Extracellular matrix in muscle-invasive bladder cancer: Identification and validation of ECM related mechanisms in MIBC: A multi-omics analysis of a novel interpreted 3D in vitro model

Bladder cancer (BC) is the second leading cause of cancer death in the world. Treatment options for muscle-invasive bladder cancer (MIBC) are limited, with poor prognosis and incomplete molecular tumor classification posing a significant problem. BC onset and progression is highly dependent on extracellular matrix (ECM) remodeling. Moreover, the use or development of translational 3D in vitro models is rare in BC research, thus the mechanistically background underlying the interaction between MIBC tumor cells and ECM, as well as the identification of novel therapeutic targets and biomarker needs to be further investigated. In this study, the BC cell lines (UROtsa, RT4, T24/83, SCaBER) were applied to the 3D in vitro ultra-low-attachment (ULA) method under Matrigel-free conditions. Intact spheroids were analyzed by a transcriptomic microarray and proteomic approach, and were compared to the regular used 2D model. Based on the protein level, gene set enrichment analysis was performed and pathways related to ECM proteins were identified. Resulting targets were further validated by public available mRNA sequencing data from MIBC patients. The targets gene expression was related to clinicopathological parameters, overall survival (OS), and disease-free survival (DFS). Intact spheroid formation and growth was realized for T24/83 and RT4, while T24/83 needed the addition of 50% unconditioned fibroblast basal medium (UCM) or fibroblast conditioned medium (CM). A combination of ECM-based and -interacting targets (n=10) were significantly increased on protein level and mRNA level for the invasive T24/83 spheroids, compared to 2D culture. 6(10) targets were elevated under CM (CALU, CD109, FBN1, HTRA1, LAMC1, LRP1), 2(10) in both (P4HA2 and PLOD1), and 2(10) under UCM condition (DPYSL3 and SUMF2). Furthermore, increased levels of these targets correlated with worse OS, DFS and advanced MIBC features. Mechanistically, these targets were found to act in epithelial to mesenchymal transition (EMT), embryonic stem cell signature (ESC), neurological activity, cancer pathways, and posttranslational modification (PTM). For RT4, no ECM-related target was valid. Spheroids of the invasive T24/83 BC cell line expressed malignant features more abundant compared to the 2D standard condition. Under CM, more targets were validated compared to UCM conditions, indicating more distinct MIBC properties under CM condition. Thus, T24/83 spheroids cultured under CM conditions can serve as a valid in vitro 3D model to study invasive BC mechanisms (EMT, ESC, RAF1/MAP2K1/ERK pathway, ERBB2) and treatment options for future projects. Furthermore, follow up the 10 identified ECM-related targets could lead to promising molecular biomarker or future therapeutic targets, which is urgent to improve personalized stratification, and therapy for MIBC patients

Predictive value of lymphangiogenesis and proliferation markers on mRNA level in urothelial carcinoma of the bladder after radical cystectomy

Objective: To evaluate the mRNA expression of lymphangiogenesis and proliferation markers and to examine its association with histopathological characteristics and clinical outcome in patients with urothelial carcinoma of the bladder (UCB) after radical cystectomy (RC). Patients and methods: Gene expression analysis of the vascular endothelial growth-C and-D (VEGF-C/-D), its receptor VEGF receptor-3 (VEGFR-3), MKI67, and RACGAP1 was performed in 108 patients after radical cystectomy and their correlation with clinical-pathological parameters was investigated. Uni- and multivariate regression analyses were used to identify predictors for cancer -specific survival (CSS), recurrence-free survival (RFS) and overall survival (OS) after RC. Results: The expression of RACGAP1 and VEGFR-3 showed an association with a higher pT stage (P = 0.049; P = 0.009). MKI67 showed an association with a high-grade urothelial carcinoma of the bladder (P = 0.021). VEGFR-3 expression was significantly associated with the presence of lymphovascular invasion (LVI) (P = 0.016) and lymph node metastases (pN+) (P = 0.028). With the univariate analysis, overexpression of VEGFR-3 (P = 0.029) and the clinical-pathological parameters pT stage (P < 0.0001), pN+ (P = 0.0004), LVI (P < 0.0001) and female gender (P = 0.021) were significantly associated with a reduced CSS. Multivariate analysis identified a higher pT stage (P = 0.017) and LVI (P = 0.008) as independent predictors for reduced CSS. Independent predictors for reduced OS were a higher pT stage (P = 0.0007) and LVI (P = 0.0021), while overexpression of VEGF-D was associated with better OS (P < 0.0001). Conclusions: The mRNA expression of the investigated markers showed associations with common histopathological parameters. Increased expression of VEGF-D is independently associated with better overall survival. (C) 2018 Elsevier Inc. All rights reserved

Evaluating the value of intrapartum fetal scalp blood sampling to predict adverse neonatal outcomes: A UK multicentre observational study.

ObjectiveTo evaluate the value of fetal scalp blood sampling (FBS) as an adjunct test to cardiotocography, to predict adverse neonatal outcomes.Study designA multicentre service evaluation observational study in forty-four maternity units in the UK. We collected data retrospectively on pregnant women with singleton pregnancy who received FBS in labour using a standardised data collection tool. The primary outcome was prediction of neonatal acidaemia diagnosed as umbilical cord arterial pH < 7.05, the secondary outcomes were the prediction of Apgar scores<7 at 1st and 5th minutes and admission to the neonatal intensive care unit (NICU). We evaluated the correlation between the last FBS blood gas before birth and the umbilical cord blood and adjusted for time intervals. We constructed 2 × 2 tables to calculate the sensitivity, specificity, positive (PPV) and negative predictive value (NPV) and generated receiver operating curves to report on the Area Under the Curve (AUC).ResultsIn total, 1422 samples were included in the analysis; pH values showed no correlation (r = 0.001, p = 0.9) in samples obtained within an hour (n = 314), or within half an hour from birth (n = 115) (r=-0.003, p = 0.9). A suboptimal FBS pH value (<7.25) had a poor sensitivity (22%) and PPV (4.9%) to predict neonatal acidaemia with high specificity (87.3%) and NPV (97.4%). Similar performance was noted to predict Apgar scores <7 at 1st (sensitivity 14.5%, specificity 87.5%, PPV 23.4%, NPV 79.6%) and 5th minute (sensitivity 20.3%, specificity 87.4%, PPV 7.6%, NPV 95.6%), and admission to NICU (sensitivity 20.3%, specificity 87.5%, PPV 13.3%, NPV 92.1%). The AUC for FBS pH to predict neonatal acidaemia was 0.59 (95%CI 0.59–0.68, p = 0.3) with similar performance to predict Apgar scores<7 at 1st minute (AUC 0.55, 95%CI 0.51–0.59, p = 0.004), 5th minute (AUC 0.55, 95%CI 0.48–0.62, p = 0.13), and admission to NICU (AUC 0.58, 95%CI 0.52–0.64, p = 0.002).Forty-one neonates had acidaemia (2.8%, 41/1422) at birth. There was no significant correlation in pH values between the FBS and the umbilical cord blood in this subgroup adjusted for sampling time intervals (r = 0.03, p = 0.83).ConclusionsAs an adjunct tool to cardiotocography, FBS offered limited value to predict neonatal acidaemia, low Apgar Scores and admission to NICU