3D Tumor Models in Urology.

Three-dimensional tumor models have become established in both basic and clinical research. As multicellular systems consisting of tumor and tumor-associated cells, they can better represent tumor characteristics than monocellular 2D cultures. In this review, we highlight the potential applications of tumor spheroids and organoids in the field of urology. Further, we illustrate the generation and characteristics of standardized organoids as well as membrane-based 3D in vitro models in bladder cancer research. We discuss the technical aspects and review the initial successes of molecular analyses in the three major urologic tumor entities: urinary bladder carcinoma (BCa), prostate carcinoma (PCa), and renal cell carcinoma (RCC).

Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells.

The differential activation of Wnt pathways (canonical: Wnt/ß-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca2+) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD-co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6-LRP6 and FZD6-ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6-LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of ß-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6-ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/ß-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.

Cost-Constrained feature selection in binary classification: adaptations for greedy forward selection and genetic algorithms.

BACKGROUND: With modern methods in biotechnology, the search for biomarkers has advanced to a challenging statistical task exploring high dimensional data sets. Feature selection is a widely researched preprocessing step to handle huge numbers of biomarker candidates and has special importance for the analysis of biomedical data. Such data sets often include many input features not related to the diagnostic or therapeutic target variable. A less researched, but also relevant aspect for medical applications are costs of different biomarker candidates. These costs are often financial costs, but can also refer to other aspects, for example the decision between a painful biopsy marker and a simple urine test. In this paper, we propose extensions to two feature selection methods to control the total amount of such costs: greedy forward selection and genetic algorithms. In comprehensive simulation studies of binary classification tasks, we compare the predictive performance, the run-time and the detection rate of relevant features for the new proposed methods and five baseline alternatives to handle budget constraints. RESULTS: In simulations with a predefined budget constraint, our proposed methods outperform the baseline alternatives, with just minor differences between them. Only in the scenario without an actual budget constraint, our adapted greedy forward selection approach showed a clear drop in performance compared to the other methods. However, introducing a hyperparameter to adapt the benefit-cost trade-off in this method could overcome this weakness. CONCLUSIONS: In feature cost scenarios, where a total budget has to be met, common feature selection algorithms are often not suitable to identify well performing subsets for a modelling task. Adaptations of these algorithms such as the ones proposed in this paper can help to tackle this problem.

Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder.

Bladder dysfunction is characterized by urgency, frequency (pollakisuria, nocturia), and dysuria and may lead to urinary incontinence. Most of these symptoms can be attributed to disturbed bladder sensitivity. There is growing evidence that, besides the urothelium, suburothelial interstitial cells (suICs) are involved in bladder afferent signal processing. The massive expansion of the bladder during the filling phase implicates mechanical stress delivered to the whole bladder wall. Little is known about the reaction of suICs upon mechanical stress. Therefore, we investigated the effects of mechanical stimulation in cultured human suICs. We used fura-2 calcium imaging as a major physiological readout. We found spontaneous intracellular calcium activity in 75 % of the cultured suICs. Defined local pressure application via a glass micropipette led to local increased calcium activity in all stimulated suICs, spreading over the whole cell. A total of 51% of the neighboring cells in a radius of up to 100 µm from the stimulated cell showed an increased activity. Hypotonic ringer and shear stress also induced calcium transients. We found an 18-times increase in syncytial activity compared to unstimulated controls, resulting in an amplification of the primary calcium signal elicited in single cells by 50%. Our results speak in favor of a high sensitivity of suICs for mechanical stress and support the view of a functional syncytium between suICs, which can amplify and distribute local stimuli. Previous studies of connexin expression in the human bladder suggest that this mechanism could also be relevant in normal and pathological function of the bladder in vivo.

IC/BPS-associated alterations of M2 and M3 muscarinic acetylcholine receptor trafficking in human detrusor.

