Mouse Urothelium Research Articles

Urinary bladder carcinogenic potential of 4,4'-methylenebis(2-chloroaniline) in humanized-liver mice.

Occupational exposure to 4,4'-methylenebis(2-chloroaniline) (MOCA) has been linked to an increased risk of bladder cancer among employees in Japanese plants, indicating its significance as a risk factor for urinary bladder cancer. To investigate the role of MOCA metabolism in bladder carcinogenesis, we administered MOCA to non-humanized (F1-TKm30 mice) and humanized-liver mice for 4 and 28 weeks. We compared MOCA-induced changes in metabolic enzyme expression, metabolite formation, and effects on the urinary bladder epithelium in the two models. At week 4, MOCA exposure induced simple hyperplasia, cell proliferation, and DNA damage in the urothelium of the humanized-liver mice, while in the non-humanized mice these effects were not observed. Notably, the concentration of 4-amino-4'-hydroxylamino-3,3'-dichlorodiphenylmethane (N-OH-MOCA) in the urine of humanized-liver mice was more than 10 times higher than that in non-humanized mice at the 4-week mark. Additionally, we observed distinct differences in the expression of cytochrome P450 isoforms between the two models. Although no bladder tumors were detected after 28 weeks of treatment in either group, these findings suggest that N-OH-MOCA significantly contributes to the carcinogenic potential of MOCA in humans.

Sex-Dependent Differences in Blood-Urine Barrier Are Subtle but Significant in Healthy and Chronically Inflamed Mouse Bladders.

The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.

O-Toluidine metabolism and effects in the urinary bladder of humanized-liver mice

Occupational exposure to aromatic amines is one of the most important risk factors for urinary bladder cancer. When considering the carcinogenesis of aromatic amines, metabolism of aromatic amines in the liver is an important factor. In the present study, we administered ortho-toluidine (OTD) in the diet to mice for 4 weeks. We used NOG-TKm30 mice (control) and humanized-liver mice, established via human hepatocyte transplantation, to compare differences in OTD-induced expression of metabolic enzymes in human and mouse liver cells. We also investigated OTD-urinary metabolites and proliferative effects on the urinary bladder epithelium. RNA and immunohistochemical analyses revealed that expression of N-acetyltransferases mRNA in the liver tended to be lower than that of the P450 enzymes, and that OTD administration had little effect on N-acetyltransferase mRNA expression levels. However, expression of CYP3A4 was increased in the livers of humanized-liver mice, and expression of Cyp2c29 (human CYP2C9/19) was increased in the livers of NOG-TKm30 mice. OTD metabolites in the urine and cell proliferation activities in the bladder urothelium of NOG-TKm30 and humanized-liver mice were similar. However, the concentration of OTD in the urine of NOG-TKm30 mice was markedly higher than in the urine of humanized-liver mice. These data demonstrate differences in hepatic metabolic enzyme expression induced by OTD in human and mouse liver cells, and consequent differences in the metabolism of OTD by human and mouse liver cells. This type of difference could have a profound impact on the carcinogenicity of compounds that are metabolized by the liver, and consequently, would be important in the extrapolation of data from animals to humans.

Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the β-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2MBq of [90Y]DBA or unlabeled DBA 3days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. Our results demonstrated that DBA is contained in the bladder for up to 4h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urotheliumat day 10, which appeared similar to the normal urothelium of healthy mice. Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

FGFR3 Mutational Activation Can Induce Luminal-like Papillary Bladder Tumor Formation and Favors a Male Sex Bias

