Bladder Cancer is the tenth most common malignancy globally, and is the thirteenth most common cause of tumor associate morbidity. Bladder cancer is largely stratified into two categories: Non-Muscle Invasive Bladder Cancer (NMIBC) and Muscle Invasive Bladder Cancer (MIBC). NMIBC represents disease localized to the urinary bladder, and can be stratified into low and high-grade disease. MIBC represents an aggressive class of bladder cancer, with invasion into the underlying muscle layers of the bladder. MIBC can be classified as either non-metastatic MIBC, with disease localized to the bladder corpus, or metastatic MIBC, with disease spreading to sites beyond the bladder corpus. High grade NMIBC presents significant risk for progression to MIBC, and collectively both high grade NMIBC and MIBC bladder cancers demonstrate poor prognostic outcomes in clinical settings in terms of responses to therapy, recurrence risks, and overall survival. Hexaminolevulinate is a precursor of Protoporphyrin IX (PpIX) in the heme biosynthetic pathway. Hexaminolevulinate has been FDA approved under the trade name Cysview for diagnostic usage in blue light cystoscopies for fluorescence mediated visualization of disease along the bladder wall. I demonstrate that in addition to its diagnostic utility, Cysview and blue light irradiation can be utilized clinical as a potential therapeutic modality. I demonstrate the significant selective cytotoxicity of Cysview in combination with blue light against patient derived organoids (PDOs) from primary bladder cancers. My results determine that Cysview and blue light induce a rapid cell death program mediated by an influx in Reactive Oxygen Species (ROS) production, resulting in less than 5% viability within 24 hrs of treatment. This massive loss in viability is observed in low and high grade NMIBC, as well as MIBC derived PDOs with diverse mutational profiles. The results of this work demonstrate that PDOs are a significant platform for assessing therapy responses for correlation with the large patient population. Furthermore, the work identifies photodynamic therapy with Cysview and blue light irradiation as a putative therapeutic modality for localized bladder cancers, with the potential for significant improvement in patient outcomes. The identification and characterization of the therapeutic effects of Cysview come at a critical time during a global shortage of conventional therapeutics for localized bladder cancer, and presents a pathway for patients affected by these shortages. Progression in bladder cancer has been understood to be driven by processes governing subpopulation and cell state and lineage transformation. Previous studies identify phenotypic plasticity within a subset of bladder cancers and have correlated this phenomenon with an increased risk for disease progression from NMIBC to MIBC. In previous work, a subset of PDOs derived from luminal primary tumors demonstrated significant degrees of luminal to basal plasticity in vitro. In my analysis of these PDOs using transcriptomic and chromatin accessibility data, I identified a transcriptomic and epigenetic signature unique to plastic PDOs. Furthermore, I identified HNF1B, GRHL2, GATA6, and SNAI2 as putative regulators of luminal to basal plasticity in bladder cancer. Using these molecular profiles, I correlated the plasticity phenotype with reduction in overall survival using data from published bladder cancer patient cohorts. Finally, I developed a novel transcriptomic subtypes classification scheme and an accompanying R package to classify epithelial heterogeneity in bladder cancer, based on the transcriptomic subtypes I identified in bladder cancer PDOs.