AIMS: To explore caveolae- and clathrin-mediated internalization of muscarinic M2 and M3 receptors, recycling and degradation in formalin-fixed paraffin-embedded detrusor sections; to study alterations possibly involved in the pathophysiology of the bladder functional disorder, interstitial cystitis/bladder pain syndrome (IC/BPS). MATERIALS AND METHODS: Samples of IC/BPS (n = 11) and cystectomy patients (n = 11) were analyzed. Proximity ligation assay (PLA) was used to detect interactions of M2 and M3 with endocytotic regulators (Cav-1, clathrin, Rab7, and Rab11) by Cy3 labeling. Analyses of three-dimensional (3D)-reconstructed z-stacks (63 × Oil 1.4) were done with Huygens software. We determined the object density for quantification and assessed membrane localization. RESULTS: Receptor/protein complexes were detected as well-demarcated 3D objects. Interactions of M2 with Cav-1, clathrin, Rab11, and Rab7 were significantly increased in IC/BPS. M3/clathrin and M3/Rab11 complexes were higher in IC/BPS, while M3/Cav-1 and M3/Rab7 were not. A significant shift of complexes from the membrane to cytoplasm was observed in conjunction with increased internalization via clathrin vesicles or caveolae in IC/BPS. CONCLUSIONS: High numbers of M3/clathrin and M3/Rab11 complexes reflect the well-documented clathrin-mediated desensitization of M3 and speak in favor with enhanced receptor protein expression in IC/BPS. Increased amounts of M2/Cav-1, M2/clathrin, and M2/Rab11 complexes represent altered M2 internalization and recycling leading to high abundance in IC/BPS. In this regard, caveolae-localized M2 could be possibly associated with the activation of nitric oxide (NO) synthase and NO production.

Comparative immunohistochemical characterization of interstitial cells in the urinary bladder of human, guinea pig and pig.

Interstitial cells (ICs) are thought to play a functional role in urinary bladder. Animal models are commonly used to elucidate bladder physiology and pathophysiology. However, inter-species comparative studies on ICs are rare. We therefore analyzed ICs and their distribution in the upper lamina propria (ULP), the deeper lamina propria (DLP) and the detrusor muscular layer (DET) of human, guinea pig (GP) and pig. Paraffin slices were examined by immunohistochemistry and 3D confocal immunofluorescence of the mesenchymal intermediate filament vimentin (VIM), alpha-smooth muscle actin (αSMA), platelet-derived growth factor receptor alpha (PDGFRα) and transient receptor potential cation channel A1 (TRPA1). Image stacks were processed for analysis using Huygens software; quantitative analysis was performed with Fiji macros. ICs were identified by immunoreactivity for VIM (excluding blood vessels). In all species ≥ 75% of ULP ICs were VIM+/PDGFRα+ and ≥ 90% were VIM+/TRPA1+. In human and pig ≥ 74% of ULP ICs were VIM+/αSMA+, while in GP the percentage differed significantly with only 37% VIM+/αSMA+ ICs. Additionally, over 90% of αSMA+ ICs were also TRPA1+ and PDGFRα+ in human, GP and pig. In all three species, TRPA1+ and PDGFRα+ ICs point to an active role for these cells in bladder physiology, regarding afferent signaling processes and signal modification. We hypothesize that decline in αSMA-positivity in GP reflects adaptation of bladder histology to smaller bladder size. In our experiments, pig bladder proved to be highly comparable to human urinary bladder and seems to provide safer interpretation of experimental findings than GP.

3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria.

AIMS: To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. METHODS: Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. RESULTS: 3View-SEM image stacks as large as 59 × 59 × 17 µm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. CONCLUSIONS: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

Computer tomographic analysis of organ motion caused by respiration and intraoperative pneumoperitoneum in a porcine model for navigated minimally invasive esophagectomy.