BackgroundBladder cancer (BCa) is more common in men and presents differences in molecular subtypes based on sex. Fibroblast growth factor receptor 3 (FGFR3) mutations are enriched in the luminal papillary muscle-invasive BCa (MIBC) and non-MIBC subtypes. ObjectiveTo determine whether FGFR3 mutations initiate BCa and impact BCa male sex bias. Design, setting, and participantsWe developed a transgenic mouse model expressing the most frequent FGFR3 mutation, FGFR3-S249C, in urothelial cells. Bladder tumorigenesis was monitored in transgenic mice, with and without carcinogen exposure. Mouse and human BCa transcriptomic data were compared. InterventionMutant FGFR3 overexpression in mouse urothelium and siRNA knockdown in cell lines, and N-butyl-N(4-hydroxybutyl)-nitrosamine (BBN) exposure. Outcome measurements and statistical analysisImpact of transgene dosage on tumor frequency, synergy with BBN treatment, and FGFR3 pathway activation were analyzed. The sex-specific incidence of FGFR3-mutated tumors was evaluated in mice and humans. FGFR3 expression in FGFR3-S249C mouse urothelium and in various human epithelia was measured. Mutant FGFR3 regulation of androgen (AR) and estrogen (ESR1) receptor activity was evaluated, through target gene expression (regulon) and reporter assays. Results and limitationsFGFR3-S249C expression in mice induced low-grade papillary BCa resembling human luminal counterpart at histological, genomic, and transcriptomic levels, and promoted BBN-induced basal BCa formation. Mutant FGFR3 expression levels impacted tumor incidence in mice, and mutant FGFR3–driven human tumors were restricted to epithelia presenting high normal FGFR3 expression levels. BCa male sex bias, also found in our model, was even higher in human FGFR3-mutated tumors compared with wild-type tumors and was associated with higher AR and lower ESR1 regulon activity. Mutant FGFR3 expression inhibited both ESR1 and AR activity in mouse tumors and human cell lines, demonstrating causation only between FGFR3 activation and low ESR1 activity in tumors. ConclusionsMutant FGFR3 initiates luminal papillary BCa formation and favors BCa male sex bias, potentially through FGFR3-dependent ESR1 downregulation. Patients with premalignant lesions or early-stage BCa could thus potentially benefit from FGFR3 targeting. FGFR3 expression level in epithelia could account for FGFR3-driven carcinoma tissue specificity. Patient summaryBy developing a transgenic mouse model, we showed that gain-of-function mutations of FGFR3 receptor, among the most frequent genetic alterations in bladder cancer (BCa), initiate BCa formation. Our results could support noninvasive detection of FGFR3 mutations and FGFR3 targeting in early-stage bladder lesions.

Structural or functional defects of PTEN in urothelial cells lacking P53 drive basal/squamous-subtype muscle-invasive bladder cancer

Muscle-invasive bladder cancer (MIBC) exhibits strong inter- and intra-tumor heterogeneity that affects biological behaviors, therapeutic responses, and prognoses. Mutations that activate RTK-RAS-PI3K and inactivate P19-P53-P21 coexist in 60–70% of MIBC. By time-controlled ablation of Tp53 and Pten, singly or combined, in adult mouse urothelium, we found that Tp53 loss alone produced no abnormality. While Pten loss elicited hyperplasia, it synergized with Tp53 loss to trigger 100% penetrant MIBC that exhibited basal/squamous features that resembled its human counterpart. Furthermore, PTEN was inactivated in human MIBC cell lines and specimens primarily by hyperphosphorylation of the C-terminus. Mutated or tailless PTEN incapable of C-terminal phosphorylation demonstrated increased inhibition of proliferation and invasion than full-length PTEN in cultured MIBC cells. In xenograft and transgenic mice, tailless PTEN, but not full-length PTEN, prevented further growth in established tumors. Collectively, deficiencies of both PTEN and P53 drive basal/squamous subtype MIBC. PTEN is inactivated by C-terminal hyperphosphorylation, and this modification may serve as a biomarker for subtyping MIBC and predicting tumor progression. Tailless PTEN is a potential molecular therapeutic for tumors, such as bladder cancer (BC), that can be readily accessed.

Abstract 919: APOBEC3 promotes tumor progression, squamous differentiation and metastasis in bladder cancer mouse model