As Photodynamic Therapy (PDT) becomes increasingly popular as a treatment modality for some solid tumours, the need for a better understanding of the mechanism(s) of action and resistance are paramount. To this end we have generated Photofrin® PDT-induced resistant variants to numerous cell lines including the colon cancer cell line HT29. There is significant evidence indicating that stress proteins play an important role in determining the outcome of PDT on a cell. In this thesis the roles of the mitochondrial Heat Shock Protein 60 (Hsp60) as well as the endoplasmic Glucose Related Protein 78 (GRP78) were examined in the HT29 cells and their Photofrin induced resistant variant HT29-P14. The expression and role of these two stress proteins were also examined in T24 Bladder carcinoma cells and their GRP 78 stable-overexpressing clones Hsp60 protein was expressed at slightly higher basal levels in the resistant HT29-P14 cells relative to the parental HT29 cells. After incubation alone or PDT action, a temporal and dose dependent induction of Hsp60 was observed and this too was found to be significantly greater in the resistant cells. In the T24 model, no Hsp60 induction was observed following drug incubation or PDT. GRP78 protein levels were increased by PDT action but not by Photofrin® incubation alone in all cell lines tested. In the T24 model, GRP78 transfection resulted in a stable 2-fold increase in protein levels and a 10-20-fold increase in cell survival after PDT at the highest dose tested. A temporal and dose dependent response was noted in all cells and induction of GRP78 protein was lower in the stable overexpresser such that all cell lines had similar post induction levels. In the HT29 and HT29-P14 resistant cells, GRP78 protein levels were similar at basal level, and, both cell lines exhibited the same temporal and dose dependent increases in expression post PDT. Finally, broad scale expression profiling using a "stress" microarray in the HT29 and HT29-P14 resistant variants revealed a very similar expression profile for the 168 of the 169 stress proteins tested with the exception of the small Heat Shock Protein 27 (Hsp27). As confirmed by northern and western blot analysis, Hsp27 is over 20 fold greater at the transcriptional level and 10-15 fold greater at the translational level in the HT29-P14 resistant variant. These findings implicate Hsp27, Hsp60 and GRP78 as possible mediators of cellular sensitivity to Photofrin-mediated PDT. Specifically, Hsp27 appears to play a role in the increased resistance of our induced resistant HT29-P14 cells. / Thesis / Master of Science (MS)

Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer.  Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up.  Biomarkers will become more important for treatment choices in bladder cancer management.

Sources of arsenic exposure include air, water, and food from both natural and anthropogenic sources. Arsenic is categorized as a human carcinogen, and is associated with pleiotropic toxicities including cancers of the skin, lung, and bladder. Despite arsenic's long recognition as a human carcinogen, the exact mechanisms of arsenical-induced carcinogenesis are unknown. Arsenic exposure has been shown to cause DNA damage. However, because arsenic does not directly react with DNA, genotoxicity is generally considered to result from indirect mechanisms. The generation of arsenical-induced reactive oxygen species and the inhibition of critical DNA repair systems are believed to contribute to arsenical-induced carcinogenicity. The DNA damaging effects of arsenical exposure and alterations in DNA repair processes were examined within the human bladder urothelial cell line, UROtsa, following continuous exposure to the arsenic metabolite, monomethylarsonous acid [MMA(III)]. Chronic, low-level MMA(III) exposure results in the induction of DNA damage that remains elevated following the removal of MMA(III). Furthermore, data presented herein, defines the critical period in which continuous low-level MMA(III) exposure causes the malignant transformation of the UROtsa cell line. Results indicate that malignant transformation of UROtsa cells is irreversible following 12 wk of low-level MMA(III) exposure. Assessment of the MMA(III)-induced biological alterations leading to the malignant transformation of UROtsa cells following 12 wk of exposure suggest two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation. These mechanisms include MMA(III)-induced DNA damage via the production of reactive oxygen species and the MMA(III)-induced inhibition of poly(ADP-ribose) polymerase-1 as a result of the direct MMA(III)-mediated displacement of zinc.