BACKGROUND: Navigation systems have the potential to facilitate intraoperative orientation and recognition of anatomical structures. Intraoperative accuracy of navigation in thoracoabdominal surgery depends on soft tissue deformation. We evaluated esophageal motion caused by respiration and pneumoperitoneum in a porcine model for minimally invasive esophagectomy. METHODS: In ten pigs (20-34 kg) under general anesthesia, gastroscopic hemoclips were applied to the cervical (CE), high (T1), middle (T2), and lower thoracic (T3) level, and to the gastroesophageal junction (GEJ) of the esophagus. Furthermore, skin markers were applied. Three-dimensional (3D) and four-dimensional (4D) computed tomography (CT) scans were acquired before and after creation of pneumoperitoneum. Marker positions and lung volumes were analyzed with open source image segmentation software. RESULTS: Respiratory motion of the esophagus was higher at T3 (7.0 ± 3.3 mm, mean ± SD) and GEJ (6.9 ± 2.8 mm) than on T2 (4.5 ± 1.8 mm), T1 (3.1 ± 1.8 mm), and CE (1.3 ± 1.1 mm). There was significant motion correlation in between the esophageal levels. T1 motion correlated with all other esophagus levels (r = 0.51, p = 0.003). Esophageal motion correlated with ventilation volume (419 ± 148 ml) on T1 (r = 0.29), T2 (r = 0.44), T3 (r = 0.54), and GEJ (r = 0.58) but not on CE (r = - 0.04). Motion correlation of the esophagus with skin markers was moderate to high for T1, T2, T3, GEJ, but not evident for CE. Pneumoperitoneum led to considerable displacement of the esophagus (8.2 ± 3.4 mm) and had a level-specific influence on respiratory motion. CONCLUSIONS: The position and motion of the esophagus was considerably influenced by respiration and creation of pneumoperitoneum. Esophageal motion correlated with respiration and skin motion. Possible compensation mechanisms for soft tissue deformation were successfully identified. The porcine model is similar to humans for respiratory esophageal motion and can thus help to develop navigation systems with compensation for soft tissue deformation.

The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder.

The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT+ ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody-related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin-1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT+ cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT+ ICC are present in bladder. In this perspective, functional concepts of KIT+ ICC being involved in sensory and/or motor aspects of bladder physiology should be revised.

Comparative study of the organisation and phenotypes of bladder interstitial cells in human, mouse and rat.

With most research on interstitial cells (IC) in the bladder being conducted on animal models, it remains unclear whether all structural and functional data on IC from animal models can be translated to the human context. This prompted us to compare the structural and immunohistochemical properties of IC in bladders from mouse, rat and human. Tissue samples were obtained from the bladder dome and subsequently processed for immunohistochemistry and electron microscopy. The ultrastructural properties of IC were compared by means of electron microscopy and IC were additionally characterized with single/double immunohistochemistry/immunofluorescence. Our results reveal a similar organization of the IC network in the upper lamina propria (ULP), the deep lamina propria (DLP) and the detrusor muscle in human, rat and mouse bladders. Furthermore, despite several similarities in IC phenotypes, we also found several obvious inter-species differences in IC, especially in the ULP. Most remarkably in this respect, ULP IC in human bladder predominantly displayed a myoid phenotype with abundant presence of contractile micro-filaments, while those in rat and mouse bladders showed a fibroblast phenotype. In conclusion, the organization of ULP IC, DLP IC and detrusor IC is comparable in human, rat and mouse bladders, although several obvious inter-species differences in IC phenotypes were found. The present data show that translating research data on IC in laboratory animals to the human setting should be carried out with caution.

Acute focal bacterial nephritis is associated with invasive diagnostic procedures - a cohort of 138 cases extracted through a systematic review.

