COMPARISON OF 2D AND 3D PRIMARY CELL CULTURES OBTAINED FROM EXPLANT OF HIGH-GRADE UROTHELIAL BLADDER CANCER.

Studying the biological characteristics of bladder cancer in primary culture can be an effective way for diagnostic and prognostic purposes, as well as choosing a scheme for personalized therapy. AIM: To characterize and compare 2D and 3D primary cell cultures obtained from the same tumor sample resected from a patient with high-grade bladder cancer. MATERIALS AND METHODS: 2D and 3D primary cell cultures were obtained from explants of resected bladder cancer. Glucose metabolism, lactate dehydrogenase (LDH) activity, and level of apoptosis were studied. RESULTS: Multicellular tumor spheroids (3D) differ from planar culture (2D) by more pronounced consumption of glucose from the culture medium (1.7 times higher than 2D on Day 3 of culture), increased lactate dehydrogenase activity (2.5 times higher on Day 3 vs. Day 1 of cultivation, while in 2D culture LDH activity is constant), stronger acidification of the extracellular environment (pH dropped by 1 in 3D and by 0.5 in 2D). Spheroids demonstrate enhanced resistance to apoptosis (1.4 times higher). CONCLUSION: This methodological technique can be used both for tumor characterization and for selection of optimal postoperative chemotherapeutic schemes.

Expression of TLR4 and major inflammatory cytokines in patients with bladder cancer of different grade and stage.

BACKGROUND: The bladder cancer is immunogenic, and neoantigens generated by tumor cells trigger a notable immune response in the host. On the other hand, multiple immune escape mechanisms allow for avoiding the recognition by the host immune system. Toll-like receptor type 4 and inflammatory cytokines play major role in the immune response to bladder cancer. AIM: To assess the expression of TLR4 and the genes of major inflammatory cytokines in tumor cells and in unaffected tissue of the bladder. MATERIALS AND METHODS: The pairs of samples from the urinary bladder tumor and unaffected adjacent tissue were obtained from 50 surgically treated patients with bladder cancer. The level of expression of TLR4, TGF-ß1, INF-γ, TNF-α genes was evaluated by real-time polymerase chain reaction. RESULTS: Bladder cancer cells are characterized by lower expression levels of TLR4, TGF-ß1, INF-γ, TNF-α as compared to unaffected tissue. In patients with recurrent cancer, expression of TLR-4 and cytokines does not change both in tumor and in unaffected tissue of the bladder. Expression of TLR4 is identically low both in low- and high-grade cancer. Expression levels of the INF-γ and TNF-α are remarkably low in muscle-invasive cancer compared to the unaffected bladder tissue. The level of TGF-ß1 in bladder cancer is comparable to the unaffected tissue of the bladder, while in the intact and metastatic lymph nodes it is significantly upregulated. CONCLUSION: Bladder cancer tissue differs from the unaffected part of the bladder wall in the level of TLR4, TGF-ß1, INF-γ, TNF-α expression.

ROS production by circulating phagocytes and Guerin carcinoma resistance to cisplatin.

BACKGROUND: Tumor drug resistance remains a primary cause of unsuccessful cancer therapy. The search for biological markers of the sensitivity/resistance of malignant neoplasms to drug therapy is an urgent and important task, the solution of which will increase the effectiveness of anticancer chemotherapy. AIM: To study the relationship between the functional activity (parameters of the phagocytosis and reactive oxygen species (ROS) production) of neutrophils and monocytes in the peripheral blood of rats with transplanted Guerin carcinoma and the degree of its sensitivity to cisplatin (Cpt). MATERIALS AND METHODS: The original and Cpt-resistant variants of Guerin carcinoma were transplanted to female Wistar rats 2.5 months old. The parameters of the phagocytic activity of circulating neutrophils and monocytes were determined by the degree of ingestion of inactivated and FITC-labeled staphylococci using flow cytometry. The number of ROS-generating cells and the intensity of ROS production by phagocytes were determined by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: The growth of both variants of Guerin carcinoma caused a statistically significant decrease in the intensity of neutrophil phagocytosis by more than 47% with a tendency to the reduction of the intensity of phagocytosis by monocytes. The phagocytic activity of circulating neutrophils and monocytes did not differ significantly between the groups of animals with the original and Cpt-resistant variant of Guerin carcinoma. In contrast, the intensity of ROS generation by both monocytes and neutrophils in the peripheral blood of animals with Cpt-resistant tumor increased by more than 86% as compared to original carcinoma-bearing rats. CONCLUSION: This study provides evidence that the intensity of ROS production by circulating monocytes and neutrophils may reflect the degree of tumor sensitivity to Cpt. Increased intensity of ROS production could serve as a pretreatment predictor of the formation of tumor drug resistance.

