Basal Urothelial Cells Research Articles

Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

Abstract PR001: TRIM29 is required for basal bladder cancer invasive progression

Abstract TRIM29 (Tripartite Motif 29) also known ATDC, is an E3 ubiquitin ligase frequently upregulated in muscle-invasive bladder cancer (MIBC). We have previously shown TRIM29 global overexpression in a genetically engineered mouse model (GEMM) is sufficient to ` bladder cancer development and progression to MIBC. Other studies strongly suggest cytokeratin-5/14 (Krt5+/Krt14+) expressing basal-stem progenitor cells in the urothelium are the cells of origin for MIBC in mice. We therefore hypothesized that Trim29 expression in Krt5+ basal urothelial cells is required for development of MIBC in mice. To test this, we generated a GEMM harboring a Trim29 conditional KO allele and tamoxifen responsive Cre under control of the bovine Krt5promoter. Exposure of Krt5-CreERT2 Trim29flox/flox mice to tamoxifen resulted in activation of Cre and loss of Trim29 expression only in the Krt5+ basal urothelium. To induce bladder tumorigenesis, we utilized the urothelial specific carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water at 0.05%. After 6 months, tumor invasion was staged by a genitourinary pathologist based on depth of invasion: T1 (stromal invasive), T2 (muscle invasive) and T3 (deep muscle/peri-vesicular invasion). In agreement with previous studies, BBN exposure resulted in loss of Krt5-negative intermediate and superficial cells, and expansion of Krt5+/Krt14+ basal cells across the urothelium. In combination with a tamoxifen treatment this led to total Trim29 KO in the urothelium. All male Trim29 WT mice exposed to BBN developed Trim29+ bladder tumors, >50% of which demonstrated extensive progression to muscle-invasion (57% n=23). In contrast, only 16% of mice with Trim29 loss in Krt5+ basal cells developed MIBC (n=18). As an E3 ubiquitin ligase TRIM29 is known to target immune adaptor molecules, notably the stimulator of interferon genes (STING), for proteasomal degradation. We hypothesized TRIM29 targets STING during invasive progression and supports the establishment of an immune-privileged niche at the bladder cancer invasive front. We find TRIM29 expression in human bladder tissues is upregulated in the urothelium under stress and in MIBC tumors. In mice, BBN exposure upregulated Trim29 expression in the bladder urothelium which correlated with low urothelial Sting. In contrast, Krt5+ Trim29 loss resulted in significantly elevated Sting in the bladder urothelium. Multiplex immunofluorescence imaging and immunotyping of Trim29 KO mouse tissues revealed peritumoral enrichment of CD4+ and CD8+ T cells, CD206+ immunosuppressive macrophages, and CD4+FoxP3+ regulatory T cells. Taken together, these results suggest that TRIM29-mediated downregulation of STING in the bladder urothelium results in decreased immune surveillance at the tumor-stromal interface which expedites invasion and tumor burden. Citation Format: Alan Kelleher, Luke Broses, Yin Wang, Marian Henderson, Mark Day, Aaron Udager, Phillip Palmbos. TRIM29 is required for basal bladder cancer invasive progression [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR001.