BACKGROUND: Acute focal bacterial nephritis (AFBN) is a rare disease currently described only in case reports and small case series. In this study we summarize the clinical features of AFBN as has been documented in the literature and draw recommendations on the proper diagnosis and therapy. METHODS: A systematic literature review was undertaken in PUBMED, Web of Science and The Cochrane Library online databases for relevant literature on AFBN in adults. RESULTS: Literature review revealed a total of 38 articles according to our inclusion criteria, of which we could extract data from 138 cases of AFBN. Fever (98%) and flank pain (80%) were most commonly reported symptoms. E. coli was the most frequent pathogen. Diagnosis was set by CT and/or MRI (52%) with or without sonography or by sonography alone (20%) as well as by sonography combined with IVU. In total, sonography was applied in 83% of cases. All but one patient received antibiotic treatment. Kidney lesions were occasionally mistaken for neoplasms or renal abscesses and as a result, cases were subjected to percutaneous puncture (12.3%), surgical exploration (5.1%) and partial or radical nephrectomy (4.4%). Four cases (2.9%) developed a renal abscess. CONCLUSIONS: The diagnosis of AFBN is set by characteristic clinico-radiological findings. Differential diagnoses of this interstitial bacterial infection include renal abscess and tumor. Correct diagnosis is occasionally impeded by atypical symptoms. Invasive diagnostic and therapeutic procedures should be limited as the majority of cases respond well to conservative treatment.

Striated Muscle in Radical Prostatectomy Specimens: A Marker of Apical Dissection Quality and an Independent Predictor of Urinary Continence after Endoscopic Extraperitoneal Radical Prostatectomy.

INTRODUCTION: The study aimed to determine if the presence and amount of striated muscle on the apical sections of the cruciate sections of laparoscopic radical prostatectomy (LRP) specimens predict early and long-term urinary continence outcomes. PATIENTS AND METHODS: We conducted a retrospective review of our prospectively collected single surgeon LRP database. We identified patients based on their continence outcomes (continent (0 pads) or incontinent at 12 months), with an approximate even spread early continent and incontinent patients). An uropathologist separate from the urology team was blinded to outcome and assessed each patients' apical cruciate sections (H&E stained) for the presence, percentage and maximal diameter of muscle and extraprostatic tissue on these sections. Specifically 2 scoring systems were used: (1) semi-quantitative estimation of percentage of muscle on the apical cruciate sections (low <5% and high >5%) and (2) percentage of total extraprostatic tissue on cruciate section (low <10% and high >10%). Logistic regression and classification and regression tree analyses were performed to identify the predictors of urinary incontinence (UI). RESULTS: In total 80 patients were analyzed, 38 were continent and 42 were incontinent at 12 months follow-up. The percentage of extraprostatic tissue/muscle being an independent predictor of being wet at 12 months (p = 0.002) on multivariate regression along with age (p = 0.04). Using percentage of extraprostatic tissue in cruciate section (high >10%) to predict UI at 12 months, it yielded 71% sensitivity, 82% specificity, 81% PPV, 72% NPV and 76% accuracy. CONCLUSION: The use of simple additional reporting of muscle and extraprostatic tissue on the apical sections of RP specimens can help to better predict the likelihood of continence return.

Protease Expression Levels in Prostate Cancer Tissue Can Explain Prostate Cancer-Associated Seminal Biomarkers-An Explorative Concept Study.

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

Anatomical study of pelvic nerves in relation to seminal vesicles, prostate and urethral sphincter: immunohistochemical staining, computerized planimetry and 3-dimensional reconstruction.