Changes in expression of TLR-4, TGF-ß, INF-γ, TNF-α in cultured T24/83 cells of invasive bladder cancer treated with cisplatin and/or polyphenolic adjuvant melanin.

BACKGROUND: Toll-like receptor 4 (TLR4) is known to be involved in carcinogenesis and cancer progression. Changes in TLR4 expression are associated with changes in the expression of key cellular cytokines (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)), which affect cancer progression and metastasis. AIM: To study changes in the expression of TLR4, TGF-ß, TNF-α, IFN-γ genes, the level of apoptosis and cell cycle distribution in human invasive urothelial carcinoma T24/83 cells under the treatment with polyphenolic adjuvant compound of fungal origin melanin, cytotoxic drug cisplatin, and combination of both. MATERIALS AND METHODS: T24/83 cells were incubated with cisplatin (0.05 mM), melanin (5 µg/ml), or their combination. The expression level of TLR-4, TGF-ß, INF-γ, TNF-α was evaluated by the real time polymerase chain reaction. The flow cytometry was used to study cell cycle distribution, proliferative activity and level of apoptosis. Morphological analysis of the Т24/83 cells was performed as well. RESULTS: Melanin, cisplatin, and their combination downregulate TLR4 expression (2.67; 1.28; and 2.73-fold decrease, respectively) and TNF-α expression (6.5; 1.4; and 1.7-fold decrease, respectively). Melanin did not affect TGF-ß expression while cisplatin caused 13-fold downregulation of TGF-ß. The combined use of cisplatin and melanin decreased TGF-ß expression by 6.5 times. The upregulation of IFN-γ by melanin, cisplatin, and their combination was demonstrated (4.3; 6.7; and 2-fold increase, respectively). All treatment modalities increased the level of apoptosis in T24/83 cells. Melanin treatment increased significantly the proportion of fibroblast-like cells in T24/83 culture with decreased cell adhesion to the substrate. CONCLUSIONS: Melanin, cisplatin, and combination of both agents affect significantly TLR4, TNF-α, TGF-ß, INF-γ expression, cell cycle distribution and morphology in T24/83 cells suggesting their transition to less aggressive phenotype.

Tumor microenvironment changes tumor cell sensitivity to action of energy metabolism modifiers.