MP66-15 PATIENT-CENTRIC 3 DIMENSIONAL-BIOSYSTEMS FOR DEVELOPING PERSONALIZED IMMUNOTHERAPY FOR PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-15 PATIENT-CENTRIC 3 DIMENSIONAL-BIOSYSTEMS FOR DEVELOPING PERSONALIZED IMMUNOTHERAPY FOR PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA Sanghee Lee, Michelle Muldong, Jonathan Hsu, Fady Ghali, Sung Gu Kang, Andrew Karim Kader, Amirali Salmasi, and Christina Jamieson Sanghee LeeSanghee Lee More articles by this author , Michelle MuldongMichelle Muldong More articles by this author , Jonathan HsuJonathan Hsu More articles by this author , Fady GhaliFady Ghali More articles by this author , Sung Gu KangSung Gu Kang More articles by this author , Andrew Karim KaderAndrew Karim Kader More articles by this author , Amirali SalmasiAmirali Salmasi More articles by this author , and Christina JamiesonChristina Jamieson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper urinary tract urothelial carcinoma (UTUC) affects both men and women and accounts for approximately 5% of urothelial malignancies and 5 to 10% of renal cancers. Although surgery remains the mainstay of UTUC therapy, there is an increasing need for multi-modality approaches. Recent studies reviewed the genetic landscape of UTUC and revealed significant recurrent mutations. These observations emphasize the great potential of personalized immunotherapy which requires a thorough understanding of the adoptive and innate immune response to tumor development and progression. Therefore, we aimed to establish 3D-Biosystems in which a patient’s tumor infiltrating lymphocytes (TILs) were combined with their own ureter-derived organoids. METHODS: Ureter tissue samples from a patient with UTUC were collected immediately after surgical removal. 3D-organoids were established as described in Mullenders et al (2019). In parallel, TILs were isolated and expanded as described in Dudley et al (2003) and Tran et al (2007). 3D-Biosystems were established by adding 48 x 104 TILS to the 3D-organoids. Time course imaging of organoids was performed before and after the addition of TILs (1, 4, 28, 56, 84 and 91 hr) utilizing an EVOS microscope. Organoids and TILs in 3D-Biosystems were processed for histology as described in Lee et al (2020) and stained using Immuno-fluorescent cytochemistry (IFC) for markers: Cytokeratin 5 (CK5, basal urothelial cells), Uroplakin III (UPK III, intermediate and superficial urothelial cells) and P63 (basal and intermediate urothelial cells). Gene expression profiles were obtained by RNA-Seq analysis. RESULTS: In 3D-Biosystems, TILs migrated to 3D-organoids by 1 hr after co-culture. Sub-populations of 3D-organoids had extruded compartments by 91 hr after interacting with TILs. The patient’s ureter-derived organoids were CK5 and P63 positive but UPK III negative as was previously seen in urothelial organoids by Clevers group. Transcriptomic data revealed TILs expression profiles. CONCLUSIONS: Our 3D-Biosystems demonstrated functional responsiveness of TILs to tumor-derived organoids from the same patient. The TILs recognized and interacted with 3D-organoids that retain their original characteristics. Therefore, this personalized 3D-Biosystem has great potential as a screening platform to determine immunotherapy responses in combination with existing therapies and as a research platform to determine mechanisms of action. Source of Funding: This study is supported by the JM Foundation. © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1135-e1135 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sanghee Lee More articles by this author Michelle Muldong More articles by this author Jonathan Hsu More articles by this author Fady Ghali More articles by this author Sung Gu Kang More articles by this author Andrew Karim Kader More articles by this author Amirali Salmasi More articles by this author Christina Jamieson More articles by this author Expand All Advertisement Loading ...

Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

To study the progression of bladder cancer from non-muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. SIGNIFICANCE: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Abstract A29: Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer

Abstract Next-generation sequencing has led to substantial insights into the genetic and molecular heterogeneity of bladder cancer. However, the functional consequences of many of these genetic abnormalities have not been explored. Current genetically defined models of tumorigenesis are poorly suited for systematic functional interrogation of the diverse genetic abnormalities in bladder cancer. To address this issue, we are developing an approach that is readily scalable and leverages an established organoid transformation assay, multiplex transduction of organoids by a lentiviral pool, and digital profiling by single-cell sequencing. We found that C57BL/6J mouse basal urothelial cells (Lin−EpCAM+CD49fhigh) efficiently initiated organoids in 3D culture in defined media conditions. Urothelial organoids were readily transduced with lentivirus to achieve viral copy numbers (VCNs) of up to 20 with a linear relationship between VCN and lentiviral dose. As lentiviral transduction follows a Poisson distribution, we reasoned that a lentiviral pool could be used to introduce random combinations of genetic alterations into urothelial organoids. We selected 33 recurrently altered genes in bladder cancer and categorized them as gain of function (genomic amplification and activating mutations) and loss of function (genomic deletion and inactivating mutations). We cloned lentiviral constructs that express open reading frames to recapitulate gain-of-function events and short hairpin RNAs to model loss-of-function events. Each lentiviral clone has also been barcoded with matching 10-nucleotide sequences at both the 5' and 3' ends to facilitate barcode enumeration by DNA amplicon sequencing and RNA sequencing. In preliminary experiments, we have transduced mouse urothelial organoids with the barcoded lentiviral pool to target a mean viral copy number of 4. After a brief period of culture, organoids were grafted subcutaneously into immune-deficient mice to biologically select for tumorigenic clones. Resultant tumors will be isolated for single-cell DNA amplicon sequencing to enable the enumeration of combinations of oncogenic events within each tumor clone that induce bladder cancer tumorigenesis. Optimization of single-cell RNA sequencing is ongoing to allow direct, massively parallel association of genotype to bladder cancer phenotype within this assay. Overall, we are establishing a functional cancer genomics platform to provide rapid functional annotation of genetic alterations and their roles in bladder cancer initiation. Another goal of these studies is the generation of multiple genetically defined models of bladder cancer that recapitulate the spectrum of human disease. Citation Format: Alicia Wong, Huiyun Sun, Sujata Jana, Andrew C. Hsieh, John K. Lee. Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A29.

