The urothelium is a crucial stratified epithelial barrier that protects the urinary tract. It consists of basal cells in the lower layers and intermediate and superficial cells in the luminal layer. These urothelial cells can be identified by their distinct gene expression patterns. Superficial cells are terminally differentiated, binucleated, post-mitotic cells that are responsible for the barrier function of the urothelium via the production of uroplakin proteins. Intermediate cells act as the progenitor cells for superficial cells during development, homeostasis, and after acute injury. Basal cells consist of two populations, K14-basal cells and K5-basal cells. K14- basal cells have been shown to be progenitors that can repopulate the urothelium after chronic injury and are the cells of origin that produce bladder cancer. Bladder cancer can be classified as basal subtype or luminal subtype. The basal subtype is generally immune infiltrated, aggressive, and invasive with a poor prognosis. The luminal subtype is generally immune poor, less aggressive, and non-invasive with a better prognosis compared to basal tumors. Pparg is a nuclear hormone receptor that has been described as a master regulator of adipogenesis and cellular differentiation that also carries out important anti-inflammatory functions (in part by antagonizing the NFKB pathway). Pparg is downregulated in basal subtype muscle invasive bladder cancer and amplified in luminal subtype bladder cancer. In vivo we find that Pparg is a master regulator of cell specification during urothelial development, homeostasis, regeneration, and cancer. When Pparg is ablated in the entire urothelium, Pparg KO mutants lack mature superficial cells and undergo squamous differentiation, with an expansion of the K14-basal cell population. These Pparg KO mutants also display persistent inflammation and squamous metaplasia after injury by urinary tract infection (UTI), due to unregulated NFKB signaling. However, the squamous differentiation in the Pparg KO mutants did not progress to bladder cancer. Constitutive activation of Pparg in basal cells using a novel VP16;Pparg transgenic mouse line crossed to an Krt5CreERT2 driver induces basal cells to undergo a luminal differentiation program towards post-mitotic S-cells during homeostasis. Not surprisingly, these cells did not progress to form bladder cancer on their own. Interestingly, expression of VP16;Pparg in basal cells only drives tumor formation when the basal cells are in an “activated state,” induced by 1 month of BBN treatment. In a BBN mouse model which produces basal subtype bladder cancer in wild type animals, expression of the VP16;Pparg transgene in activated basal cells drives the formation of luminal tumors with papillary morphology, suggesting that this transcription factor is a master regulator of urothelial luminal differentiation, as has been suggested from previous in vitro studies. Like their human counterparts, these VP16;Pparg luminal tumors are immune cold. Additionally, these VP16;Pparg luminal tumors have different domains; a top domain that is “luminal,” and a bottom domain that is “basal”, suggesting the luminal tumors produced by activation of Pparg are not homogenous and undergo a phenotypic shift that mimics what has previously been reported in patient-derived organoids. Understanding the molecular mechanism that drives luminal bladder cancer provides critical information in bettering our approach in diagnosing and treating MIBCs.

The two studies follow free nucleic acids in urine in search for biomarkers to distinguish urinary bladder cancer patients from controls. Bladder cancer forms 4 % of newly diagnosed oncological diseases in the Czech Republic. Nowadays, there is no accredited non-invasive method for its diagnosis, which is sufficiently accurate. Urine supernatant, which is washing the bladder mucosa and which does not contain cell debris, seems to be an appropriate source of biomarkers for non-invasive diagnosis. miRNAs, as a non-invasive biomarker of urinary bladder cancer, were studied in one of the studies. miRNAs are short noncoding RNA, which block the process of translation. miRNAs occur in all body fluids and are relatively stable. A study with three phases was assessed to find a suitable miRNA marker. 109 individuals were examined in total (36 controls and 73 bladder cancer patients). The analysis of miRNAs was based on RT-PCR (Reverse Transcription Polymerase Chain Reaction). In the first phase, the urine of 59 individuals was analyzed on TaqMan array card with 381 miRNAs. In the second phase, the results of the first phase were confirmed on the same cohort by a single miRNA assay. In the third phase, a new cohort was used (23 controls and 27 bladder cancer patients), analyzed by a single miRNA assay again....

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Bladder cancer, as one of the most common cancer types and with high recurrence risk, is considered a candidate for novel immunotherapy strategies. An important aspect of the research for immunotherapy drug development for bladder cancer is to study the tumor microenvironment (TME) and it’s immune contexture. Besides tumor-infiltrating lymphocytes (TILs) as the main drivers of anti-tumor response, recent studies revealed the importance of tumor-associated tertiary lymphoid structures (TLSs) and high endothelial venules (HEVs) in the TME. Structures similar to these were found to spontaneously form in the orthotopic MB49 model used for bladder cancer research in our group. The aim of this study was to perform a deeper characterization of the TME in this model, by using immunofluorescent staining and microscopy. Specifically, the co-localization of tumor infiltrating lymphocytes (CD8+ and CD4+ T cells, CD19+ B cells), CD11c+ dendritic cells and HEVs along with CCL21 signaling were analyzed within orthotopic MB49 tumors, with and without immune stimulation. The quantification of cells expressing CD8, CD19 and CD11c immune markers, CCL21 levels, vascular density and numbers of HEVs, showed higher densities within the immune-stimulated tumors, indicating a rapid effect of immune stimulation on increasing immune cell infiltration and vascular density after only 24 hours post CpG therapy. Also, the highest frequency of TILs, CCL21 chemokine and vascular density was located in regions of the tumor border indicating that these regions should be studied further in depth as a potential target for entry of cells to the tumor with immunotherapy or as a model of the tumor microenvironment since tumor cell density is maintained high in these locations.

