Context: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics. Objective: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018. Evidence acquisition: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review. Evidence synthesis: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models. Conclusions: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development. Patient summary: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes. (C) 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Exsosomes are nano-vesicles secreted by most types of cells, and substantially increased secretion in cancer cells. Their role in tumorigenesis/cancer progression have been extensively studied but their role in regulating therapy response have been suggested without in depth investigation. We are particularly interested in exosome9s roles in cisplatin resistant, since cisplatin is one of the most widely used chemodrugs for many cancer types. To investigate the role of exosomes in cisplatin resistance in bladder cancer, we examined the cisplatin sensitivity among five bladder cancer cell lines. The expression levels of exosome associated genes that are involved in exosome biogenesis and secretion/internalization were examined by quantative PCR. Interestingly, cisplatin resistant bladder cancer cells express relative high level of exosome associated genes as compared to cisplatin sensitive cells. More importantly, cisplatin treatment induces the expression of exosome associated genes’ expression in cisplatin resistant cells, but not in sensitive cell. Among all those genes, we are particularly interested in Rab gene family, and mechanistic study of targeting those Rab proteins is undergoing. The correlative exosome genes profiles with cisplatin resistance suggested exosome production might facilitate cancer cells escaping from cisplatin-induced cell death; thereby, inhibition of exosome might sensitize cancer cells response to cisplatin. To test this, we have screened several compounds that are known to inhibit exosome biogenesis and uptake, and. found that inhibition of exosomes by dynasore + NSC23766, indeed can sensitize bladder cancer cells to cisplatin. In summary, we concluded that cancer exosome play a critical role of promoting cisplatin resistance in bladder cancer, and inhibition of exosomes sensitizes bladder cancer cell response to cisplain. The exosome related profiles potentially can serve as therapy biomarkers Citation Format: Chiahao Wu, Chris Silvers, Elizabeths Guancial, Jong-Wei Hsu, Edward Messing, Yi-Fen Lee. Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2015-5475

Abstract Background: Elevated serum levels of interleukin-6 (IL-6) in lung cancer patients have been observed and have been implicated in lung cancer progression. However, the exact role and the mechanism of IL-6 actions have not been clearly dissected. Especially, the role of IL-6 in lung cancer stem cells (CSCs) has never been addressed. Methods: In this study, we isolated CD133+ cancer stem cells (CSCs) and CD133- non-CSCs from 3 non-small cell lung cancer (NSCLC) cell lines, A549, H1299, and H157, and investigated the roles of IL-6 in modulating the growth of these two cell populations separately. After isolation of CD133+ and CD133- cells by magnetic sorting, and after identification of CSC characteristics in the isolated CD133+ cells, cells were treated with IL-6 for 2 weeks and their growth analyzed by direct cell counting for non-CSCs and by sphere formation assay for CSCs. To investigate the role of IL-6 in the IL-6-expressing A549 and H157 cell lines further, we manipulated IL-6 expression by lentiviral infection prior to the isolation of the CD133+ and CD133- cells. As the 3rd approach, we long-term treated H1299 cells with IL-6 and then isolated CD133+ and CD133- cells from this culture, as well as from the passage-matched non-treated parental cells. We further confirmed the in vitro results by performing in vivo xenografted mouse studies. We subcutaneously injected mice with CD133+ cells isolated from the IL-6 knocked-down A549 (A549IL-6si) or control (A549sc) cells and monitored tumor growth. Finally, we dissected the mechanism by which IL-6 promoted self-renewal of CSCs. Results: Results of these 3 approaches clearly indicated that IL-6 played opposite roles in regulating the growth of the two different cell populations; an inhibitory role in the growth of the CD133- cells, but a promoting role in the self-renewal of CD133+ CSCs. In mouse studies, we observed higher tumor incidence and higher tumor growth in mice injected with the A549sc-CD133+ cells compared to mice injected with A549IL-6si-CD133+ cells, confirming the promoter role of IL-6 in CD133+ cell growth in vivo. Further mechanism dissection studies using Western blot analyses revealed that IL-6 modulated the activation of the Hedgehog (hhg) and MEK/Erk signaling pathways, and increased the expression of anti-apoptotic proteins, bcl-2. Inhibitor studies using the inhibitors of these signaling pathways confirmed that increased self-renewal of CD133+ cells were blocked by inhibitions of hhg and bcl-2. Conclusions and Impacts: Together, these results, for the first time, showed differential effects of IL-6 in regulating non-CSCs vs. CSCs of NSCLC that are paradoxical, which may explain why previous researchers had reported inconsistent results of IL-6 effects. The target molecules revealed in the mechanistic studies could be further utilized for developing future therapeutic approaches to target CSCs of NSCLC. Citation Format: Soo Ok Lee, Ying Tsai, Laura Stronjy, Jong-Wei Hsu, Shan-Zhou Duan, Peter Keng, Yuhchyau Chen. Paradoxical roles of IL-6 in modulating growth of stem vs. non-stem cells of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-57. doi:10.1158/1538-7445.AM2014-LB-57