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.

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 ...

Compensatory regrowth of the mouse bladder after partial cystectomy.

Cystectomy is the removal of all or part of the urinary bladder. It has been observed that there is significant regrowth of the bladder after partial cystectomy and this has been proposed to be through regeneration of the organ. Regrowth of tissue in mammals has been proposed to involve compensatory mechanisms that share many characteristics of true regeneration, like the growth of specialized structures such as blood vessels or nerves. However, the overall structure of the normal organ is not achieved. Here we tested if bladder growth after subtotal cystectomy (STC, removal of 50% of the bladder) was compensatory or regenerative. To do this we subjected adult female mouse bladders to STC and assessed regrowth using several established cellular parameters including histological, gene expression, cytokine accumulation and cell proliferation studies. Bladder function was analyzed using cystometry and the voiding stain on paper (VSOP) technique. We found that STC bladders were able to increase their ability to hold urine with the majority of volume restoration occurring within the first two weeks. Regenerating bladders had thinner walls with less mean muscle thickness, and they showed increased collagen deposition at the incision as well as throughout the bladder wall suggesting that fibrosis was occurring. Cell populations differed in their response to injury with urothelial regeneration complete by day 7, but stromal and detrusor muscle still incomplete after 8wks. Cells incorporated EdU when administered at the time of surgery and tracing of EdU positive cells over time indicated that many newborn cells originate at the incision and move mediolaterally. Basal urothelial cells and bladder mesenchymal stem cells but not smooth muscle cells significantly incorporated EdU after STC. Since anti-inflammatory cytokines play a role in regeneration, we analyzed expressed cytokines and found that no anti-inflammatory cytokines were present in the bladder 1wk after STC. Our findings suggest that bladder regrowth after cystectomy is compensatory and functions to increase the volume that the bladder can hold. This finding sets the stage for understanding how the bladder responds to cystectomy and how this can be improved in patients after suffering bladder injury.

Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia

Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.

The New Immortalized Uroepithelial Cell Line HBLAK Contains Defined Genetic Aberrations Typical of Early Stage Urothelial Tumors.

Background: Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells.Objective: To characterize utility and limitations of HBLAK cells as an urothelial cell culture model.Methods: Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting.Results: HBLAK cells proliferated for >50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in p16INK4A loss. A C228T TERT promoter mutation was present, but no other mutation typical of urothelial carcinoma. TP53 was wild-type and the cell cycle was arrested in response to genomic stress.Conclusions: HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial in vitro carcinogenesis model.

Noninvasive Papillary Basal-like Urothelial Carcinoma: A Subgroup of Urothelial Carcinomas With Immunohistochemical Features of Basal Urothelial Cells Associated With a High Rate of Recurrence and Progression.

