Recurrence risk assessment for stage III colorectal cancer based on five methylation biomarkers in plasma cell-free DNA.

For stage III colorectal cancer (CRC) patients with a high risk of recurrence, intensified adjuvant chemotherapy can improve overall survival. We aimed to develop a circulating tumor DNA (ctDNA) methylation marker model for predicting the relapse risk of stage III CRC patients. Differentially methylated markers identified between 53 normal mucosa samples and 165 CRC tissue samples, as well as between plasma samples from 75 stage I/II (early-stage) CRC patients and 55 stage IV (late-stage) CRC patients, were analyzed using Student's t-tests. The overlapping methylation markers shared by plasma and tissue samples were used to establish a methylation marker model to evaluate the tumor burden in the peripheral blood of CRC patients using the random forest method. This model was verified in the validation cohort (n = 44) and then applied to predict recurrence risk in 50 stage III CRC patients and monitor the clinical disease course in serial samples from four CRC patients. We built a five-marker-based ctDNA methylation model that had high sensitivity (84.21%) and specificity (84%) in identifying late-stage CRC in a validation cohort containing 24 stage I/II CRC patients and 20 stage IV CRC patients. The model achieved high sensitivity (87.5%) and specificity (94.12%) in predicting tumor relapse in an independent cohort of 50 stage III CRC patients and could be an independent recurrence risk factor for stage III patients [Hazard ratio (HR), 60.4; 95% confidence interval (CI): 7.68-397; p = 9.73e-5]. Analysis of serial blood samples of CRC showed that the model could monitor disease relapse earlier than imaging examination and serum carcinoembryonic antigen (CEA) and so may provide an opportunity for the early adjustment of therapeutic strategies. Moreover, the model could potentially monitor the clinical course and treatment response dynamically. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PPARγ activation serves as therapeutic strategy against bladder cancer via inhibiting PI3K-Akt signaling pathway.

BACKGROUND: Heterogeneity in bladder cancer results in variable clinical outcomes, posing challenges for clinical management of this malignancy. Recent studies suggest both tumor suppressive and oncogenic role of PPARγ in bladder cancer. The fuction of PPARγ signaling pathway in modulating carcinogenesis is controversial. METHODS: The expression of PPARγ and association with overall survival were analyzed in patients from two cohorts. The effect of PPARγ activation on cell proliferation, cell cycle, and cell apoptosis were determined with the agonists (rosiglitazone and pioglitazone), the inverse agonist (T0070907), and the antagonist (GW9662) in Umuc-3 and 5637 bladder cancer cells. The correlation of PPARγ activation with PI3K-Akt pathway was evaluated with RNA sequencing data from the TCGA cases and 30 human bladder cancer cell lines. The effect of PPARγ activation on tumor growth was validated with subcutaneous tumor models in vivo. The effect of PPARγ activation on PI3K-Akt signaling transduction was determined with multiple assays including immunohistochemistry, flow cytometry, proteomic array, and western blotting. RESULTS: We showed that PPARγ was a favorable prognostic factor in patients with bladder cancer. PPARγ activation by rosiglitazone and pioglitazone markedly induced cell cycle G2 arrest and apoptosis in bladder cancer cells, which resulted in inhibition of cell proliferation in vitro and suppression of tumor growth in vivo. The underlying mechanism involved marked inhibition of PI3K-Akt pathway. CONCLUSIONS: This study reported the tumor-suppressive effect of PPARγ agonists in bladder cancer, suggesting that transactivation of PPARγ could be served as a potential strategy for the chemoprevention and therapeutic treatment of bladder cancer.

A modified method for isolation of bladder cancer stem cells from a MB49 murine cell line.

BACKGROUND: The vaccine was efficiently effective against bladder cancer in earlier studies. However, a part of the mouse bladder tumour regrew due to regression after a period of time as the cancer stem cells could not be eliminated. In this study, we showed a modified method for the isolation of MB49 bladder cancer stem cells (MCSCs). METHODS: Through a comparison of different serum-free culture mediums (SFM), MCSCs were isolated by a combination of the limited dilution method and the optimal SFM method. The characterizations of MCSCs were verified by the fluorescence activated cell sorting, the quantitative polymerase chain reaction, the western blotting, the cell proliferation assay, the soft agar assay, the transwell assay, the resistance to chemotherapy assay and the tumor xenograft formation assay. RESULTS: The optimal SFM contained a RPMI1640+ epidermal growth factor (20 ng/ml), a basic fibroblast growth factor (20 ng/ml), a leukemia inhibitory factor (20 ng/ml), a B-27 serum-free supplement (20 µl/ml), and a bovine serum albumin (4 µg/ml). MCSCs possessed the high expression of cancer stem cell markers (CD133, CD44, OCT4, NANOG, and ABCG2) and the ability of differentiation. In functional comparisons, MCSCs had higher proliferative abilities, lower susceptibility to chemotherapy, greater migration in vitro, and stronger tumorigenic abilities in vivo. CONCLUSION: MCSCs displayed specific cancer stem cells properties. Our study showed MCSCs were isolated successfully with a modified method using a combination of limited dilution and SFM methods.