O carcinoma da bexiga urinária (CB) é a segunda doença maligna mais frequente do trato urinário. Devido a baixa eficácia dos tratamentos intravesicais atuais (imunoterapia com BCG e quimioterapia), seja pelo baixo tempo de residência do fármaco na bexiga ou pela baixa permeabilidade no urotélio, novas estratégias que aumentem esse tempo de residência do fármaco e sua penetração na bexiga têm sido investigadas. Dentre estas estratégias pode-se citar os sistemas de liberação sustentada nanoestruturados, que liberam o fármaco gradativamente, protegem o fármaco encapsulado, aumenta a biodisponibilidade, aumentando a eficácia da terapia e diminuindo os efeitos adversos. Nesta linha, o objetivo desse trabalho foi produzir e utilizar carreadores lipídicos nanoestruturados (CLN) como sistema de carreamento de doxorrubicina (DOXO) e a sua avaliação biológica em células de câncer de bexiga. Os CLN, compostos por manteiga de Illipê (lipídeo sólido), ácido oleico (óleo) e o estabilizante Pluronic F68, foram preparados pelo método de emulsão a quente e sonicação. A caracterização físico-química do CLN foi realizada determinando o diâmetro hidrodinâmico médio e potencial zeta (carga superficial) por espalhamento de luz dinâmico (DLS), cristalinidade por calorimetria exploratória diferencial (DSC), eficiência de encapsulamento por espectrofotometria UV-vis, ensaio de citotoxicidade em células RT4 e análise de permeação ex vivo e in vivo das formulações aplicadas em bexiga de porco por microscopia confocal. O diâmetro hidrodinâmico médio dos CLN sem o fármaco foi de 103 nm e seu PdI (índice de polidispersão) igual a 0,2. O encapsulamento da DOXO aumentou o diâmetro dos CLN para 112 nm e o valor de PdI foi de 0,2. O baixo valor de PdI indica formulações com baixa polidispersão. Os valores de potencial zeta dos CLN sem e com DOXO foram ambos negativos, variando de -5 mV a -25 mV. Nos termogramas das amostras de CLN-DOXO não foi observado o pico referente a fusão da DOXO em 197,93ºC, indicando que o fármaco provavelmente está molecularmente disperso na matriz lipídica. Nos ensaios de citotoxicidade, a formulação CLN-DOXO mostrou-se mais citotóxica do que a DOXO livre em baixas concentrações (31-250 ng/mL). O valor de IC50 reduziu 2,1 vezes quando a DOXO foi encapsulada. Essa maior atividade antitumoral in vitro pode estar relacionada ao aumento do uptake celular como confirmado pelos ensaios de citometria de fluxo. No estudo de permeação ex vivo em bexiga de porco foram observadas permeações muito semelhantes das formulações de DOXO livre e encapsulada em CLN. Porém no ensaio in vivo a DOXO encapsulada permeou mais que a DOXO livre. Os resultados obtidos apontam para um promissor sistema de liberação de doxorrubicina para a terapia do câncer de bexiga. / Urinary bladder carcinoma (BC) is the second most common malignant disease of the urinary tract. Due to the low efficacy of current intravesical treatments (BCG immunotherapy and chemotherapy), either because of the low residence time of the drug in the bladder or due to low permeability in the urothelium, new strategies that increase the residence time of the drug and its penetration into the bladder have been investigated. These strategies include nanostructured sustained release systems, which release the drug gradually, protect the encapsulated drug, increase the bioavailability; increasing the effectiveness of the therapy and reducing side effects. In this line, the aim of this work was to produce and apply nanostructured lipid carriers (NLC) as a doxorubicin delivery system (DOXO) and its biological evaluation in bladder cancer cells. The NLC, composed by Illipe butter (solid lipid), oleic acid (oil) and Pluronic F68 stabilizer, were prepared by the hot emulsion and sonication method. The physical-chemical characterization of these NLC was performed by measured the mean hydrodynamic diameter and zeta potential (surface charge) by dynamic light scattering (DLS), crystallinity by differential scanning calorimetry (DSC), encapsulation efficiency by UV-vis spectrophotometry, cytotoxicity assay in RT4 cells and ex vivo and in vivo permeation assay of formulations applied to pig bladder by confocal microscopy. The mean hydrodynamic diameter of NLC without the drug was 103 nm and its PdI (polydispersity index) was 0.2. The encapsulation of DOXO increased the NLC diameter to 112 nm and the PdI value was 0.2. The low value of PdI indicates formulations with low polydispersion. The zeta potential values of the NLC without and with DOXO were both negative (-5 mV to -25 mV). In the thermograms of the NLC-DOXO sample the fusion peak of DOXO at 197.93 was not observed, indicating that the drug is molecularly dispersed in the lipid matrix. In the cytotoxicity assays, the NLC-DOXO formulation was more cytotoxic than free DOXO at low concentrations (31-250 ng/mL). The IC50 value was reduced 2.1 fold when DOXO was encapsulated. This increased in vitro antitumor activity may be related to increased cell uptake as confirmed by flow cytometry analysis. In the ex vivo permeation study in the pig bladder, a very similar permeation was observed between free and encapsulated DOXO. However, in the in vivo assay the encapsulated DOXO permeated more than free DOXO. The results indicates a promising release system of doxorubicin to bladder cancer therapy.