Abstract Bladder cancer is the sixth most common type of cancer in the United States. In 2021, there are an estimated 83,730 new cases and 17,200 deaths from bladder cancer. Only about 15% of patients with metastatic disease survive past five years. The apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) is overexpressed in bladder tumors compared to corresponding healthy tissues. This enzyme catalyzes the conversion of cytidine to uracil playing an important physiologic role in restricting viral replication. When aberrantly over-expressed, APOBEC3B can damage single-stranded DNA present during DNA replication and transcription, resulting in an increased tumor mutational burden. Strikingly, APOBEC mutagenesis accounts for 70% of all single-nucleotide variants found in bladder cancer. Despite the strong evidence of APOBEC mutagenesis in bladder cancer, the effects of APOBEC3B on the disease progression remain unclear. To address this question, we generated the UPPA (urothelial specific over-expression of mouse Apobec3 along with Pten and p53 knock-out) and control UPP mouse models to interrogate the role Apobec3 in bladder cancer progression. Apobec3 over-expression shortened tumor latency in the UPPA model with the median tumor free survival time of 43.3 weeks compared to 53.6 weeks in the UPP model. Histological analysis showed that bladder epithelial cells in the UPPA mice underwent squamous differentiation which is associated with aggressive disease in human. Additionally, immunofluorescence analysis showed up-regulation of krt6a (a squamous marker) in UPPA urothelium compared to a complete lack of krt6a signal in the urothelium of UPP mice. Necropsy revealed the presence of metastatic nodules in lung, liver and peritoneum in UPPA mice. Cell lines derived from a matched pair of UPPA primary tumor and peritoneal metastatic tumor formed lung and liver nodules in wild-type C57BL/6J mice via tail vein injection. Our results so far suggest that Apobec3 promotes tumor progression, squamous differentiation and metastasis. Additional work utilizing bladder organoids and scRNAseq are underway to unravel the mechanism by which Apobec3 promotes these observed phenotypes. Currently, single-cell RNAseq analysis on primary UPPA tumor demonstrated high degree to T cell infiltration. Approximately 60% of cells sequenced were annotated as T-cells and natural killer T-cells by SingleR. We treated mice bearing UPPA primary bladder tumors with anti-PD-1 antibody and observed significant tumor growth inhibition. Citation Format: Andrew S. Truong, Mi Zhou, Michael S. Sturdivant, John D. Raupp, Ujjawal Manocha, William Y. Kim. APOBEC3 promotes tumor progression, squamous differentiation and metastasis in bladder cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 919.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Challenges Facing an EM Core Laboratory: Mitochondria Structural Preservation and 3DEM Data Presentation

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Abstract B12: Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium

Abstract In the urinary bladder, the normal urothelium is divided into three subpopulations of cells: superficial umbrella cells, intermediate cells, and a basal layer, each with distinct expression patterns of uroplakins (UP) and keratin 5 (K5). Many current transgenic mouse models (Tg) for urinary bladder cancer rely on uroplakin or keratin promoters to drive inducible Cre-lox recombination in basal or luminal bladder urothelial cells. Further, many models utilize the chemical carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), to drive or accelerate tumorigenesis. A major limitation in Tg model systems where genes are knocked out is the requirement for highly penetrant and reproducible Cre activity and subsequent gene deletion in order to see a phenotype. This problem is compounded in long-term experiments such as BBN carcinogenesis that can run for up to six months after Cre induction. We have previously shown that Trim29 (tripartite motif containing 29) is sufficient to drive invasive bladder cancer formation in Tg models and in human bladder tumors. We have now developed a Tg system utilizing K5-Cre-ERT2 to target Trim29 for deletion in K5-expressing basal urothelial cells. In this model, tamoxifen exposure results in Cre expression and Trim29 deletion in K5 basal cells. Whether this would translate into complete loss of Trim29 in the urothelium over time and what effect BBN might have on tamoxifen induction of K5-Cre and loss of Trim29 were unclear. We found that exposure of K5-Cre-ERT2, Trim29 flox/flox mice to tamoxifen for six weeks resulted in loss of Trim29 expression only in the K5+ basal urothelium and that these K5+ Trim29 -/- basal cells did not give rise to intermediate cells or umbrella cells during the six months of follow-up. In contrast, mice treated with 0.05% BBN in drinking water for six months after induction of Cre produced full thickness of Trim29 knockout (KO) in 50% of mice (8/16 mice). Our results indicate that in a nonstressed state, K5 basal cells give rise to other basal cells but do not repopulate intermediate or umbrella cell layers. Furthermore, exposure to BBN resulted in the proliferation of basal cells and the replacement of intermediate cells by basal precursors in the urothelium. These results suggest that K5-Cre promoters may have different effects on transgene recombination in the bladder in the presence or absence of BBN, which induces more universal expression of Cre and its subsequent recombination events. These findings should be considered when interpreting and developing BBN-induced bladder cancer Tg models. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Sylvia Zelenka-Wang, Phillip L. Palmbos. Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B12.

Does the Urothelium of Old Mice Regenerate after Chitosan Injury as Quickly as the Urothelium of Young Mice?

