A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells.

OBJECTIVE: In this study, we showed a modified method for the isolation of cancer stem cells (CSCs) using a combination of differential adhesion method and serum-free culture medium (SFM) method. MATERIALS AND METHODS: Trypsin-sensitive cells and trypsin-resistant cells were isolated from MB49, EJ, and SK-OV-3 cells using a combination of differential adhesion method and SFM method. The CSCs markers expression of trypsin-resistant cells was verified by the flow cytometry, the Western blotting, and the quantitative polymerase chain reaction. Functional comparisons were verified by the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. RESULTS: Trypsin-resistant cells were isolated successfully. They were identified with high expression of CSCs markers and possessed higher resistance to chemotherapy, greater migration in vitro and stronger tumorigenic abilities in vivo. CONCLUSION: Trypsin-resistant cells showed specific CSCs characterizations. They were able to be isolated successfully with a modified method by a combination of differential adhesion method and SFM method.

[Three-dimensional versus two-dimensional imaging systems in laparoscopic radical prostatectomy for prostate cancer: a retrospective cohort study].

OBJECTIVE: To compare the perioperative, functional and oncologic outcomes of patients with prostate cancer receiving laparoscopic radical prostatectomy (LRP) using three-dimensional (3D) versus two-dimensional (2D) imaging systems. METHODS: From February, 2014 to January 2016, 72 consecutive patients with clinically localized prostate cancer underwent LRP with 2D or 3D imaging systems performed by a single experienced surgeon. The baseline characteristics, perioperative data, and functional and oncologic outcomes of the patients were collected and analyzed. RESULTS: Thirty-six patients underwent 3D LRP and the other 36 patients underwent 2D LRP. Compared with 2D LRP group, 3D LRP group had a significantly shorter operative time (167 vs 218 min, P<0.001), a smaller volume of intraoperative blood loss (86.11 vs 177.78 mL, P<0.001) and a better early urinary continence outcome (88.89% vs 63.89%, P=0.026). No significant differences were found between the two groups in terms of complications, potency outcome or biochemical recurrence-free rate. CONCLUSION: Compared with 2D LRP, 3D LRP shortens the operative time, reduces intraoperative blood loss and is associated with a better early urinary continence outcome in patients with clinically localized prostate cancer.

A modified method by differential adhesion for enrichment of bladder cancer stem cells.

PURPOSE: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. MATERIALS AND METHODS: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. RESULTS: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. CONCLUSION: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.

A modified method by differential adhesion for enrichment of bladder cancer stem cells

ABSTRACT Purpose: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. Materials and Methods: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. Results: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. Conclusion: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.

Characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression.

The aim of the present study was to examine the characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression. An immunofluorescence assay was used to detect E-cadherin and N-cadherin expression in infiltrative bladder cancer tissues, and immunofluorescence and western blot analysis were used to detect E-cadherin and N-cadherin expression in human urinary bladder grade II carcinoma 5637, transitional cell carcinoma UMUC-3 and invasive bladder carcinoma EJ cells. Cell proliferation, migration, invasion and plate colony formation assays were used to detect the proliferative, migratory and invasive abilities and the efficiency of plate colony formation of 5637, UMUC3 and EJ cells. A tumor xenograft formation assay was used to evaluate the tumorigenic abilities of 5637, UMUC-3 and EJ cells in vivo. E-cadherin and N-cadherin double-negative expression was identified in various pathological grades of infiltrative bladder cancers. E-cadherin positive and N-cadherin negative expression was exhibited by 5637 cells. By contrast, E-cadherin negative and N-cadherin positive expression was exhibited by EJ cells, and E-cadherin and N-cadherin double-negative expression was exhibited by UMUC-3 cells. The ability of cells to proliferate, migrate, invade, and the efficiency of plate colony formation and tumorigenic abilities of the cells were significantly different among 5637, UMUC-3 and EJ cells. These cell characteristics were significantly increased in UMUC-3 cells compared with 5637 cells; however, the characteristics were significantly decreased compared with EJ cells. The biological characteristics of bladder cancer cells with E-cadherin and N-cadherin double-negative expression was between bladder cancer cells that exhibited a E-cadherin positive and N-cadherin negative expression, and bladder cancer cells that exhibited E-cadherin negative and N-cadherin positive expression. The present study deduces that the status of E-cadherin and N-cadherin double-negative expression may participate in the process of epithelial-mesenchymal transition in the pathogenesis of bladder urothelial carcinoma.