[Microanatomy of blood vessels in spermatic cords and its clinical implication].

OBJECTIVE: Both microsurgical subinguinal varicocelectomy (MSIV) and microsurgical high inguinal varicocelectomy (MHIV) are recommended for the treatment of varicocele, but they differ in technical complexity. This study aimed to determine the microanatomy of spermatic blood vessels in the two surgical approaches. METHODS: We recorded the numbers of spermatic veins, arteries and lymphatics in 80 cases of MSIV and 20 cases of MHIV. We also examined the spermatic cords from 10 adult male cadavers by histological staining. RESULTS: The numbers of medium spermatic veins (2 -5 mm in diameter) were 1.80 +/- 0.83 and 3.98 +/- 1. 99 in MHIV and MSIV, respectively, with significant difference between the two groups (t = -7.536, P < 0.01), and the total numbers of spermatic veins were 6.40 +/- 1.67 and 9.01 +/- 2.70, also with significant difference between the two (t = -4.071, P < 0.01). However, there were no significant differences between MHIV and MSIV in the numbers of small spermatic veins (diameter < or = 2 mm), large spermatic veins (diameter > or = 5 mm), arteries and lymphatics, nor in the numbers of spermatic veins and arteries of the cadavers. CONCLUSION: The total number of spermatic veins and the number of medium spermatic veins may be larger in MSIV than in MHIV, but the medium spermatic veins do not increase surgical difficulty, and MSIV is not more complicated than MHIV.

Seven DNA Methylation Biomarker Prediction Models for Monitoring the Malignant Progression From Advanced Adenoma to Colorectal Cancer.

Advanced adenoma (AA) holds a significantly increased risk for progression to colorectal cancer (CRC), and we developed a noninvasive DNA methylation prediction model to monitor the risk of AA progression to CRC. We analyzed the differential methylation markers between 53 normal mucosa and 138 CRC tissues, as well as those in cfDNA (cell-free DNA) between 59 AA and 68 early-stage CRC patients. We screened the overlapping markers between tissue DNA and cfDNA for model variables and optimized the selected variables. Then, we established a cfDNA methylation prediction model (SDMBP model) containing seven methylation markers that can effectively discriminate early-stage CRC and AA in the training and validation cohorts, and the AUC (area under the curve) reached 0.979 and 0.918, respectively. Our model also reached high precision (AUC=0.938) in detecting advanced CRC (stage III/IV) and presented better performance than serum CEA and CA199 in screening CRC. The cd-score of the SDMBP model could also robustly predict the TNM stage of CRC. Overall, our SDMBP model can monitor the malignant progression from AA to CRC, and may provide a noninvasive monitoring method for high-risk populations with AA.

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.

Leukemia Inhibitory Factor Receptor Suppresses the Metastasis of Clear Cell Renal Cell Carcinoma Through Negative Regulation of the Yes-Associated Protein.

The role of leukemia inhibitory factor receptor (LIFR), which is important in the signal transduction of the interleukin-6 cytokine family, is still undefined in clear cell renal cell carcinoma (ccRCC). Thus, we examined the function and mechanism of LIFR in ccRCC. Low LIFR expression correlated with a poor prognosis and an aggressive tumor phenotype. Moreover, integrated LIFR DNA and mRNA analysis revealed that promoter methylation and copy number variation contributed to the reduced LIFR expression. LIFR knockdown increased 786-O and Caki-2 cell invasion and migration. Notably, the Hippo pathway was highlighted as a potential downstream target of LIFR, where loss of LIFR inhibited the kinase activity of the pathway and increased the intracellular Yes-associated protein (YAP) level. Conversely, YAP inhibition impaired the LIFR-silencing promotion of cell migration, invasion, and cancer stem cell marker expression. Moreover, drug sensitivity analysis and the Cancer Cell Line Encyclopedia database revealed that LIFR-deficient cells had high sensitivity to a YAP inhibitor and to two other anticancer drugs (PHA-665752, PF2341066). Our study revealed that LIFR attenuates tumor metastasis by suppressing YAP expression, suggesting that LIFR may serve as a potential target for ccRCC treatment.

Histone methyltransferase KMT2D sustains prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4.

Abnormalities in epigenetic modifiers are emerging as driving events in prostate cancer (PCa). The histone methyltransferase KMT2D, a frequently aberrant epigenetic modifier in various tumors, has an undefined role in PCa. Moreover, little is known regarding KMT2D's mutation in Chinese patients or its downstream signaling pathways and targets. Here, we profiled the mutational spectrum of 32 significantly PCa-associated genes by using disease-targeted sequencing, and found that KMT2D was highly mutated (63.04%, 29/46) in Chinese patients. Moreover, high KMT2D transcription was also associated with poor prognosis in an independent cohort (n = 51). In KMT2D-knockdown PC-3 and DU145 cells, cell proliferation (P < 0.01), invasion (P < 0.001), and migration (P < 0.01) were consequently suppressed. KMT2D depletion effectively suppressed tumor growth by 92.21% in vivo. Notably, integrative analyses of RNAseq and ChIPseq characterized two crucial genes downregulated by KMT2D, leukemia inhibitory factor receptor (LIFR) and Kruppel-like factor-4 (KLF4), which are regulators in PI3K/Akt and EMT, respectively. Our present study revealed that KMT2D epigenetically activates PI3K/Akt pathway and EMT by targeting LIFR and KLF4 and thus serves as a putative epigenetic-based target for treating PCa.