We investigated the clinical and pathologic significance of a subgroup of noninvasive papillary urothelial carcinomas (UCs) expressing reactivity to urothelial basal cell markers. In total, 302 consecutive cases of noninvasive papillary UC were evaluated immunohistochemically with cytokeratin 5 (CK5)/CD44. Any UC that was reactive for greater than 25% thickness of the urothelium was designated as basal-like urothelial carcinoma (BUC); remaining UC cases were designated as non-BUC. The follow-up period was up to 3 years. Historical review of UC was extended for up to 3 retrospective years. Among 302 noninvasive UC, BUC was identified in 33 of 256 (12.9%) low-grade UC and 8 of 46 (17%) high-grade UC (P=0.041). Immunoreactivity for CD44 was similar to that of CK5, but displayed weaker and more diffuse staining. CK20 was reactive in 9 cases, primarily high-grade BUC. Other basal cell markers (34bE12, p63, bcl2, and EP4) were found to be neither sensitive nor specific in detecting UC with high CK5 expression. In comparison with non-BUC, BUC was associated with increased multifocality, larger tumor size, higher recurrence rate, and more frequent upgrading and stage progression. In the follow-up period of 3 years, distant metastasis occurred in 6 cases of which 5 were in the BUC subgroup. Our results showed that noninvasive papillary BUC represents a small subset associated with increased risk of tumor recurrence and progression. The aggressive behavior is likely associated with basal-like features of BUC, as seen in carcinomas with basal cell features in other body sites.

Sensory Hyperinnervation Distinguishes Bladder Pain Syndrome/Interstitial Cystitis from Overactive Bladder Syndrome

Pain is the key symptom that distinguishes bladder pain syndrome/interstitial cystitis from overactive bladder syndrome but overlap occurs. To find a discriminating marker for these bladder diseases we examined sensory hyperinnervation and neurotrophin receptor expression in bladder biopsies as well as nerve growth factor levels in urine. Bladder biopsies from patients with bladder pain syndrome/interstitial cystitis, including 12 with and 19 without Hunner lesions, 13 with overactive bladder syndrome and 12 healthy controls, were analyzed by immunohistochemistry with antibodies to the nerve cell marker PGP9.5 (neuron-specific protein gene product 9.5), p75NTR (p75 neurotrophin receptor), the B-lymphocyte marker CD20 and mast cell tryptase. Urinary nerve growth factor was quantified by enzyme-linked immunosorbent assay. Subepithelial sensory hyperinnervation on PGP9.5 staining had 97% sensitivity and 76% specificity, increased lymphocytic infiltration had 90% sensitivity and 80% specificity, and urothelial defects had 97% sensitivity and 76% specificity to distinguish bladder pain syndrome/interstitial cystitis with and without Hunner lesions from overactive bladder syndrome and healthy controls. Increased sensory innervation was associated with submucosal mast cell localization. Staining of p75NTR in basal urothelial cells was indicative of bladder pain syndrome/interstitial cystitis. Urinary nerve growth factor levels were below the detection level and did not differentiate bladder diseases from healthy controls. Sensory hyperinnervation and basal urothelial p75NTR staining together with assessment of inflammatory lymphocytes and urothelial integrity allow for the differentiation of bladder pain syndrome/interstitial cystitis and overactive bladder syndrome even in the absence of Hunner lesions. Furthermore, these histopathological criteria enable the identification of early disease stages or oligosymptomatic/asymptomatic cases and may permit timely treatment to prevent disease progress.

MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL Bernadette Zwaans, Sarah Bartolone, Peter Levanovich, Monica Liebert, Michael Chancellor, and Laura Lamb Bernadette ZwaansBernadette Zwaans More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , Peter LevanovichPeter Levanovich More articles by this author , Monica LiebertMonica Liebert More articles by this author , Michael ChancellorMichael Chancellor More articles by this author , and Laura LambLaura Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.770AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radiation cystitis (RC) is a chronic inflammatory condition of the bladder that results from extensive exposure to radiation therapy for the treatment of pelvic malignancies. RC is a severely debilitating condition due to its symptoms (e.g. hematuria) and the lack of reliable treatment options. The underlying mechanism that contributes to RC development is not well understood. In this study, we aim to investigate the contribution of radiation-induced urothelial damage to the development of RC. We hypothesize that radiation exposure weakens the urothelial layer by reducing cellular proliferation, survival and differentiation ability of basal urothelial cells. Consequently, the damaged urothelium becomes more permeable to urine, which can cause irritation to the underlying tissue. METHODS Normal human basal urothelial (UR15) cells, developed by Jim Bassuk at the University of Washington, were used to study the effect of radiation therapy on the urothelium. UR15 cells, immortalized using HPV E6/E7, were cultured in serum-free keratinocyte media and differentiated in 10% DMEM. Cells were irradiated and the effect of radiation on cellular proliferation, survival, and differentiation was determined. RESULTS Exposure of undifferentiated UR15 cells to radiation treatment resulted in a dose-dependent decline in cellular proliferation that lasted beyond 2 weeks in culture. This decline in cellular proliferation was both due to an increase in apoptosis (Annexin V) as well as a decrease in the number of cell divisions. Additionally, irradiated UR15 cells had reduced differentiation capacity as measured by the formation of tight junctions (E-cadherin), a marker of urothelial differentiation. Ongoing studies are determining cell survival of differentiated urothelial cells as well as identifying changes to urothelial permeability in response to radiation. CONCLUSIONS Using a novel urothelial radiation model our studies concluded that radiation exposure to the bladder damages the urothelium, at least in part by reducing proliferation and differentiation of basal urothelial cells. Furthermore, a decrease in E-cadherin localized to the cell membrane suggests an increase in bladder permeability. This model may be advantages to investigate therapeutic options that may treat or prevent radiation cystitis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e275 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bernadette Zwaans More articles by this author Sarah Bartolone More articles by this author Peter Levanovich More articles by this author Monica Liebert More articles by this author Michael Chancellor More articles by this author Laura Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibition of urothelial P2X3 receptors prevents desensitization of purinergic detrusor contractions in the rat bladder.

To evaluate whether P2X3 receptors (P2X3R) are expressed in the bladder urothelium and to determine their possible function in modulating purinergic detrusor contractions in the rat urinary bladder. The expression of urothelial receptors was determined using conventional immunohistochemistry in bladders from normal Sprague-Dawley rats. The urothelial layer was removed by incubation with protamine, and disruption of the urothelium was confirmed using haematoxylin and eosin staining on bladder sections. Open cystometry was used to determine the effects of both urothelial removal as well as intravesical application of a specific P2X3R antagonist on bladder properties from intact and protamine-treated rats. Isometric contractile responses to potassium chloride (KCl) depolarization, electrical field stimulation (EFS) or chemical P2X activation were determined in normal and urothelium-denuded bladder strips, with and without application of the P2X3R antagonist. Immunohistochemical staining showed high expression of P2X3R in the medial and basal layers of the urothelium. Removal of the urothelial layer disturbed normal bladder performance in vivo and eliminated the effects of the P2X3R antagonist on increasing the contractile interval and reducing the amplitude of voiding contractions. Removal of the urothelium did not affect bladder strip contractile responses to KCl depolarization or EFS. Pharmacological inhibition of P2X3R prevented desensitization to P2X-mediated detrusor muscle contractions during EFS only in the strips with an intact urothelium. A concentration-dependent, specific inhibition of P2X3R also prevented desensitization of purinergic contractile responses in intact bladder strips. In the rat bladder, medial and basal urothelial cells express P2X3R, and specific inhibition of the receptor leads to a more hyporeflexive bladder condition. This pathway may involve P2X3R driving a paracrine amplification of ATP released from umbrella cells to increase afferent transmission in the sub-urothelial sensory plexus and desensitization of P2X1-mediated purinergic detrusor contractions.

Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumor progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma-in-situ. Shh-expressing basal cells within this precursor lesion become tumor-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumor cell phenotype can diverge significantly from that of the cancer cell-of-origin.