The aging of organisms leads to a decreased ability of tissue to regenerate after injury. The regeneration of the bladder urothelium after induced desquamation with biopolymer chitosan has been studied in young mice but not in old mice. Chitosan is a suitable inducer of urothelial desquamation because it is known to be non-toxic. We used chitosan for desquamation of urothelial cells in order to compare the dynamics of urothelial regeneration after injury between young and old mice. Our aim was to determine whether the urothelial function and structure of old mice is restored as fast as in young mice, and to evaluate the inflammatory response due to chitosan treatment. We discovered that the urothelial function restored comparably fast in both age groups and that the urothelium of young and old mice recovered within 5 days after injury, although the onset of proliferation and differentiation appeared later in old mice. Acute inflammation markers showed some differences in the inflammatory response in young versus old mice, but in both age groups, chitosan caused short-term acute inflammation. In conclusion, the restoration of urothelial function is not impaired in old mice, but the regeneration of the urothelial structure in old mice slightly lags behind the regeneration in young mice.

Re: Acute Spinal Cord Injury is Associated with Mitochondrial Dysfunction in Mouse Urothelium.

No AccessJournal of UrologyUrological Survey1 Jul 2020Re: Acute Spinal Cord Injury is Associated with Mitochondrial Dysfunction in Mouse Urothelium Alan J. WeinMD, PhD (hon), FACS Alan J. WeinAlan J. Wein More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001059AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Re: Acute Spinal Cord Injury is Associated with Mitochondrial Dysfunction in Mouse Urothelium." The Journal of Urology, 204(1), pp. 189–190 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 204Issue 1July 2020Page: 189-190 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alan J. Wein More articles by this author Expand All Advertisement PDF downloadLoading ...

MP28-17 FOXA1 EXPRESSION IS REQUIRED FOR MAINTENANCE OF SUPERFICIAL UMBRELLA CELLS IN THE UROTHELIUM

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I (MP28)1 Apr 2020MP28-17 FOXA1 EXPRESSION IS REQUIRED FOR MAINTENANCE OF SUPERFICIAL UMBRELLA CELLS IN THE UROTHELIUM Lauren Shuman*, Jenna Buckwalter, Thomas Wildermuth, Klaus Kaestner, Cathy Mendelsohn, and David DeGraff Lauren Shuman*Lauren Shuman* More articles by this author , Jenna BuckwalterJenna Buckwalter More articles by this author , Thomas WildermuthThomas Wildermuth More articles by this author , Klaus KaestnerKlaus Kaestner More articles by this author , Cathy MendelsohnCathy Mendelsohn More articles by this author , and David DeGraffDavid DeGraff More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000867.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chronic injury of the urothelium is associated with bladder pain syndrome and voiding dysfunction for which there are presently no cures. Therefore, understanding mechanisms responsible for the maintenance of urothelial differentiation and regeneration is important. Moreover, identifying molecular contributors to these processes could result in novel approaches to enhance healing. Forkhead Box A1 (Foxa1) is a transcription factor important for the regulation of tissue-specific gene expression in the urothelium and other cell types. We previously reported that inducible Foxa1 knockout (KO) in adult mice results in hyperplasia, squamous differentiation, and reduced expression of tissue-specific genes in the urothelium. Here, we use a Upk2Cre driven KO of Foxa1 to determine the impact of early Foxa1 inactivation in urothelial development and differentiation. METHODS: The Upk2Cre mouse line (expressed at embryonic day 13) was bred with previously described Foxa1loxp/loxp mice. Control and Upk2Cre/Foxa1loxp/loxp mice were sacrificed at 3 months of age and bladders were analyzed using hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence (IF). RESULTS: While urothelium from control mice exhibited a normal number of superficial umbrella, intermediate, and basal urothelial cells, the number of umbrella cells within the bladders of a subset of Upk2Cre/Foxa1loxp/loxp mice was significantly reduced in number (p=0.003; Student’s t-test), with IHC confirming KO in remaining superficial urothelial cells. The urothelium of Foxa1 KO mice also often appeared dysmorphic and umbrella cells were undetectable in a subset of bladders. In addition, while IF confirmed decreases in the number of Upk3+/Krt20+/Tp63- superficial urothelial cells, we also show that Foxa1 KO bladders have a reduced number of Upk3+/Tp63+ intermediate cells. CONCLUSIONS: These results suggest Foxa1 expression is required for normal urothelial development and/or maintenance of superficial and intermediate urothelial populations. However, it is still unknown if Foxa1 expression plays a role in the maintenance of basal urothelium, or if Foxa1 is required for urothelial regeneration following injury. Therefore, future studies will focus on these and other areas of importance. Source of Funding: 5U54DK104309 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e426-e426 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lauren Shuman* More articles by this author Jenna Buckwalter More articles by this author Thomas Wildermuth More articles by this author Klaus Kaestner More articles by this author Cathy Mendelsohn More articles by this author David DeGraff More articles by this author Expand All Advertisement PDF downloadLoading ...