PURPOSE: Studies of male pelvic neuroanatomy are mandatory to improve functional outcome after radical prostatectomy. We performed a topographical investigation of nerves on the course from the seminal vesicles along the prostate toward the striated urethral sphincter. MATERIALS AND METHODS: Serial whole mount sections (1 mm intervals) of pelvic blocks of human adult male autopsy cadavers were investigated after immunohistochemical nerve staining. Computerized nerve quantification and planimetry of the total nerve surface area were performed within defined regions (ventral, ventrolateral, dorsolateral and dorsal) at the levels of the seminal vesicles and prostate, and at the striated urethral sphincter. The distance between the seminal vesicles and the nerves was measured. For improved topographical understanding 3-dimensional reconstructions were created. Differences between 3 independent variables were tested with the nonparametric Kruskal-Wallis test. RESULTS: We studied a total of 969 whole mount sections of 5 cadavers. Nerves were arranged in a vertical plate lateral to the seminal vesicles. Mean ± SD distance to the seminal vesicles was 1.68 ± 0.84, 1.50 ± 0.12 and 1.76 ± 0.37 mm at the tip, middle and base, respectively. Periprostatic nerves were mainly found dorsolaterally. At the striated urethral sphincter 38.9% of nerves had shifted to the dorsal region. The total nerve surface area decreased significantly from the seminal vesicle tip (50.2 mm(2)) to the striated urethral sphincter level (13.3 mm(2)) (p = 0.0004). CONCLUSIONS: Our findings underline that during nerve sparing prostatectomy nerve damage might occur during mobilization of the entire seminal vesicles, apical dissection and posterior reconstruction of the rhabdosphincter. Nerve planimetry revealed that 75% of the nerves from the seminal vesicles do not reach the striated urethral sphincter level and seem to innervate structures other than the corpora cavernosa.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Navigation system for minimally invasive esophagectomy: experimental study in a porcine model.

BACKGROUND: Navigation systems potentially facilitate minimally invasive esophagectomy and improve patient outcome by improving intraoperative orientation, position estimation of instruments, and identification of lymph nodes and resection margins. The authors' self-developed navigation system is highly accurate in static environments. This study aimed to test the overall accuracy of the navigation system in a realistic operating room scenario and to identify the different sources of error altering accuracy. METHODS: To simulate a realistic environment, a porcine model (n = 5) was used with endoscopic clips in the esophagus as navigation targets. Computed tomography imaging was followed by image segmentation and target definition with the medical imaging interaction toolkit software. Optical tracking was used for registration and localization of animals and navigation instruments. Intraoperatively, the instrument was displayed relative to segmented organs in real time. The target registration error (TRE) of the navigation system was defined as the distance between the target and the navigation instrument tip. The TRE was measured on skin targets with the animal in the 0° supine and 25° anti-Trendelenburg position and on the esophagus during laparoscopic transhiatal preparation. RESULTS: On skin targets, the TRE was significantly higher in the 25° position, at 14.6 ± 2.7 mm, compared with the 0° position, at 3.2 ± 1.3 mm. The TRE on the esophagus was 11.2 ± 2.4 mm. The main source of error was soft tissue deformation caused by intraoperative positioning, pneumoperitoneum, surgical manipulation, and tissue dissection. CONCLUSION: The navigation system obtained acceptable accuracy with a minimally invasive transhiatal approach to the esophagus in a realistic experimental model. Thus the system has the potential to improve intraoperative orientation, identification of lymph nodes and adequate resection margins, and visualization of risk structures. Compensation methods for soft tissue deformation may lead to an even more accurate navigation system in the future.

Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization-A New Standardized Approach in Bladder Cancer Research.

Three-dimensional tumor models have gained significant importance in bladder cancer (BCa) research. Organoids consisting of different cell types better mimic solid tumors in terms of 3D architecture, proliferation, cell-cell interaction and drug responses. We developed four organoids from human BCa cell lines with fibroblasts and smooth muscle cells of the bladder, aiming to find models for BCa research. The organoids were characterized in terms of cytokeratins, vimentin, α-actin and KI67 by immunoreactivity. Further, we studied ligand-dependent activation of the Wnt/ß-catenin pathway and investigated the responses to anti-tumor therapies. The organoids mimicked the structure of an inverse bladder wall, with outside urothelial cells and a core of supportive cells. The cytokeratin staining patterns and proliferation rate were in conjunction with the origins of the BCa cells. RT-112 even showed stratification of the epithelium. Treatment with Wnt10B led to increased ß-catenin (active) levels in high-grade organoids, but not in low-grade BCa cells. Doxorubicin treatment resulted in clearly reduced viability (10-30% vs. untreated). In contrast, the effectivity of radiotherapy depended on the proliferation status of BCa cells. In conclusion, cell-line-based organoids can form bladder-like structures and reproduce in vivo features such as urothelial differentiation and stratification. Thus, they can be useful tools for functional studies in BCa and anti-cancer drug development.