O carcinoma da bexiga urinária (CB) é a segunda doença maligna mais frequente do trato urinário. Devido a baixa eficácia dos tratamentos intravesicais atuais (imunoterapia com BCG e quimioterapia), seja pelo baixo tempo de residência do fármaco na bexiga ou pela baixa permeabilidade no urotélio, novas estratégias que aumentem esse tempo de residência do fármaco e sua penetração na bexiga têm sido investigadas. Dentre estas estratégias pode-se citar os sistemas de liberação sustentada nanoestruturados, que liberam o fármaco gradativamente, protegem o fármaco encapsulado, aumenta a biodisponibilidade, aumentando a eficácia da terapia e diminuindo os efeitos adversos. Nesta linha, o objetivo desse trabalho foi produzir e utilizar carreadores lipídicos nanoestruturados (CLN) como sistema de carreamento de doxorrubicina (DOXO) e a sua avaliação biológica em células de câncer de bexiga. Os CLN, compostos por manteiga de Illipê (lipídeo sólido), ácido oleico (óleo) e o estabilizante Pluronic F68, foram preparados pelo método de emulsão a quente e sonicação. A caracterização físico-química do CLN foi realizada determinando o diâmetro hidrodinâmico médio e potencial zeta (carga superficial) por espalhamento de luz dinâmico (DLS), cristalinidade por calorimetria exploratória diferencial (DSC), eficiência de encapsulamento por espectrofotometria UV-vis, ensaio de citotoxicidade em células RT4 e análise de permeação ex vivo e in vivo das formulações aplicadas em bexiga de porco por microscopia confocal. O diâmetro hidrodinâmico médio dos CLN sem o fármaco foi de 103 nm e seu PdI (índice de polidispersão) igual a 0,2. O encapsulamento da DOXO aumentou o diâmetro dos CLN para 112 nm e o valor de PdI foi de 0,2. O baixo valor de PdI indica formulações com baixa polidispersão. Os valores de potencial zeta dos CLN sem e com DOXO foram ambos negativos, variando de -5 mV a -25 mV. Nos termogramas das amostras de CLN-DOXO não foi observado o pico referente a fusão da DOXO em 197,93ºC, indicando que o fármaco provavelmente está molecularmente disperso na matriz lipídica. Nos ensaios de citotoxicidade, a formulação CLN-DOXO mostrou-se mais citotóxica do que a DOXO livre em baixas concentrações (31-250 ng/mL). O valor de IC50 reduziu 2,1 vezes quando a DOXO foi encapsulada. Essa maior atividade antitumoral in vitro pode estar relacionada ao aumento do uptake celular como confirmado pelos ensaios de citometria de fluxo. No estudo de permeação ex vivo em bexiga de porco foram observadas permeações muito semelhantes das formulações de DOXO livre e encapsulada em CLN. Porém no ensaio in vivo a DOXO encapsulada permeou mais que a DOXO livre. Os resultados obtidos apontam para um promissor sistema de liberação de doxorrubicina para a terapia do câncer de bexiga. / Urinary bladder carcinoma (BC) is the second most common malignant disease of the urinary tract. Due to the low efficacy of current intravesical treatments (BCG immunotherapy and chemotherapy), either because of the low residence time of the drug in the bladder or due to low permeability in the urothelium, new strategies that increase the residence time of the drug and its penetration into the bladder have been investigated. These strategies include nanostructured sustained release systems, which release the drug gradually, protect the encapsulated drug, increase the bioavailability; increasing the effectiveness of the therapy and reducing side effects. In this line, the aim of this work was to produce and apply nanostructured lipid carriers (NLC) as a doxorubicin delivery system (DOXO) and its biological evaluation in bladder cancer cells. The NLC, composed by Illipe butter (solid lipid), oleic acid (oil) and Pluronic F68 stabilizer, were prepared by the hot emulsion and sonication method. The physical-chemical characterization of these NLC was performed by measured the mean hydrodynamic diameter and zeta potential (surface charge) by dynamic light scattering (DLS), crystallinity by differential scanning calorimetry (DSC), encapsulation efficiency by UV-vis spectrophotometry, cytotoxicity assay in RT4 cells and ex vivo and in vivo permeation assay of formulations applied to pig bladder by confocal microscopy. The mean hydrodynamic diameter of NLC without the drug was 103 nm and its PdI (polydispersity index) was 