Inflammation-like changes in the urothelium of Lifr-deficient mice and LIFR-haploinsufficient humans with urinary tract anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of end-stage kidney disease in children. While the genetic aberrations underlying CAKUT pathogenesis are increasingly being elucidated, their consequences on a cellular and molecular level commonly remain unclear. Recently, we reported rare heterozygous deleterious LIFR variants in 3.3% of CAKUT patients, including a novel de novo frameshift variant, identified by whole-exome sequencing, in a patient with severe bilateral CAKUT. We also demonstrated CAKUT phenotypes in Lifr-/- and Lifr+/- mice, including a narrowed ureteric lumen due to muscular hypertrophy and a thickened urothelium. Here, we show that both in the ureter and bladder of Lifr-/- and Lifr+/- embryos, differentiation of the three urothelial cell types (basal, intermediate and superficial cells) occurs normally but that the turnover of superficial cells is elevated due to increased proliferation, enhanced differentiation from their progenitor cells (intermediate cells) and, importantly, shedding into the ureteric lumen. Microarray-based analysis of genome-wide transcriptional changes in Lifr-/- versus Lifr+/+ ureters identified gene networks associated with an antimicrobial inflammatory response. Finally, in a reverse phenotyping effort, significantly more superficial cells were detected in the urine of CAKUT patients with versus without LIFR variants indicating conserved LIFR-dependent urinary tract changes in the murine and human context. Our data suggest that LIFR signaling is required in the epithelium of the urinary tract to suppress an antimicrobial response under homeostatic conditions and that genetically induced inflammation-like changes underlie CAKUT pathogenesis in Lifr deficiency and LIFR haploinsufficiency.

Monitoring of Active Notch Signaling in Mouse Bladder Urothelium.

Notch signaling plays a crucial role in differentiation and homeostasis in a wide variety of epithelia. The tumor suppressor role of Notch in bladder urothelium is well accepted as the inactivation of this pathway due to damaging mutations in its components is associated with neoplastic transformation. Monitoring Notch signaling is therefore critical to understand how the deregulation of cell-cell communication can lead to differentiation loss and carcinogenesis. In this chapter, we provide a method to visualize active Notch signaling by the detection of the nuclear levels of Notch intracellular domain in mouse urothelium. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human tumor specimens. In this context, this technique could be used to characterize the molecular profile of Notch-deficient tumors and analyze the clonal expansion dynamics and the heterogeneity patterns of Notch inactivation.

Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Serotonin exerts a direct modulatory role on bladder afferent firing in mice.

Functional disorders (i.e. interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel, respectively. Various non-5-HT3 receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5-HT. Whilst 5-HT3 receptors were not detected in mouse urothelium, 5-HT3 receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability both under normal conditions and in a mouse model of chronic visceral hypersensitivity. These data suggest that the role 5-HT3 receptors play in bladder afferent signalling warrants further study as a potential therapeutic target for functional bladder disorders. Serotonin (5-HT) is an excitatory mediator that in the gastrointestinal (GI) tract plays a physiological role in gut-brain signalling and is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross-sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5-HT in bladder afferent signalling and its role in colon-bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5-HT on bladder afferent signalling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid-induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5-HT receptor subtypes, the serotonin transporter (SERT) and 5-HT-producing enzymes was determined in the urothelium using RT-PCR. Retrograde labelling of bladder-projecting dorsal root ganglion neurons was used to investigate expression of 5-HT3 receptors using single cell RT-PCR, while sensory neuronal and urothelial responses to 5-HT were determined by live cell calcium imaging. 5-HT elicited bladder afferent firing predominantly via 5-HT3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5-HT bioavailability. Granisetron, a 5-HT3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5-HT exerts a direct effect on bladder afferents to enhance signalling. 5-HT3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders.