Cytokeratin 15 Marks Basal Epithelia in Developing Ureters and Is Upregulated in a Subset of Urothelial Cell Carcinomas

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Constitutive β-Catenin Activation Induces Male-Specific Tumorigenesis in the Bladder Urothelium

The incidence for bladder urothelial carcinoma, a common malignancy of the urinary tract, is about three times higher in men than in women. Although this gender difference has been primarily attributed to differential exposures, it is likely that underlying biologic causes contribute to the gender inequality. In this study, we report a transgenic mouse bladder tumor model upon induction of constitutively activated β-catenin signaling in the adult urothelium. We showed that the histopathology of the tumors observed in our model closely resembled that of the human low-grade urothelial carcinoma. In addition, we provided evidence supporting the KRT5-positive;KRT7-negative (KRT5(+); KRT7(-)) basal cells as the putative cells-of-origin for β-catenin-induced luminal tumor. Intriguingly, the tumorigenesis in this model showed a marked difference between opposite sexes; 40% of males developed macroscopically detectable luminal tumors in 12 weeks, whereas only 3% of females developed tumors. We investigated the mechanisms underlying this sexual dimorphism in pathogenesis and showed that nuclear translocation of the androgen receptor (AR) in the urothelial cells is a critical mechanism contributing to tumor development in male mice. Finally, we carried out global gene profiling experiments and defined the molecular signature for the β-catenin-induced tumorigenesis in males. Altogether, we have established a model for investigating sexual dimorphism in urothelial carcinoma development, and implicated synergy between β-catenin signaling and androgen/AR signaling in carcinogenesis of the basal urothelial cells.

Stem cells in the biology of normal urothelium and urothelial carcinoma

Urothelium is a special type of stratified epithelium that lines the distal portion of the urinary tract. For a long time, basal urothelial cells have been suspected to include a population of urothelial stem cells. Recent experiments identifying label-retaining cells as well as lineage tracing analyses corroborate this notion. There are striking morphological and antigenic similarities between basal or differentiated urothelial cells and the corresponding cells in some urothelial carcinomas. In this respect, basal cell-specific markers provide good candidates to identify urothelial carcinoma stem cells, e.g. specific cytokeratins (CK5, CK14, CK17) or adhesion molecules (specific integrin subspecies, CD44). Common properties of the stem cells of normal urothelium and urothelial cancer have thus emerged. Both are characterized by a remarkable plasticity and both rely on reciprocal interactions with stromal fibroblasts. However, the stem cells of individual urothelial carcinomas appear to differ considerably and may contribute to the heterogeneity of this disease. The presence, quantity, and particular biological nature of urothelial carcinoma stem cells in each case may thus carry important clinical information that might allow a rationale stratification of urothelial cancer patients for treatment in the near future.

Prostaglandin Receptor EP1 and EP2 Site in Guinea Pig Bladder Urothelium and Lamina Propria

Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Differential Localizations of the Transient Receptor Potential Channels TRPV4 and TRPV1 in the Mouse Urinary Bladder

We studied the localization and physiological functions of the transient receptor potential (TRP) channels TRPV1 (TRP vanilloid 1) and TRPV4 (TRP vanilloid 4) in the mouse bladder, because both channels are thought to be mechanosensors for bladder distention. RT-PCR specifically amplified TRPV4 transcripts from the urothelial cells, whereas TRPV1 transcripts were barely detectable. ISH experiments showed that TRPV4 transcripts were abundantly expressed in the urothelium, whereas TRPV1 transcripts were not detectable in the urothelial cells. Immunoblotting and IHC studies showed that TRPV4 proteins were mainly localized at the basal plasma membrane domains of the basal urothelial cells. In contrast, TRPV1-immunoreactivities were found not in the urothelial cells but in the nerve fibers that innervate the urinary bladder. In Ca(2+)-imaging experiments, 4alpha-phorbol 12,13-didecanoate, a TRPV4 agonist, and hypotonic stimuli induced significant increases in intracellular calcium ion concentration ([Ca(2+)](i)) in isolated urothelial cells, whereas capsaicin, a TRPV1 agonist, showed no marked effect on the cells. These findings raise the possibility that, in mouse urothelial cells, TRPV4 may contribute to the detection of increases in intravesical pressure related to the micturition reflex.