Development of a therapy against metastatic bladder cancer using an interleukin-2 surface-modified MB49 bladder cancer stem cells vaccine.

INTRODUCTION: In previous study the streptavidin interleukin-2 (SA-IL-2)-modified MB49 vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate MB49 bladder cancer stem cells (MCSCs). Accordingly, we developed a SA-IL-2-modified MCSCs vaccine and evaluated its antitumor effects. METHODS: MCSCs were isolated and identified in cancer stem cells (CSCs) characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. The SA-IL-2 MCSCs vaccine was prepared and its bioactivity was evaluated. The protective, therapeutic, specific and memory immune response in animal experiments were designed to identify whether the vaccine elicited antitumor immunity and acted against metastatic bladder cancer. RESULTS: MCSCs had higher level of CD133 and CD44, less susceptibility to chemotherapy, more pronounced migration and greater tumorigenic ability. The successfully prepared SA-IL-2 MCSCs vaccine inhibited the tumor volume and prolonged mice survival in animal experiments. The expression of IgG, the population of dendritic cells, CD8(+) and CD4(+) T cells were highest in the experimental group than in the four control groups. CONCLUSIONS: The SA-IL-2 MCSCs vaccine induced an antitumor immune response and was used to eliminate MCSCs to prevent tumor regrowth.

The YAP1 oncogene contributes to bladder cancer cell proliferation and migration by regulating the H19 long noncoding RNA.

BACKGROUND: Yes-associated protein 1 (YAP1) and long noncoding RNA H19 act as potent oncogenes in many human cancers, but little is known about their roles in bladder cancer or their relationship with each other. METHODS: Quantitative real-time polymerase chain reaction and western blotting were performed retrospectively on human bladder cancer specimens and on bladder cancer cell lines (UMUC-3, EJ, and 5637). YAP1 and H19 expression levels were detected and correlated with clinical and pathologic grades. To determine whether YAP1 regulates H19 expression, their genes were overexpressed or suppressed in 5637 and UMUC-3 cells. The effects of YAP1/H19 on proliferation and migration were determined by viability, colony formation, transwell migration, and wound-healing assays. RESULTS: YAP1 and H19 expression levels were markedly elevated in bladder cancer tissues and cells, and H19 expression was found to be significantly associated with YAP1 expression. Determination of their clinicopathologic significance in 40 human bladder cancer tissues showed that specimens in which YAP1 and H19 were overexpressed were associated with poorer clinicopathologic prognosis. In addition, YAP1 was found to enhance H19 expression, whereas H19 had no significant effect on YAP1 expression in bladder cancer cells. Furthermore, the results of in vitro analyses suggested that this association regulates cell proliferation and migration. CONCLUSION: Our results emphasize the importance of YAP1 and H19 in bladder cancer progression and indicate that H19, at least in part, is induced by YAP1 overexpression.

The granulocyte macrophage-colony stimulating factor surface modified MB49 bladder cancer stem cells vaccine against metastatic bladder cancer.

The MB49 bladder cancer cell vaccine was effective against bladder cancer in the mice model in previous studies. However, part of the tumors regrew as the vaccine could not eliminate the cancer stem cells (CSCs). MB49 bladder cancer stem cells (MCSCs) were isolated by a combination of the limited dilution method and the serum free culture medium method. MCSCs possessed higher expression of CD133, CD44, OCT4, NANOG, and ABCG2, the ability of differentiation, higher proliferative abilities, lower susceptibility to chemotherapy, greater migration in vitro, and stronger tumorigenic abilities in vivo. Then streptavidin-mouse granulocyte macrophage-colony stimulating factor (SA-mGM-CSF) MCSCs vaccine was prepared. SA-mGM-CSF MCSCs vaccine extended the survival of the mice and inhibited the growth of tumor in protective, therapeutic, memorial and specific immune response experiments. The level of immunoglobulin G and the ratio of dendritic cells and CD4(+) and CD8(+) T cells were highest in the experimental group when compared to those in other four control groups, as well as for the cytotoxicity assay. We demonstrated that SA-mGM-CSF MCSCs vaccine induces an antitumor immune response to metastatic bladder cancer.