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.

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.

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.

Reduction of Muscle-Invasive Tumors by Photodynamic Therapy with Tetrahydroporphyrin-Tetratosylat in an Orthotopic Rat Bladder Cancer Model.

Photodynamic therapy (PDT) is a promising option for minimal-invasive treatment of bladder cancer. Efficacy of PDT in muscle-invasive urothelial cancer is still hampered by low tissue penetration of most photosensitizers due to short excitation wavelength. The novel light reactive agent tetrahydroporphyrin-tetratosylat (THPTS) is excitable at near-infrared (760 nm), allowing tissue penetration of up to 15 mm. Here, we established an orthotopic rat bladder cancer model and examined the effects of THPTS-PDT on tumor growth in vivo, and analyzed molecular mechanisms in vitro We examined pharmacokinetics and subcellular localization, and evoked cell death mode in cultured rat urothelial carcinoma cells (AY-27). We used female F344 Fischer rats for in vivo studies. Ten rats each were used for THPTS-PDT and light-only control. Bladders were evaluated by macroscopy and histology. Temperature-dependent THPTS uptake resulted in endosomal/lysosomal localization. PDT (0-50 µmol/L THPTS; 10 J/cm2) induced early onset of apoptosis leading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 µmol/L THPTS; 10 J/cm2) in F344 rats led to significant reduction of muscle-invasive tumor number (2/10 vs. 7/10 in controls) and total tumor volume (60% reduction) 2 weeks after PDT, while sparing healthy tissue. Here, we report for the first time effective tumor growth control by PDT in vivo THPTS is a promising new photosensitizer with the advantage of higher therapeutic depth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allows minimal-invasive bladder preserving treatment of bladder cancer without systemic side effects.

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

PURPOSE: Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. MATERIAL AND METHODS: Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (Gαq/11, Gαs, Gαi) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor®488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. RESULTS: Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. CONCLUSION: PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.

Tetrahydroporphyrin-tetratosylat (THPTS): A near-infrared photosensitizer for targeted and efficient photodynamic therapy (PDT) of human bladder carcinoma. An in vitro study.

BACKGROUND: Efficacy of PDT in muscle-invasive bladder cancer is hampered by low tissue penetration of most photosensitizers by short excitation wavelength. THPTS is excitable at near-infrared (760nm) allowing tissue penetration up to 15mm. We examined the cellular effects of THPTS-PDT in human bladder cancer cells. MATERIAL AND METHODS: We used four human transitional carcinoma cell lines, epithelial bladder progenitors (HBLAK) and bladder smooth muscle cells (HBSMC). We used flow cytometry to examine pharmacokinetics of THPTS, confocal laser scanning microscopy to analyze subcellular localization and production of reactive oxidative species (ROS), examined cytotoxicity and cell death pathways (qRT-PCR). RESULTS: Total uptake varied between cell lines and was significantly high in HBLAK and HBSMC. Lysosomal localization was mainly seen in cancer cells and HBLAK, while THPTS was distributed throughout the cytoplasm in HBSMC. Significant ROS production was detected 30min after THPTS-PDT. Growth arrest occurred within 4h and resulted in apoptotic and necrotic cytotoxicity after 24h. Cytotoxicity was dose-dependent and specifically high in cancer cells and HBLAK and significantly low in HBSMC. CONCLUSION: THPTS-PDT induces cellular mechanisms leading to cellular growth arrest, apoptosis and necrosis in human bladder cancer cells. These effects are only partly dependent on the total amount of THPTS uptake and rather dependent on its subcellular compartmentalization. HBSMC are hardly affected by THPTS-PDT confirming tumor specificity and safety. THPTS is a promising new photosensitizer with the unique advantage of deep tissue penetration allowing the treatment of solid tumors and warranting further animal studies.

Histamine receptors in human detrusor smooth muscle cells: physiological properties and immunohistochemical representation of subtypes.

The potent inflammatory mediator histamine is released from activated mast cells in interstitial cystitis (IC). Here, we report on the histamine receptor subtypes involved in the intracellular calcium response of cultured smooth muscle cells (cSMC). Fura-2 was used to monitor the calcium response in cSMC, cultured from human detrusor biopsies. The distribution of histamine receptor subtypes was addressed by immunocytochemistry in situ and in vitro. Histamine stimulated a maximum of 92% of the cells (n=335), being more effective than carbachol (70%, n=920). HTMT (H1R-agonist), dimaprit (H2R) and MTH (H3R) lead to significant lower numbers of reacting cells (60, 48 and 54%). Histamine receptor immunoreactivity (H1R, H2R, H3R, H4R) was found in situ and in vitro. Histamine-induced calcium increase is mediated by distinct histamine receptors. Thus, pre-therapeutic evaluation of histamine receptor expression in IC patients may help to optimize therapy by using a patient-specific cocktail of subtype-specific histamine receptor antagonists.

Smooth muscle cells from human urinary bladder express connexin 43 in vivo and in vitro.

Smooth muscle cells (SMC) of the human urinary bladder are believed to be electrically coupled. However, it has not been successfully demonstrated the nature of the underlying cell-cell communication. Here, we used Western blot technique to demonstrate the gap junction protein connexin 43 (Cx 43) in human bladder musculature and in smooth muscle cell cultures. We found expression of Cx 43 in all samples of the detrusor (Mdv, n=6) and in five of six samples of the internal sphincter (Mv). Cx 43 expression was less in cell cultures, 60% showing Cx 43 expression. Iontophoretic application of the gap junction permeable fluorescent dye lucifer yellow revealed a mean of 2.6+/-2.2 (mean+/-SD, n=8) coupled cells in cultured smooth muscle cells. Our findings support the notion that Cx 43 is constitutively expressed in human bladder SMC in vivo. Thus, the function of the bladder is likely to be influenced by the organisation of smooth muscle cells into functional syncytia.