and above) can be achieved in the tumor as a result of systemic administration of FMN (at 2 and 24 hours after injection). The effect of PDT using near IR light for UCNP-mediated excitation of FMN was demonstrated in mouse xenografts SKBR-3, with the tumor growth inhibition of 90±5%.Conclusion: The study has demonstrated the possibility to use riboflavin (vitamin B2) as a photosensitizer for PDT. The photoexcitation of FMN via the anti-Stokes photoluminescence of UCNPs allows for implementation of the PDT technique with the near IR spectral range.

Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.

Abstract Melanoma is one of the most aggressive and lethal form of cancer. Photodynamic therapy (PDT) is a clinically approved technique for cancer treatment, including non-melanoma skin cancer. However, the most of conventional photosensitizers are of low efficacy against melanoma due to the possible dark toxicity at high drug concentrations, melanin pigmentation, and induction of anti-oxidant defense mechanisms. In the current research we propose non-toxic flavin mononucleotide (FMN), which is a water-soluble form of riboflavin (vitamin B2) as a promising agent for photodynamic therapy of melanoma. We demonstrated selective accumulation of FMN in melanoma cells in vivo and in vitro in comparison with keratinocytes and fibroblasts. Blue light irradiation with dose 5 J/cm2 of melanoma cells pre-incubated with FMN led to cell death through apoptosis. Thus, the IC50 values of human melanoma A375, Mel IL, and Mel Z cells were in a range of FMN concentration 10–30 µM that can be achieved in tumor tissue under systemic administration. The efficiency of reactive oxygen species (ROS) generation under FMN blue light irradiation was measured in single melanoma cells by a label-free technique using an electrochemical nanoprobe in a real-time control manner. Melanoma xenograft regression in mice was observed as a result of intravenous injection of FMN followed by blue-light irradiation of tumor site. The inhibition of tumor growth was 85–90% within 50 days after PDT treatment.

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma.

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

The dynamics of the production of nitric oxide (NO) metabolites: nitrites, nitrates, volatile nitrosamines and iNOS expression was studied in mice with subcutaneous transplanted, spontaneous and chemical- induced tumors. Tumor growth was accompanied by increased production of nitrites + nitrates in tumors or their release with urine that not dependent on tumor histotype. The total concentration of nitrites and nitrates in tumors reached micromolar levels characteristic of nitrosative stress. The ability of peritoneal macrophages + monocytes to generates nitrites was suppressed at the stage of intensive growth of the Lewis lung carcinoma, which may indicate a decrease in the cytotoxic properties of immune cells. The possibility of formation in the Erlich carcinoma of volative N-nitrosodimethylamine and N-nitrosodiethylamine compounds with pronounced carcinogenic properties was demonstrated. A positive expression of iNOS was revealed in some areas of lung carcinoma at all investigated time points using the immunohistochemical method. The lungs metastases were not stain or weakly stained. This may indicate selection of the cells with a low activity of iNOS migrating in the lungs.

Background: To improve quality of surgery in oncology, it is necessary to completely remove the tumor, including its metastases, to minimize injury to normal tissues and to reduce duration of an intervention. Modern methods of detection based on radiological computerized tomography and magnetic resonance imaging can identify a tumor after its volume has become big enough, i.e. it contains more than 10 billion cells. Therefore, an improvement of sensitivity and resolution ability of diagnostic tools to identify early stages of malignant neoplasms seems of utmost importance. Aim: To demonstrate the potential of a new class of anti-Stokes luminescence nanoparticles for deep optical imaging with high contrast of malignant tumors. Materials and methods: Upconversion nanoparticles with narrow dispersion and a size of 70 to 80 nm, with a core/shell structure of NaYF4:Yb3+:Tm3+/NaYF4 were used in the study. The nanoparticles have an intensive band of anti-Stokes photoluminescence at a wavelength of 800 nm under irradiation with a wavelength of 975 nm (both wavelengths are within the transparency window for biological tissues). The conversion coefficient of the excitation radiation into the anti-Stokes luminescence was 9%. To increase the time during which nanoparticles can circulate in blood flow of small animals, the nanoparticles were covered by a biocompatible amphiphilic polymer shell. As a tumor model we used Lewis epidermoid carcinoma transfected to mice. Results: We were able to obtain stable water colloids of nanoparticles covered with amphiphilic polymer that could preserve their initial size at least for one month. The use of upconversion nanoparticles with a hydrophilic shell made of intermittent maleic anhydride and octadecene co-polymer with subsequent coating with diglycidyl polyethylene glycol ether allowed for reduction of non-specific reaction of nanoparticles with plasma proteins. In its turn, it resulted in an increased time of their circulation in blood flow of small animals for up to 1 hour. With the Lewis lung carcinoma transfected to mice model we demonstrated аn in-life transportation of upconversion nanoparticles into the tumor with a high degree of localization due to a passive EPR effect. The contrast of luminescent signal in the tumor compared to adjacent tissues was at least 70%. The possibility of visualization of upconverted nanoparticles up to 15 mm of biological tissue was shown. Conclusion: The optical imaging techniques with anti-Stokes photoluminescent markers ensure a high contract real-time detection of tumor tissues that allows for their use for intra-operative diagnostics.

Abstract. Involvement of dendritic cells (DC) into switching of immune response to T-independent type 2 antigens (TI2 antigens) is poorly studied. The present study addressed some interactions between DCs and TI2-type antigens, i.e., with Streptococcus pneumoniae polysaccharide type 3 (S3), and polyvinylpyrrolidone (PVP, mw. of 360 kDa), as well as a role of antigen-loaded DCs for triggering the immune response. Shortterm treatment of DCs with TI-2 antigens induced their activation manifesting as an increase in numbers of CD80- and CD86-positive cells under the cell culture conditions. DCs loaded with TI-2 antigens were then mixed with normal murine splenocytes and cultured in complete RPMI 1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells (AFCs and IFCs, respectively) were determined by ELISPOT assay. Supplementation with TI2-treated DCs induced a 2.0 to 2.7-fold increase in specific immune response. Antigen-induced polyclonal response was enhanced by 40%. These data suggest a direct interaction between DCs and TI-2 antigens, and their presentation to murine splenocytes, thus leading to both specific and polyclonal B cell activation. B-1 cells are shown to play a main role in such response. Separation of loaded DC and splenocytes by semi-permeable membranes caused inhibition of this immune response.

Abstract. Interactions between B1, B2 and bone marrow dendritic cells (DCs) of CBA mice were investigated in the course of immune response to T-independent antigen type 2 (TI 2 Ag) in model system. Splenocytes of Xid-mice CBA/N were used as “fillers”. It was shown that DCs augmented viability of B1 and B2 lymphocytes. Functional activity of the B1 and B2 lymphocytes was determined by ELISPOT method, as relative numbers of antibody- and immunoglobulin-forming cells (AFCs and IFCs, respectively) detectable after four days in culture. An increased immune response to TI 2 Ag in presence of DCs was observed mainly for B2 subpopulation, containing MZ-B lymphocytes as well. Specific effect of DC upon B2 cell subpopulation was confirmed by detection of direct in vitro interactions of DCs with T and B lymphocytes. DCs cultivated with B2 and T lymphocytes, used as fillers, decreased the numbers of AFCs and IFCs, irrespectively of absence or presence of TI-2 Ags. A decrease in AFC and IFC numbers in the cultures with B1 cells was evident in presence of the Ags only. It was shown that the inhibitory effect of DCs depended on their contact interactions. Separate cultures of B1 and B2 cells with DC under transwell conditions resulted into occurence of AFC and IFC in amounts similar to those generated in vitro without DCs.