0.2. The encapsulation of DOXO increased the NLC diameter to 112 nm and the PdI value was 0.2. The low value of PdI indicates formulations with low polydispersion. The zeta potential values of the NLC without and with DOXO were both negative (-5 mV to -25 mV). In the thermograms of the NLC-DOXO sample the fusion peak of DOXO at 197.93 was not observed, indicating that the drug is molecularly dispersed in the lipid matrix. In the cytotoxicity assays, the NLC-DOXO formulation was more cytotoxic than free DOXO at low concentrations (31-250 ng/mL). The IC50 value was reduced 2.1 fold when DOXO was encapsulated. This increased in vitro antitumor activity may be related to increased cell uptake as confirmed by flow cytometry analysis. In the ex vivo permeation study in the pig bladder, a very similar permeation was observed between free and encapsulated DOXO. However, in the in vivo assay the encapsulated DOXO permeated more than free DOXO. The results indicates a promising release system of doxorubicin to bladder cancer therapy.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

膀胱尿路上皮腫瘤發病率在泌尿道腫瘤中排第二位，它具有高複發性的特點。目前，有創性尿道膀胱鏡檢查是診斷的金標準。儘管先後有很多血液或尿液中的分子被先後用於診斷膀胱癌的診斷研究，但到目前為止尚未有任何一種方法可以取代膀胱鏡檢查。有證據表明在膀胱上皮腫瘤組織中有很多異常表達的microRNA，但是內在機制的有關研究相對缺乏。在本研究中，我們利用在尿液上清中異常表達的microRNA來評估它們在膀胱癌診斷中的價值。而且，我們揭示了其潛在的調控機理。通過microRNA基因芯片，我們結合并對比來自膀胱腫瘤病人和正常對照患者的9個尿液上清樣本，以及4對腫瘤組織及臨近正常黏膜上皮中microRNA的表達，初步篩選出10個異常的microRNA。然後我們使用定量RT-PCR的方法在另外獨立的18對腫瘤組織和正常黏膜中進一步驗證芯片結果。最後我們就6個被帥選出來的microRNA在71例的膀胱癌患者和正常對照組的尿液上清中進行檢測並評估其診斷效能。我們發現，miR-125b和miR-99a的表達在膀胱癌患者的尿液上清中明顯下調。另外，它們下調程度與腫瘤的病理分級相關。結合miR-125b和miR-99b兩者作為診斷膀胱癌的指標，靈敏度達86.7%，特異度達81.1%，同時有陽性預測值達91.8%。當作為腫瘤分級指標，miR-125b具有81.4%的敏感度，87.0%的特異度，陽性預測值達93.4%。膀胱腫瘤切除之後，和術前比較，兩個microRNA的表達水平再度上升。我們將miR-99轉染到三個膀胱腫瘤細胞株中（T24，UMUC3和J82）。我們發現miR-99a對UMUC3細胞具有輕微的抗增殖功能。同時，miR-99a在3個細胞株中顯示均顯示具有抗遷移和抗侵襲能力。為尋找miR-99a的目標mRNA，我們結合數據庫算法預測，在Western blot中驗證到miR-99a能顯著下調VLDLR蛋白。隨後我們將帶有VLDLR的3'UTR質粒轉染進入細胞中并證實VLDLR mRNA是miR-99a直接作用的目標。另外，當VLDLR siRNA被轉入3個細胞株之後，我們觀察到相似的抗遷移和抗侵襲的現象。最後我們發現N-cadherin是該通路中的下游抑制遷移和侵襲的分子。本項研究證實研究尿液上清中的microRNA是可行的。MiR-125b和miR-99a是膀胱腫瘤的診斷和分級的有效指標。此外，miR-99a能夠通過和VLDLR mRNA直接結合從而抑制膀胱腫瘤遷移和侵襲功能。 / Urothelial carcinoma of the bladder (UCB) is the second most common malignancy in the urological system with high recurrence rate. Current gold standard examination for diagnosis is urethrocystoscopy, which is an invasive procedure. Although numerous molecular markers in blood or urine have been proposed as diagnostic biomarkers for bladder cancer, none of them could replace urethrocystoscopy in clinical practice. There are accumulating evidences suggesting microRNA dysregulation might be related to the pathogenesis of UCB. However, the exact functions of these microRNAs in UCB remain unknown. In this thesis, the role of selected microRNAs in urine supernatant was investigated in the diagnosis of UCB and also the carcinogenesis of UCB. / In brief, a high-throughput microarray was carried out on nine supernatants of urine from UCB and normal subjects, and also four pairs of tissue from UCB and normal mucosa. Ten microRNA candidates were then identified. Quantitative RT-PCR was used to validate these microRNAs on a set of 18 pairs of tumor tissue and normal mucosa. Eventually, six potential candidate microRNAs were selected and then validated as diagnostic tools on the samples of urine supernatants from 71 patients (50 of known UCB and 21 of normal subjects). The expression levels of these selected microRNAs were further evaluated in the urine supernatants of 20 patients after tumors resections. MiR-125b and miR-99a were the two most significantly down-regulated microRNAs in the urine supernatants of patients with UCB. Moreover, the degree of down-regulation was associated with the pathological grade of the tumor. A combined index of miR-125b and miR-99a in urine supernatant had a sensitivity of 86.7%, specificity of 81.1%, and a positive predicted value of 91.8% for diagnosing UCB. When used to discriminate high-grade from low-grade UCB, miR-125b alone had a sensitivity of 81.4%, specificity of 87.0% and PPV of 93.4%. After transurethral resections, the expression levels of both microRNAs were significantly increased compared to pre-operative levels. / In further studies on the role of microRNAs on the development of UCB, miR-99a was selected