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway. We show that inhibition of the COX-2/PGE₂ pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.

Abstract Background: Lung cancer is the leading cause of cancer-related deaths among both men and women. Long-term survival of lung cancer patients remains low despite aggressive therapies including surgery, radiation, and chemotherapy. Ionizing radiation (IR) is the standard therapy for patients diagnosed with localized unresectable non-small cell lung cancer (NSCLC). However local tumor control by standard fractionated radiotherapy (RT) remains poor due to tumor resistance to IR. Accumulating evidences have indicated that cancer stem cells (CSCs, also known as cancer initiating cells) exist as a very minor population in NSCLC tumors, which are composed of heterogeneous cell populations. Our laboratory and others found that CSC population increases upon irradiation, which may contribute to radiation resistance. The role of IL-6 in NSCLC progression has been suggested since high IL-6 levels were detected in sera of lung cancer patients, but not detectable in most patients with benign lung diseases. However, the exact mechanism by which IL-6 influences lung cancer progression and its role in CSCs mediated radioresistance remain unresolved. Methods: We isolated CD133+ CSC population from 3 NSCLC cell lines (NSCLC-CSCs) and studied the role of IL-6 in CD133+ CSCs growth by treating cells with IL-6. In addition, we manipulated IL-6 expression levels in the IL-6 expressing A549 and H157 cells by the knockdown of IL-6 using lentiviral infection system carrying IL-6siRNA and isolated CD133+ cells for further analyses. The growth and stem cell marker expressions of these CSCs, IL-6 treated or non-treated, or IL-6 expressing or knocked down cells upon irradiation were then investigated. The differences in the activation of signaling pathways and neuroendocrine differentiation were also studied. Results and Conclusions: We found that IL-6 increased self-renewal of NSCLC-CSCs significantly and thereby increased NSCLC-CSC population. We also found that IL-6 increased stemness of the isolated CD133+ CSCs when CD133+ cells were treated with IL-6. More importantly, when we irradiated the CD133+ CSCs isolated from the IL-6 knocked down and scramble control A549 cells, we found that the CSCs isolated from the IL-6 knocked down cells exhibited lower survival than the CSCs of control cells. Therefore, we suggest that IL-6 may contribute to NSCLC-CSCs mediated-radioresistance via (i) direct triggering radioresistance, and (ii) increasing self-renewal and stemness of the survived CSCs. Mechanistic dissection studies revealed that the increased self-renewal of CSCs by IL-6 is due to the activation of the hedgehog signaling and the increased expression of bcl-2, while the IL-6 mediated increase of radioresistance is due to the activation of neuroendocrine differentiation, Stat 3 signaling pathway, and increased expression of bcl-2. Citation Format: Yuhchyau Chen, Ying Tsai, Jong-Wei Hsu, Shan-Zhou Duan, Peter Keng, Soo Ok Lee. IL-6 promotes stemness of cancer stem cells and may contribute to radiation resistance of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-210. doi:10.1158/1538-7445.AM2014-LB-210

The underglycosylated form of the MUC1 glycoprotein, uMUC1, has been identified as a ligand for both E-selectin and ICAM-1 and can play multiple potential roles during rolling and firm adhesion events in the metastatic cascade. Using flow cytometry and confocal microscopy, the T47D and ZR-75-1 cell lines were verified to highly express uMUC1, however it was found that only ZR-75-1 cells expressed the E-selectin binding moiety sialyl Lewis x (sLe x ). Furthermore, perfusing T47D cells through E-selectin coated microtubes resulted in fast rolling velocities and low numbers of interacting cells and blocking uMUC1 with the SM3 antibody had no effect. ZR-75-1 cells, on the other hand, were highly dependent on the E-selectin:uMUC1 interaction as exemplified by significant increases in cell rolling velocities and decreases in the number of interacting cells when blocking with SM3 or when uMUC1 expression was knocked down via siRNA transfection. Whereas uMUC1 interactions with E-selectin supported cell rolling, P-selectin: uMUC1 interactions exclusively facilitated cell tethering, while L-selectin surfaces supported no cell adhesive interactions. These experimental observations are consistent with molecular dynamics simulations of uMUC1 bound to E-, P-, and L-selectin where the degree of residue contact correlated with the differential adhesion of uMUC1 to each selectin. Finally, an E-selectin and SM3 combined surface coating captured approximately 30% of the total number of interacting cancer cells comparable to the number of adhered cells when utilizing E-selectin and ICAM-1 combined surfaces. The E-selectin/SM3 surface strategy offers a viable method to selectively capture cancer cells from whole blood samples.