BACKGROUND: Taking into account differences in the bioenergetics between malignant and normal cells a search of antitumor drugs among the modifiers of tumor metabolism has a reasonable excuse. Earlier it was found that the cytotoxic/cytostatic action of sodium dichloroacetate (DCA) against Lewis lung carcinoma (LLC) cells in vitro was enhanced in the case of its combination with metformin (MTF). AIM: To study the antitumor action of DCA in combination with MTF against LLC in vivo. MATERIALS AND METHODS: LLC/R9, a low metastatic variant of LLC cells, was used. LLC/R9 bearing mice were treated with MTF (at a total dose 0.15 g/kg b.w.) alone or in combination with DCA (at a total dose of 0.75 g/kg b.w.). LLC/R9 growth kinetics and the primary tumor growth and metastasis indices on the 23rd day after tumor cell inoculation were evaluated by routine procedures. The state of the electron transport chain of mitochondria in tumor cells was studied using electron paramagnetic resonance. The content of lactate and glucose in blood plasma from mice was measured by enzymatic methods using biochemical analyzer. The number of tumor-associated macrophages (TAMs) and their distribution by M1/M2 phenotype were estimated by flow cytometry using antibodies against CD68 and CD206. RESULTS: In LLC/R9-bearing mice treated with DCA in combination with MTF, tumor growth and metastasis indices, as well as circulating glucose and lactate levels were not significantly different from those in the control group. The level of nitrosylation of non-heme and heme proteins and the content of iron-sulfur centers in the mitochondria of tumor cells in LLC/R9-bearing mice administered with DCA in combination with MTF did not also differ from the corresponding indices in control. Instead, in tumors treated with MTF alone and in combination with DCA the total CD68+ TAMs count was almost 27% (p < 0.05) and 43% lower (p < 0.05) correspondingly than that in control, but this decrease was not accompanied by redistribution of CD68+/CD206+ and CD68+/D206- subsets. CONCLUSION: DCA in combination with MTF, at least in doses applied, did not affect LLC/R9 growth and metastasis in vivo. The complete absence of an antitumor effect of DCA in combination with MTF was simultaneously associated with the absence of significant changes in the functional state of electron transport chain of mitochondria in tumor cells, circulating glucose and lactate levels, and the decrease of the TAMs amount in tumors. It suggests that the antitumor activity of DCA and MTF could be determined by both their local effects within a tumor and their multiple systemic impacts.

Prognostic significance of MDM2 gene expression in childhood neuroblastoma.

AIM: To investigate the association of MDM2 expression at the mRNA levels in neuroblastoma with clinical features and unfavorable disease factors to determine the possibility of it usage as a prognostic marker of neuroblastoma. MATERIALS AND METHODS: Total RNA and DNA were extracted from tumor tissue samples of total 91 neuroblastoma patients (mean age: 39.45 ± 4.81 months). MDM2 mRNA levels were detected with Q-PCR. TP53 gene deletion was detected with FISH method. MYCN amplification was detected with -Q-PCR analysis in fresh tumor samples and FISH in FFPE samples. RESULTS: We investigated the association of MDM2 mRNA expression with clinical outcome in neuroblastoma patients (n = 91). Kaplan - Meier curves showed a significant association of high MDM2 expression with poor event-free survival (p < 0.001). Clinical outcome of patients without MYCN amplification with low MDM2 expression was associated with better event-free survival than with high MDM2 expression (p < 0.001). Overexpression of MDM2 can be used as significant prognostic marker for patient stratification on risk groups and treatment optimization. CONCLUSION: Our results showed that the high expression of MDM2 at mRNA levels is an important factor of neuroblastoma prognosis. It may be a valuable additional molecular marker in guiding specific therapy in MYCN non-amplified NB patients without TP53 gene deletion.

Immunological markers of anti-tumor dendritic cells vaccine efficiency in patients with non-small cell lung cancer.

AIM: To investigate the quantitative and functional status of peripheral blood lymphocytes in patients with non-small cell lung cancer during DC-vaccine therapy and identify the most informative immunological parameters which are associated with clinical outcome. MATERIALS AND METHODS: The study was conducted within the framework of randomized phase III clinical trial of DC-vaccine efficacy in patients with non-small cell lung cancer. Quantitative composition of peripheral blood lymphocytes was determined by flow cytometry. Cytokines mRNA expression level was estimated using real-time RT-PCR. RESULTS: In our study the most pronounced changes in the immune system have been defined after fourth DC-vaccine injection. Immunologic features such as reduction the MIP-1α mRNA expression level, increasing the RANTES mRNA expression level and NK-cells count, retention CD4/CD8 ratio at physiological level were associated with favorable clinical outcome after DC-immunotherapy. CONCLUSIONS: Immunological markers established in our investigation can be used for estimation of DC-immunotherapy efficiency. The results of our research are very promising, but these data should be confirmed in further studies with a large cohort of patients.