Real-time multi-circle detection has been a challenging problem in the field of biomedical image processing, due to the variable sizes and non-ideal shapes of cells in microscopic images. In this study, two new multi-circle detection algorithms are developed to facilitate an automatic bladder cancer diagnosis system: one is a modified circular Hough Transform algorithm integrated with edge gradient information; and the other one is a stochastic search approach based on real valued artificial immune systems. Computer simulation results show both algorithms outperform traditional methods such as the Hough Transform and the geometric feature based method, in terms of both precision and speed.

Background: Bladder cancer (BCa) is the most frequent malignancy in the urinary tract. Despite great progress in our understanding of BCa in the past decades, we still lack significant improvement in the development of new BCa chemotherapeutics, which is largely attributed to the fact that 2D tumor models are used as the predominant platform for cell-based assays. The conventional 2D tumor models, although simple and convenient, fail to recreate an in vivo-like tumor microenvironment (TME). 3D tumor models more faithfully recapitulate the complexity of TME than 2D-based models. 3D-organoid models reproduce many biological characteristics of the real solid tumor, including biochemical gradients, different proliferating states, complex cell-cell interaction, ECM deposition, and chemo-resistance, thus providing a promising tool in tumor biology research. There have been many studies about the patient-derived BCa organoids. However, we still lack a research about the development and characterization of multiple bladder organoid models that mimicked both normal bladder tissue and bladder tumors representing the entire range of malignant grades. Aims: The study aimed to investigate the formation and characteristics of hetero-typed bladder organoids derived from the three major cell types in the human bladder, including either non-cancerous urothelial cells or different bladder cancer cell lines. Hypotheses: 1. Bladder-like organoids can formed by self-organization from mixed bladder cell suspension 2. The degree of histological organization depends on the urothelial cells used 3. The different cell types can be identified in the bladder organoids by immunohistochemistry Materials and Methods: We used RT4, RT112, T24, and CAL29 cells (transitional cancer cell lines, histological grade: G1-G4), non-malignant HBLAK (bladder epithelium progenitors), primary human bladder fibroblasts (hBF), and human bladder smooth muscle cells (hBSMC) in this study. The following figure displays the construction process of hetero-typed bladder organoids by ultra-low attachment (ULA) 96-well microplate method. At day 4 post seeding, bladder organoids were harvested, fixed, processed, and sectioned. Then, we characterized the bladder organoids by histology and immunohistochemistry (IHC) staining. Bladder organoids were stained for panCK, Vimentin, α-SMCA, CK7, CK13, CK20, Ki67, Claudin4, ZO-1, and fibronectin and the immunoreactivity (IR) of specific antigen was analyzed using ImageJ plus IHC profiler plugin. We also did H&E and Crossmon staining to investigate the histology and ECM deposition of the bladder organoids. Results: Mixed cell suspensions self-organized into compact organoids in the ULA plate within 24 hours after seeding. At day 4 post seeding, the organoids were grown to 650-1,000 μm in diameter, with BCa organoids significantly bigger than HBLAK organoids. The morphology of bladder organoids greatly varied depending on the urothelial cells used. Besides, urothelial cells were mainly located in the periphery and supportive cells (hBF and hBSMC) were in the core of the organoids. High-grade RT112, T24 and CAL29 organoids showed significantly thicker urothelial cell layers and more urothelial cells in the periphery than low-grade RT4 organoids and non-malignant HBLAK organoids. The CK7, CK13 and CK20-IR greatly varied between the organoid-cultured urothelial cells. HBLAK cells showed significant lower panCK, CK7, CK13 and CK20-IR than BCa cells. CK-IR was higher in RT4 and RT112 cells than in T24 cells. CAL29 organoids showed a stratified layering: CK7-IR in superficial cells, CK13-IR in intermediate cells, and CK20 in all cells. The urothelial cells in organoid culture consisted of not only proliferating cells but also a large portion of quiescent cells. High-grade RT112, T24 and CAL29 cells (30-50%) showed a significantly higher proliferation index than the low-grade RT4 (2%) and non-malignant HBLAK (6.6%) cells. All urothelial cells in organoid culture expressed Claudin4(CLDN4) positively. In addition, high-grade T24 and CAL29 cells showed higher Claudin4-IR than low-grade RT4 and RT112 cells. In contrast, all bladder organoids showed very low ZO-1-IR. Fibronectin and Crossmon staining indicated fibronectin, collagen, and reticular fibers deposition in the bladder organoids. Besides, high-grade T24 and CAL29 cells showed remarkably higher fibronectin-IR than low-grade RT4 and RT112 cells. Discussion: All urothelial cells were able to form compact and reproducibly sized organoids with hBSMC and hBF by spontaneous cell aggregation in ULA 96-well plate. The compactness of bladder organoids might reflect the adhesion between the cells and the expression of underlying adhesion molecules. The size of organoids and number of urothelial cells in the periphery could be influenced by multiply