Enhanced urothelial expression of human chorionic gonadotropin beta (hCGß) in bladder pain syndrome/interstitial cystitis (BPS/IC).

PURPOSE: Bladder pain syndrome/interstitial cystitis (BPS/IC) is associated with urothelial lesions. Pathomechanisms of urothelial damage and factors for urothelial restoration are unknown. hCG is a factor for cellular differentiation, angiogenesis and immune competence of the endometrium during pregnancy. Clinical observations demonstrate improvement of BPS/IC symptoms during pregnancy or during infertility treatment with hCG. Our research aims were to examine the expression of hCG and luteinizing hormone receptor (LHR) in the urothelium of BPS/IC patients and compare the levels of hCGß with healthy controls. METHODS: Bladder biopsies of BPS/IC (CLSM: n = 10; qPCR: n = 15); Tumour-free control tissue from cystectomies (n = 12). hCGα, hCGß and LHR expression were examined by confocal laser scanning microscopy (CLSM), and hCGß expression was quantified. hCGß5 and hCGß7 mRNA splice variants were quantified in real-time polymerase chain reaction. RESULTS: We found constitutive expression of hCGα, hCGß and LHR in healthy controls. HCGß was significantly upregulated in BPS/IC patients in CLSM. PCR analysis revealed higher levels of hCGß7 than hCGß5 in controls and BPS/IC patients. CONCLUSIONS: The constitutive expression of hCG and LHR speaks in favour for a functional signalling in urothelial cells without any association with either pregnancy or tumour. We show for the first time that hCGß is upregulated in BPS/IC urothelium and that hCGß7 is the dominant splice variant in those cells. Our findings imply a major role of hCG for urothelial integrity and a disturbance of hCG signalling in case of BPS/IC. We conclude that hCG could gain therapeutical relevance in the future.

Individual receptor profiling as a novel tool to support diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC).

PURPOSE: Dysregulation of neurotransmitter receptors may contribute to bladder overactivity (OAB) symptoms. To address the question whether specific receptor expression patterns are associated with bladder pain syndrome/interstitial cystitis (BPS/IC), we examined the expression of muscarinic, purinergic and histamine receptors in the detrusor. METHODS: Detrusor receptor expression was investigated in bladder biopsies of female BPS/IC patients (n = 44; age 60.64 ± 13.78, mean ± SD) and carcinoma patients (n = 11; age 58.91 ± 12.72) undergoing cystectomy. Protein expression of muscarinic (M2, M3), purinergic (P2X1-3) and histamine receptors (H1, H2) was analysed by confocal immunofluorescence, and gene expression was quantified by real-time polymerase chain reaction (qPCR). RESULTS: M2, P2X1, P2X2 and H1 receptor immunoreactivity (-IR) was significantly enhanced in BPS/IC compared to the control group, while there was no difference for M3-, P2X3- and H2-IR. We calculated a score, which separated BPS/IC from control patients with an AUC of 89.46%, showing 84.09% sensitivity and 90.91% specificity. Patients had a 9.25 times enhanced calculated risk for BPS/IC. In addition, two patient subgroups (M2 > M3 and M3 > M2) were observed, which differed in associated purinergic and histamine receptor expression. CONCLUSIONS: M2, P2X1, P2X2 and H1 were significantly upregulated in BPS/IC patients, and H2 was occasionally highly overexpressed. There was no significant correlation between receptor protein and gene expression, implying posttranslational mechanisms being responsible for the altered receptor expressions. On the basis of individual receptor profiles, upregulated receptors could be targeted by monotherapy or combination therapy with already approved receptor inhibitors, thereby promoting tailored therapy for patients suffering from BPS/IC-like symptoms.

Editorial: Special Issue "Biomarkers in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS)".

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a disabling chronic disease of still unknown origin and complex pathophysiology [...].