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.

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.

Bladder sparing surgery in high-grade bladder cancer.

The gold standard for managing muscle-invasive bladder cancer is radical cystectomy (RCE). The RCE is a treatment, which carries high burden of perioperative morbidity and mortality. As biomolecular markers make muscle-invasive high-grade bladder cancer (HGBC) an entity different from non-invasive papillary disease, we tested a hypothesis that alternative bladder preserving surgery (BPS) approaches, such as partial cystectomy and transurethral resection of the bladder would not compromise the oncological results of treating HGBC in selected patients. Aim: To study the cancer specific survival of HGBC patients depending on the mode of surgical treatment - RCE, partial en-block cystectomy, and transurethral resection of the bladder in the practice of the Departments of Urology and Oncology of Bogomolets National Medical University, and to assess the prevalence of bladder sparing surgical management of HGBC in local practice as a part of trimodal treatment approach to bladder preservation. Materials and Methods: Retrospectively we studied the medical records of 3597 urothelial bladder cancer patients, of whom 346 (10%) had high-grade disease and who underwent surgical treatment in 2004-2017. All patients were studied with contact computed tomography of the chest, abdomen, pelvis, and biopsy of the tumor. Based on the results of the diagnostic workup the choice of surgical treatment between RCE, partial cystectomy and transurethral resection was made considering the size of the tumor, location of the tumor in the bladder in relation to the bladder neck, and technical and oncological feasibility of performing the bladder sparing surgery. Kaplan - Meier survival curves were built to compare the results of survival per cancer stage and type of surgical treatment. Survival data of the patients were collected from the cancer registry maintained at the Kyiv Municipal Clinical Oncological Center. Results of data analysis were controlled for confounding parameters, such as adjuvant treatment: perioperative radiotherapy, and chemotherapy. Results: Median follow-up was 93 months (1-226 months). Males were 276 (80%). Average age at diagnosis was 62 ± 4.5 years. By the time of the study 61% of patients have died due to the progression of the disease. All patients with stage I disease (7% or 24 patients) were managed with bladder-sparing surgery. In muscle-invasive disease (309 patients), the RCE was performed in 109 (35.3%) patients, partial cystectomy was performed in 79 (25.6%) patients, and transurethral resection - in 121 (39.1%) patients. The overall 5-year survival of HGBC patients after radical surgical treatment (RCE/BPS) for stage I patients was 0%/83%, for stage II - 43%/58%, for stage III - 37%/42%, and for stage IV - 10%/40%. A total of 44 patients (12.7% of all treated, and 19.6% of treated with bladder sparing) received postoperative radiotherapy after bladder-sparing surgery. A total of 14 patients (4% of all treated) received postoperative chemotherapy. Conclusion: Bladder sparing surgery (partial en-block cystectomy, and transurethral resection of the bladder) in selected patients is not inferior to RCE in terms of cancer-specific survival when treating patients with HGBC of all stages. The bladder sparing surgery was performed in 64.7% of patients with high grade bladder cancer. Utilization of adjuvant treatment is low, 12.7% for postoperative radiotherapy, and 4% for perioperative chemotherapy.

Effect of androgen suppression on bone mineral density in patients with prostate cancer.

UNLABELLED: The androgen-suppressive therapy (AST) in patients with prostate cancer (PC) may dramatically affect the bone mineral density (BMD), which puts patients at risk of severe adverse effects, such as weight-bearing bone fractures. AIM: To study the effect of AST on BMD in patients with non-metastatic hormone-sensitive PC treated with intermittent hormonal therapy, and effect of different total testosterone level on BMD. MATERIALS AND METHODS: From 2011 to 2013 we treated 56 patients with non-metastatic hormone-naïve PC. Intermittent hormonal treatment with flutamide at a dose of 250 mg 3 times per day with nine monthly injections of luteinizing gonadotropic releasing hormone (LGnRH) ["treatment" period] followed by period of observance ("no treatment") was administered. We evaluated the BMD of lumbar spine and both proximal thighs by means of dual-energy x-ray densitometry at the end of "treatment" period and at the end of "no treatment" period. RESULTS: During the first treatment period, 44 of 56 patients (78.6%) experienced the reduction in BMD in both lumbar spine and thighs. Total testosterone level in all patients dropped to castration level. During the first period of "no treatment" there was an increase in BMD (p < 0.05) in 30 (68.2%) of 44 patients. The median time to recovery of total testosterone level to the level > 50 ng/dl was 91 days (from 30 to 308 days), and > 100 ng/dl was 110 days (from 49 to 343 days). The changes in BMD positively correlated with the changes in total testosterone level (correlation 0.18 [95% CI, 0.04-0.27], p = 0.009). The decline in total testosterone level in serum was followed by the decline in BMD value in the studied areas, and vice versa. CONCLUSIONS: The changes in BMD positively correlated with changes in total testosterone level. The BMD decreases during the androgen suppression and increases during the pause in the treatment. This demonstrates the benefit of intermittent AST in preventing osteoporosis, pathological bone fractures and possibly, bone metastases.