factors. The organoids formed a bladder-like structure with outside urothelial cells and a core of supportive cells, indicating they could be useful tools in the testing of anti-cancer drugs. The cytokeratin expression profiles reflect the differentiation status of tumors. All urothelial cells retained the CK7/CK13/CK20 expression patterns of their original tissues in organoid culture, supporting that the bladder organoids mimicked both the normal bladder urothelium and bladder tumors of different grades. In addition, RT112 and CAL29 organoids formed a stratified urothelium. Urothelial cells in organoid culture were at different proliferation stages, better mimicking the in vivo bladder tumors in terms of proliferation and cell heterogeneity than 2D-based BCa models. Besides, the organoid-cultured urothelial cells retained the proliferation characteristics of their original tissues. Bladder organoids showed abundant fibronectin deposition, which could affect their response to drug treatments. Besides, the fibronectin expression level in BCa cells was correlated with their primary origins, supporting the view that the BCa cells in organoid culture retained the invasive and metastatic feature of their parental bladder tumors. The fibronectin, collagen, and reticular fibers deposition indicated bladder organoids mimicked the in-situ situation of bladder tumors in respect to ECM deposition. The expression of CLDN4 in urothelial cells indicated the formation of para-cellular barriers in the bladder organoids, which could limit the penetration and diffusion of anticancer drugs into bladder organoids. Especially, the RT112 and CAL29 organoids showed high expression of CLDN4 on the apical membrane, indicating that they could be helpful in the investigation of drug penetration into tumor tissues. The ULA microplate method is easy, fast, and suitable for massive production of reproducibly sized organoids. The imageJ plus IHC profiler plugin method was able to perform fast and automatic quantitative analysis for DAB-stained IHC images and comparisons of the expression of specific antigens in formalin-fixed tissues Conclusion: The bladder organoids represented a bladder-like architecture by self-organization, with a peripheral urothelium surrounding a supportive core of hBF and SMCs. These organoids exhibit characteristics of the in situ normal urothelium and bladder tumors of different grades in cell composition, proliferation, stratified urothelium, epithelial diferentiation, and ECM deposition. Thus, they can be useful tools in cancer biology research and anti-cancer drug development.:1. Title page 2. Table of contents 3. List of Abbreviations 4. Introduction 4.1 Bladder cancer 4.2 Currently available bladder cancer models 4.3 Advantages of 3D cell culture over 2D cell culture 4.4 3D tumor spheroid/organoid models 4.5 Spheroids/organoids construction methods 4.6 Application of 3D organoid culture in bladder cancer research 4.7 Limitations of previous research of bladder cancer organoids 5. Aims 5.1 Hypothesis 5.2 Tasks 6. Materials and Methods 6.1 Cell lines used in the study 6.2 Monolayer culture of the cell lines 6.3 Bladder organoids construction by ULA microplate method 6.4 Fixation, processing, sectioning, and size measurement 6.5 Histology and immunohistochemistry 6.6 Image acquisition 6.7 Image analysis with ImageJ plus IHC profiler plugin 6.8 Statistical analysis 7. Results 7.1 Spontaneous formation of packed bladder organoids in the ULA plate 7.2 Bladder-like self-organization of the organoids 7.3 Immunohistochemical characterization of the organoids 7.4 Expression of urothelial cell differentiation makers 7.5 Proliferation of urothelial cells in 3D organoid culture 7.6 Tight junction protein expression in the organoids 7.7 ECM deposition in bladder organoids 8. Discussion 8.1 Organoid models in cancer research 8.2 Morphology and Size 8.3 Bladder-like internal structure 8.4 Formation of a stratified urothelium in bladder organoids 8.5 Proliferation characteristics of the urothelial cells in organoid culture 8.6 ECM deposition in bladder organoids 8.7 TJ protein expression in bladder organoids 8.8 Possible application of bladder organoids 8.9 Review of methods used in this project 8.10 Limitations of the research 9. Summary of the work 10. References 11. Appendix 12. Declaration of independence 13. Curriculum vitae 14. Acknowledgment

Our goal was to find new diagnostic and prognostic biomarkers in bladder cancer (BCa), and to predict molecular mechanisms and processes involved in BCa development and progression. Notably, the data collection is an inevitable step and time-consuming work. Furthermore, identification of the complementary results and considerable literature retrieval were requested. Here, we provide detailed information of the used datasets, the study design, and on data mining. We analyzed differentially expressed genes (DEGs) in the different datasets and the most important hub genes were retrieved. We report on the meta-data information of the population, such as gender, race, tumor stage, and the expression levels of the hub genes. We include comprehensive information about the gene ontology (GO) enrichment analyses and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. We also retrieved information about the up- and down-regulation of genes. All in all, the presented datasets can be used to evaluate potential biomarkers and to predict the performance of different preclinical biomarkers in BCa.

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