Abstract 1932: Cationic amphiphilic drugs as a potential new treatment option for patients with high risk non-muscle invasive bladder cancer

Abstract Urothelial carcinoma of the bladder (UCB) is the fifth most common cancer in the western world. UCB present either as non-muscle-invasive BC (NMIBC) or as muscle-invasive carcinomas (MIBC). NMIBC is characterized by a high risk of recurrence and even progression to MIBC based on clinicopathological risk factors. Despite radical surgery and extensive chemotherapy, the outcome of MIBC patients is poor. High-risk NMIBC is treated with adjuvant intravesical instillations with Bacillus Calmette-Guérin (BCG). However, a substantial number of patients do not tolerate BCG or do not respond. Novel therapy for patients who do not tolerate BCG or in who are BCG unresponsive is a clear unmet clinical need. Drug repositioning of existing FDA-approved drugs could be a promising opportunity. Recently, cationic amphiphilic drugs (CADs), including commonly used antidepressants, antihistamines and antipsychotics were found to preferentially induce lysosomal cell death in transformed cells. In this study, we evaluated the potential anti-tumor efficacy and toxicity of these drugs in in vitro assays, in vivo preclinical models, and in and ex-vivo cultured patient-derived bladder cancer tissue slices. Treatment with CADs resulted in a significant dose-dependent decrease in proliferation and viability of multiple human UCB cell lines (a.o. RT4, TCC-SUP, and UM-UC-3) in vitro. Consistent with previous data regarding the ability of CADs to induce lysosomal membrane permeabilization in other solid cancer cells, we observed lysosomal puncta formation, which is a hallmark of lysosomal leakage. Strikingly, instillation of CADs in preclinical in vivo models of orthotopically growing human UM-UC-3 UCB resulted in a significant reduction in orthotopic as well as metastatic tumor burden. In line with these data, treatment of 39 patient-derived ex vivo cultured UCB slices with penfluridol resulted in significant anti-tumor responses ranging from complete regression to a significant reduction of the tumor.In vivo experiments revealed that normal mouse urothelium is not affected upon bladder instillation of CADs (weekly instillation of penfluridol for 5 weeks) in non-tumor bearing nude mice. In addition, penfluridol treatment of ex vivo cultured normal human urothelium only marginally affected the tissue. In conclusion, we demonstrated that CADs can target UCB cells and might be a promising novel approach for treatment of UCB. Citation Format: Geertje van der Horst, Arjanneke F. van de Merbel, Eline Ruigrok, Maaike H. van der Mark, Emily Ploeg, Marja Jäätelä, Janneke van Uhm, Rob C. Pelger, Chris H. Bangma, Joost Boormans, Gabri van der Pluijm, Ellen C. Zwarthoff. Cationic amphiphilic drugs as a potential new treatment option for patients with high risk non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1932.

Acute spinal cord injury is associated with mitochondrial dysfunction in mouse urothelium.

To characterize the effects of acute spinal cord injury (SCI) on mitochondrial morphology and function in bladder urothelium and to test the therapeutic efficacy of early treatment with the mitochondrially targeted antioxidant, MitoTempo. We used a mouse model of acute SCI by spinal cord transection between the T8-T9 vertebrae with or without MitoTempo delivery at the time of injury followed by tissue processing at 3 days after SCI. Control, SCI, and SCI-MitoTempo-treated mice were compared in all experimental conditions. Assessments included analysis of markers of mitochondrial health including accumulation of reactive oxygen species (ROS), morphological changes in the ultrastructure of mitochondria by transmission electron microscopy, and Western blot analysis to quantify protein levels of markers for autophagy and altered mitochondrial dynamics. SCI resulted in an increase in oxidative stress markers and ROS production, confirming mitochondrial dysfunction. Mitochondria from SCI mice developed large electron-dense inclusions and these aberrant mitochondria accumulated throughout the cytoplasm suggesting an inability to clear dysfunctional mitochondria by mitophagy. SCI mice also exhibited elevated levels of dynamin-related protein 1 (DRP1), consistent with a disruption of mitochondrial dynamics. Remarkably, treatment with MitoTempo reversed many of the SCI-induced abnormalities that we observed. Acute SCI negatively and severely affects mitochondrial health of bladder urothelium. Early treatment of SCI with MitoTempo may be a viable therapeutic agent to mitigate these deleterious effects.

Mouse and human urothelial cancer organoids: A tool for bladder cancer research

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.