for further studies. The precursor of miR-99a was temporally transfected into 3 bladder cancer cell lines: T24, UMUC3 and J82. The proliferation ability was noticed to be suppressed mildly in UMUC3, but not the other. Meanwhile, migration and invasion abilities were inhibited by miR-99a in the all 3 cell lines. Potential targets of miR-99a were predicted from several prediction databases. Subsequently, in Western Blot study, the protein level of very low density lipoprotein receptor (VLDLR) was showed to be down-regulated by miR-99a. Thereafter, a plasmid constructed with 3’UTR of VLDLR was transfected into cytoplasm, which confirmed VLDLR mRNA was a direct target of miR-99a. All 3 cells lines showed the same effect on suppression of migration and invasion after knockdown of VLDLR. N-cadherin was identified as a down-stream molecule responsible for the migration and invasion suppression in this pathway. / This study confirmed microRNA expression in urine supernatants was a feasible approach for the assessment of biomarkers, and miR-125b and miR-99a showed promising results in the diagnosis and grading of UCB. Furthermore, we showed that miR-99a suppressed tumor migration and invasion by directly targeting VLDLR. / Detailed summary in vernacular field only. / Zhang, Dingzuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 107-131). / Abstract and appendix also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgments --- p.V / Abbreviations --- p.VII / List of figures --- p.IX / List of Tables --- p.XI / Content --- p.XII / Chapter Chapter I: --- General Introduction / Chapter 1.1 --- Bladder cancer --- p.1 / Chapter 1.1.1 --- The incidence of bladder cancer / Chapter 1.1.2 --- The burden of bladder cancer to the health care system / Chapter 1.1.3 --- Risk factors for bladder cancer / Chapter 1.1.4 --- Pathology grading system in bladder cancer / Chapter 1.1.5 --- Current diagnostic methods and treatment for bladder cancer / Chapter 1.2 --- Biomarkers for bladder cancer --- p.7 / Chapter 1.2.1 --- The advantages of biomarkers in blood and urine for the diagnosis of bladder cancer / Chapter 1.2.2 --- Biomarkers in blood for bladder cancer / Chapter 1.2.3 --- Biomarkers in the urine for bladder cancer / Chapter 1.2.4 --- Current concerning problems with biomarkers / Chapter 1.3 --- MicroRNAs and bladder cancer --- p.11 / Chapter 1.3.1 --- Post-trancriptional function of microRNAs / Chapter 1.3.2 --- The function of microRNAs in tumor / Chapter 1.3.3 --- Prospects of detecting microRNA in cell-free fluid in tumor / Chapter 1.4 --- MicroRNA target identification --- p.15 / Chapter 1.4.1 --- Prediction of microRNA target / Chapter 1.4.2 --- Validation of microRNA target / Chapter 1.4.3 --- Validation of direct interaction between microRNA and target RNA / Chapter 1.4.4 --- Validation of direct binding of microRNA and mRNA in vivo / Chapter 1.5 --- Migration and invasion of bladder cancer --- p.19 / Chapter 1.5.1 --- The biological process of migration in bladder cancer / Chapter 1.5.2 --- Epithelial to mesenchymal transition in bladder cancer / Chapter 1.6 --- Objectives of this study --- p.21 / Chapter Chapter II --- MicroRNAs in urine supernatant: potential useful markers for bladder cancer screening / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and methods --- p.26 / Chapter 2.2.1 --- Ethics Statement / Chapter 2.2.2 --- Patients and samples / Chapter 2.2.3 --- RNA extraction / Chapter 2.2.4 --- MicroRNA microarray / Chapter 2.2.5 --- Quantitative real-time polymerase chain reaction (RT-PCR) / Chapter 2.2.6 --- Statistical methods / Chapter 2.3 --- Results --- p.31 / Chapter 2.3.1 --- MicroRNA screening by microRNA microarray / Chapter 2.3.2 --- Independent validation of the ten selected microRNAs by qRT-PCR on tissue / Chapter 2.3.3 --- Verification of the six validated microRNAs in urine supernatants as tumor markers / Chapter 2.3.4 --- MiR-125b and miR-99a in urine supernatants were useful for the diagnosis of bladder cancer / Chapter 2. --- 3.5 MiR-125b and miR-99a were two highly correlated microRNAs / Chapter 2.3.6 --- Expression levels of miR-125b and miR-99a increased after tumor resection / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter III: --- MiR-99a suppresses migration and invasion in bladder cancer by targeting