[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.

HnRNP-L mediates bladder cancer progression by inhibiting apoptotic signaling and enhancing MAPK signaling pathways.

Heterogeneous nuclear ribonucleoprotein L (hnRNP-L) is a promoter of various kinds of cancers, but its actions in bladder cancer (BC) are unclear. In this study, we investigated the function and the underlying mechanism of hnRNP-L in bladder carcinogenesis. Our results demonstrated that enhanced hnRNP-L expression in BC tissues was associated with poor overall survival of BC patients. Depletion of hnRNP-L significantly suppressed cell proliferation in vitro and inhibited xenograft tumor growth in vivo. Furthermore, downregulation of hnRNP-L resulted in G1-phase cell cycle arrest and enhanced apoptosis accompanied by inhibition of EMT and cell migration. All these cellular changes were reversed by ectopic expression of hnRNP-L. Deletion of hnRNP-L resulted in decreased expression of Bcl-2, enhanced expression of caspases-3, -6 and -9 and inhibition of the MAPK signaling pathway. These findings demonstrate that hnRNP-L contributes to poor prognosis and tumor progression of BC by inhibiting the intrinsic apoptotic signaling and enhancing MAPK signaling pathways.

The decrease of cyclin B2 expression inhibits invasion and metastasis of bladder cancer.

OBJECTIVE: It has been shown that cyclin B2 is commonly overexpressed in many malignant tumors. This study aimed to investigate the potential role of cyclin B2 in bladder cancer. METHOD AND MATERIAL: Fixed tissues for immunohistochemistry and fresh tissues for western blotting and quantitative real-time polymerase chain reaction assay were randomly selected from Nanfang hospital. Normal bladder urothelial cell and bladder cancer cell lines was stored in our laboratory, the bladder cancer cells were transfected to develop bladder cancer cell clones expressing decreased cyclin B2 levels, the clones were used for cell growth and metastasis experiments in vitro and in vivo. RESULTS: Western blot and immunohistochemical analysis both showed that the cyclin B2 protein expression was higher in bladder urothelial carcinoma than in normal bladder mucosa, especially in invasive cancer. Real-time polymerase chain reaction showed that the cyclin B2 messenger RNA expression exhibited the same trend. Results of cell lines experiments also showed higher cyclin B2 expression in cancer cells. In vitro tests the decrease of cyclin B2 expression that had little effect on cell growth and cell cycling according to the MTT assay and the Edu assay, whereas in the Boyden chamber transwell assay, the cyclin B2 low-expressing clones significantly inhibits the cells׳ invasion and metastatic abilities. This result was consistent with the scratch-wound assay result showing that the target clone needed more time for healing the wound. The in vivo experiment in nude mice produced similar results, the lung and liver target cell metastasis nodules were smaller and less than those of the negative control by the hepatic subcapsular injection assay, and the mice of the target clone group has longer survival time in no intervention observed test. CONCLUSION: These results indicate that the cyclin B2 was overexpressed in bladder cancer, and the down-regulation of cyclin B2 expression in bladder cancer greatly inhibits the cell׳s invasion and metastatic abilities, and it prolonged the survival time of nude mice in vivo.

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.

Red and processed meat intake and risk of bladder cancer: a meta-analysis.

Findings from epidemiologic studies concerning red and processed meat intake and bladder cancer risk remain conflicting. Thus, we conducted this meta-analysis to examine the associations of red and processed meat intake with bladder cancer. Eligible studies published up to May 2014 were retrieved via both computer searches and review of references. Finally, we identified 14 studies on red meat (involving 9,084 cases) and 11 studies on processed meat (7,562 cases) involving up to 1,558,848 individuals. Random-effects models were used to estimate summary relative risk estimates (SRRE) based on high vs. low intake, and heterogeneity between study results was explored through stratified analyses on the basis of red/processed meat category, gender, study design and geographical region. Overall, the SRRE for all studies regarding red meat intake was 1.15 (95% CI: 0.97-1.36). Significant positive association was observed between processed meat consumption and bladder cancer (SRRE = 1.22; 95% CI: 1.04-1.43). Interestingly, increased by 25% and 33% risk of bladder cancer were observed for red meat and processed meat intake respectively in populations from the American continent. In conclusion, our fi ndings showed that there was an absence of an association between red meat intake and bladder cancer, but suggested that high consumption of processed meat probably correlated with rising risk of bladder cancer. In addition, positive relationships were observed regarding people intake of red and processed meat in the American continent. These findings need to be confirmed in future research.

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.