Much fewer mice Lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited Less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 Led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1 alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.

UDP‐glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real‐time reverse transcription‐polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC‐AR reduced mRNA expression of all the UGT1A subtypes (19–75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC‐Vector. Of note were higher expression levels of UGT1As in SVHUC‐Vector than in SVHUC‐AR. In ARKO mice, all the Ugt1a subtypes were up‐regulated, compared to wild‐type littermates. In wild‐type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild‐type female mice had higher levels of Ugt1a than wild‐type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen‐mediated AR signals promote bladder carcinogenesis by down‐regulating the expression of UGTs in the bladder. © 2011 Wiley Periodicals, Inc.

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLex expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of ‘triple-negative’ breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012891 VITAMIN D AND BCG IMMUNOTHERAPY Jong-Wei Hsu, Edward Messing, and Yi-Fen Lee Jong-Wei HsuJong-Wei Hsu Rochester, NY More articles by this author , Edward MessingEdward Messing Rochester, NY More articles by this author , and Yi-Fen LeeYi-Fen Lee Rochester, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.986AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis (TB), has been used and proved to be one of the most effective treatments for superficial bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, which limits the improvement of BCG therapy. Vitamin D deficiency has been associated with the high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy for BCa. METHODS We applied a transwell migration assay to examine vitamin D signal¡ߣs impact on the BCG-mediated innate immune cells¡ߣ migration. The corresponding pro-inflammatory cytokines induced by vitamin D/vitamin D receptor were screened by cytokine array and confirmed by ELISA. Preclinical evaluation of efficacy of intravesical BCG with 1£\,25-dihydroxyvitamin D3 (1,25-VD) therapy was performed using Nbutyl- N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model. RESULTS ELISA assay revealed that treatment of BCa cells with 1,25-VD promotes BCG-induced interleukin 8 (IL-8) production, consequently inducing the migration of neutrophil-like cells, HL-60, and monocyte-like cells, THP-1. This 1,25-VD-mediated IL-8-induced innate immune cells migration was blocked by IL-8 neutralized antibody. These in vitro studies suggest that 1,25-VD could improve BCG responsiveness by enhancing recruitment of innate immune cells to promote cytotoxic killing of BCa cells. More importantly, our in vivo data revealed that intravesical treatment of 1,25-VD improves BCG efficacy where 1,25-VD+BCG treatment increased the survival rate of mice with BCa induced by BBN compared to BCG treatment alone. CONCLUSIONS The results of these studies indicate that 1,25-VD signaling is critical for success of BCG immunotherapy and co-treatment with 1,25-VD will enhance BCG's anti-tumor effects in BCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e362-e363 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jong-Wei Hsu Rochester, NY More articles by this author Edward Messing Rochester, NY More articles by this author Yi-Fen Lee Rochester, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effective therapies can prevent superficial bladder cancer from developing into muscle-invasive stage or more severe stages which require radical cystectomy and negatively affect life quality. In terms of therapeutic approaches against superficial bladder cancer, intravesical (regional) therapy has several advantages over oral (systemic) therapy. Though urologists can directly deliver drugs to bladder lesions by intravesical instillation after transurethral resection, the efficacy of conventional drug delivery is usually low due to the bladder permeability barrier and bladder periodical discharge. Nanoparticles have been well developed as pharmaceutical carriers. By their versatile properties, nanoparticles can greatly improve the interactions between urothelium and drugs and also enhance the penetration of drugs into urothelium with lesions, which dramatically improves therapeutic efficacy. In this review, we discuss the advances of nanotechnology in bladder cancer therapy by different types of nanoparticles with different encapsulating materials.