VLDLR / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Human tissue samples and bladder cancer cell lines / Chapter 3.2.2 --- RNA extraction and Polymerase Chain Reaction / Chapter 3.2.3 --- MicroRNA and plasmid transfection / Chapter 3.2.4 --- Western Immunoblotting / Chapter 3.2.5 --- Agarose gel electrophoresis / Chapter 3.2.6 --- Luciferase assay / Chapter 3.2.7 --- MTT proliferation assay / Chapter 3.2.8 --- Apoptosis assay / Chapter 3.2.9 --- Cell cycle analysis / Chapter 3.2.10 --- Cell migration Assay / Chapter 3.1.11 --- Cell invasion assay: / Chapter 3.2.12 --- Statistical methods: / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- MiR-99a was significantly down-regulated in bladder cancer / Chapter 3.3.2 --- Precursor microRNA was successfully transfected into bladder cancer cell lines / Chapter 3.3.3 --- MiR-99a had little effect on cell proliferation / Chapter 3.3.4 --- MiR-99a had little effect on cell apoptosis and cell cycle / Chapter 3.3.5 --- Over-expression of miR-99a suppressed cell migration in bladder cancer / Chapter 3.3.6 --- Over-expression of miR-99a also suppressed invasion ability in bladder cancer / Chapter 3.3.7 --- Target prediction for miR-99a using 8 target prediction databases / Chapter 3.3.8 --- Protein level of VLDLR was down-regulated by miR-99a in bladder cancer / Chapter 3.3.9 --- VLDLR was a direct target of miR-99a / Chapter 3.3.10 --- VLDLR mRNA was not down-regulated correspondingly by miR-99a / Chapter 3.3.11 --- MiR-99a suppressed down-stream protein of VLDLR in Reelin pathway / Chapter 3.3.12 --- Knockdown of VLDLR also suppressed cell migration and invasion / Chapter 3.3.13 --- N-cadherin was the down-stream protein responsible for the suppression of migration and invasion in miR-99a/VLDLR pathway / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter IV: --- Conclusion and prospective --- p.101 / Appendix --- p.105 / Reference --- p.107

Objectives: To establish a mouse orthotopic bladder cancer model with consistent tumor-take rate. This orthotopic model was subsequently used to evaluate small animal imaging techniques and investigate new therapeutic agents for bladder cancer treatment. / Materials and Methods: Different orthotopic implantation techniques have been tested. MBT-2 cells and syngeneic C3H/He mice were used in all experiments. Chemical bladder pre-treatment with different agents (saline, hydrochloric acid, trypsin and poly-L-lysine) and different concentration of instilled tumor cells (1 x 10⁶ or 2 x 10⁶) were investigated. In the second part of the experiment, trans-abdominal micro-ultrasound imaging (MUI) technique was investigated and validated. Bladder tumor growths were monitored with longitudinal measurement. Mice were killed at every MUI session. Bladder tumor volumes were measured and correlated with gross stereomicroscopy. Using the optimized orthotopic bladder cancer model, targeted contrast enhanced micro-ultrasound imaging has been investigated. VEGFR2 targeted contrast agent was prepared and injected intravenously before imaging sessions. The intra-tumoral perfusion, VEGFR2 expression and blood volume in real time were quantified. Contrast enhanced MUI was performed on Days 14 and 21. The feasibility of targeted contrast enhanced micro-ultrasound imaging was confirmed. After the establishment of orthotopic model and in vivo molecular imaging techniques, this robust platform was used for investigating new treatment agent in localized bladder cancer. Tumor-bearing mice were randomized into control and sunitinibtreated (40 mg/kg) groups. Tumor volume, intra-tumoral perfusion, and in vivo VEGFR2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by CD31 and Ki-67 immunohistochemistry. The clinical outcomes including total bladder weight, tumor stage, and survival were evaluated. / Results: A consistent tumor take-rate of over 90% was achieved by using poly-L-lysine pretreatment with 2 x 10⁶ MBT-2 cells in all of the experiments. MUI identified all tumors that were present on final histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). Measurements of intra-tumoral perfusion and in vivo VEGFR2 expression were also proved to be feasible. After the technical refinement and modification, complete measurements could be performed in all mice (n = 10) at 2 consecutive imaging sessions. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. This is the first report of applying targeted contrast enhanced MUI in orthotopic bladder cancer model. Finally, sunitinib was found to have significant tumor growth inhibition in both in vitro and in vivo experiments. In the orthotopic model, tumors in sunitinib-treated mice had reduced tumor volume and stage, lower proliferation index and micro-vessel density. Sunitinib prolonged survival in tumor-bearing mice as compared to control group. / Conclusions: The development of reliable orthotopic animal models assists in the discovery of novel therapeutic agents. The establishment in the methods of implantation with improved tumor-take rate and the advances in imaging technology form the important foundation of basic research in bladder cancer. Trans-abdominal MUI is proven to be a valuable tool for translational studies involving orthotopic mouse bladder cancer models. Furthermore, the first report of the application of targeted contrast enhanced MUI in deep-seated tumor in bladder has been published. It enables investigators to monitor tumor angiogenesis and vascular changes after treatment. It will be useful for direct, noninvasive, in vivo evaluation of anti-angiogenesis therapeutic agents. The preclinical study has demonstrated the activities of a new class of targeted therapy against localized bladder cancer in an orthotopic mouse model. Sunitinib inhibits tumor growth and thus decreases the tumor burden and prolongs survival compared with placebo. These results provide a rationale for future clinical trials using VEGFR-targeted treatments of localized bladder cancer in the neo-adjuvant and adjuvant settings. / Chan, Shu Yin Eddie. / Thesis (M.D) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 189-212).

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-25T16:00:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:38Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:46Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T20:58:56Z (GMT). No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O Mycobacterium bovis BCG é utilizado mundialmente como vacina contra tuberculose há mais de meio século, com altos níveis de segurança. O BCG possui características que o tornam um promissor vetor para a produção de vacinas recombinantes multivalentes. Modificações genéticas podem aprimorar as propriedades imunogênicas e adjuvantes do BCG. Uma dessas modificações permite que o BCG escape do endossoma de células apresentadoras de antígenos. Desta maneira, os imunógenos de BCG acessam vias de apresentação cruzada de antígenos, ampliando a sua imunogenicidade. Este trabalho teve como objetivo desenvolver uma cepa de BCG ΔleuD expressando listeriolisina (Hly) de Listeria monocytogenes e avaliar seu potencial citotóxico na linhagem celular 5637 de câncer de bexiga. Para isso, o BCG ΔleuD foi transformado com o plasmídeo recombinante pUP410 contendo o gene hly. A cepa obtida demonstrou um nível de citotoxicidade similar à cepa BCG Pasteur parental, quando avaliada na linhagem celular de câncer de bexiga. Esta cepa necessita ser avaliada em outras linhagens de câncer para comprovar seu possível efeito citotóxico potencializado. / Mycobacterium bovis BCG has been used worldwide as a vaccine against tuberculosis for over fifty years with an outstanding safety record. BCG has features that make it a promising vector for production of recombinant multivalent vaccines. Genetic modification can enhance the adjuvant and immunogenic properties of BCG. One of these modifications would allow BCG to escape the endosome of antigen presenting cells. Therefore, theimmunogens of BCG can access antigens cross priming routes, increasing their immunogenicity. The aim of this work was to develop a strain of BCG ΔleuD expressing listeriolysin (Hly) of Listeria monocytogenes and evaluate its cytotoxic potential on cell line 5637 of bladder cancer. Thus, BCG ΔleuD was transformed with recombinant plasmid pUP410 containing the hly coding sequence. The recombinant BCG strain expressing listeriolysin showed a similar level of cytotoxicity when compared to the parental BCG Pasteur. This strain needs to be evaluated in other cancer lines in order to confirm its possible potentiated cytotoxic effect.