Mouse Model Of Bladder Cancer Research Articles

MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER Ao Zhang, Hong Qiu, Timothy Chan, and Byron Lee Ao ZhangAo Zhang More articles by this author , Hong QiuHong Qiu More articles by this author , Timothy ChanTimothy Chan More articles by this author , and Byron LeeByron Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 6A (KDM6A) is a chromatin modifier that is frequently mutated in bladder cancer and causes epigenetic dysregulation. Recently immune check point inhibition has shown great clinical promise in bladder cancer. Because epigenetic signature has been shown to predict response to immunotherapy, here we assessed if loss of KDM6A leads to alterations of tumor immune microenvironment in a mouse bladder cancer model. METHODS: Kdm6a urothelium conditional knockout (cKO) mice were generated by crossing Kdm6aflox/flox mice with Upk3aCre mice that express Cre recombinase in the urothelium. Littermate control mice were of the same genetic background but intact Kdm6a gene. Mice at 8 weeks of age (three in each group) were fed with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Bladders were harvested after 4 weeks and analyzed with H&E staining. Single cell RNA sequencing was performed on urothelium from control and Kdm6A cKO mice without BBN treatment. RESULTS: After treatment with BBN for 4 weeks, there was marked edema and inflammatory changes with increased infiltration of immune cells in H&E stained bladder sections in Kdm6a cKO mice compared to Kdm6a intact controls (Figure A). While urothelium from control mice maintained grossly unchanged tissue architecture, urothelium from cKO mice exhibited cytologic atypia with loss of organized cell layer. Comparison of single cell RNA sequencing profile revealed enrichment of immune cell population in cKO mice (Figure B, cluster 12), which was consistent with histological findings. CONCLUSIONS: Loss of KDM6A in the urothelium creates a distinct immune microenvironment in the BBN mouse bladder cancer model. Whether this immune cell-rich microenvironment affects response to immune check point inhibition warrants further investigation. Source of Funding: This work was supported in part by the 2021 Urology Care Foundation Residency Research Award Program and the Kahlert Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e80 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ao Zhang More articles by this author Hong Qiu More articles by this author Timothy Chan More articles by this author Byron Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...

MP06-19 ANTI-TUMOR EFFECT IN BLADDER CANCER MODEL OF TUMOR VASCULATURE-TARGETED 10 B DELIVERY MEDIATED BORON NEUTRON CAPTURE THERAPY

You have accessJournal of UrologyCME1 May 2022MP06-19 ANTI-TUMOR EFFECT IN BLADDER CANCER MODEL OF TUMOR VASCULATURE-TARGETED 10B DELIVERY MEDIATED BORON NEUTRON CAPTURE THERAPY Tohru Yoneyama, Shingo Hatakeyama, Mihoko Sutoh Yoneyama, Taku Yoshiya, Tsuyoshi Uemura, Takehiro Ishizu, Minoru Suzuki, Shingo Hachinohe, Shintaro Ishiyama, Motohiro Nonaka, Michiko N. Fukuda, and Chikara Ohyama Tohru YoneyamaTohru Yoneyama More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Mihoko Sutoh YoneyamaMihoko Sutoh Yoneyama More articles by this author , Taku YoshiyaTaku Yoshiya More articles by this author , Tsuyoshi UemuraTsuyoshi Uemura More articles by this author , Takehiro IshizuTakehiro Ishizu More articles by this author , Minoru SuzukiMinoru Suzuki More articles by this author , Shingo HachinoheShingo Hachinohe More articles by this author , Shintaro IshiyamaShintaro Ishiyama More articles by this author , Motohiro NonakaMotohiro Nonaka More articles by this author , Michiko N. FukudaMichiko N. Fukuda More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: p-Boronophenylalanine (10BPA) is a powerful 10B drug used in current clinical trials of BNCT. For BNCT to be successful, a high (500 mg/kg) dose of 10BPA must be administered over a few hours. Here, we report BNCT efficacy after rapid, ultralow-dose administration of either tumor vasculature-specific annexin A1-targeting IFLLWQR (IF7)-conjugated 10BPA or borocaptate sodium (10BSH). METHODS: IF7 conjugates of either 10B drugs intravenously injected into MBT2 bladder tumor-bearing mice and biodistribution of 10B in tumors and normal organs analyzed by prompt gamma-ray analysis. Therapeutic effect of IF7-10B drug-mediated BNCT was assessed by either MBT2 bladder tumor bearing C3H/He mice and YTS-1 tumor bearing nude mice. RESULTS: Intravenous injection of IF7C conjugates of either 10B drugs into MBT2 bladder tumor-bearing mice promoted rapid 10B accumulation in tumor tissues and suppressed tumor growth. Moreover, multiple treatments at ultralow (10–20 mg/kg) doses of IF7-10B drug-mediated BNCT significantly suppressed tumor growth in a mouse model of human YTS-1 bladder cancer, with increased Anxa1 expression in tumors and infiltration by CD8-positive lymphocytes. Our findings suggest BNCT treatment induces an immune response by the host against tumor cells, triggering a strong cytotoxic reaction and also inflammatory and immune responses that increase after BNCT upregulate Anxa1 expression in tumor tissues. Moreover, we hypothesize that if the first BNCT treatment upregulates Anxa1 in tumor tissues due to an inflammatory response or induced immunogenicity, more effective 10B accumulation in tumor tissues might occur following the second injection of IF7-10B drugs. Thus, multiple ultralow doses of IF7-10B drug-mediated BNCT likely boost BNCT therapeutic potential. CONCLUSIONS: We conclude that IF7 serves as an efficient 10B delivery vehicle by targeting bladder tumor tissues via the tumor vasculature and could serve as a relevant vehicle for BNCT drugs. Source of Funding: This study was supported by a Center of Innovation program grant for the young scientist collaborative research fund project (no. R02W16) from the Japan Science and Technology Agency and supported by the Japan Society for the Promotion of Science KAKENHI (grants nos. 17K11119, 20K09517, and 19H05556). The study was also supported by the Kobayashi Foundation for Cancer Research and by the Aomori Prefecture Quantum Science Center © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e84 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tohru Yoneyama More articles by this author Shingo Hatakeyama More articles by this author Mihoko Sutoh Yoneyama More articles by this author Taku Yoshiya More articles by this author Tsuyoshi Uemura More articles by this author Takehiro Ishizu More articles by this author Minoru Suzuki More articles by this author Shingo Hachinohe More articles by this author Shintaro Ishiyama More articles by this author Motohiro Nonaka More articles by this author Michiko N. Fukuda More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement PDF DownloadLoading ...

MP06-04 EXPLORING A NOVEL SYNERGISTIC INTRAVESICAL ONCOLYTIC VIROTHERAPY FOR TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-04 EXPLORING A NOVEL SYNERGISTIC INTRAVESICAL ONCOLYTIC VIROTHERAPY FOR TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER Woodson Smelser, Jian Wang, Sam Chang, and Austin Kirschner Woodson SmelserWoodson Smelser More articles by this author , Jian WangJian Wang More articles by this author , Sam ChangSam Chang More articles by this author , and Austin KirschnerAustin Kirschner More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: For BCG refractory NMIBC, systemic anti-PD-1 immune checkpoint inhibition is FDA approved as second line therapy. Prior published work by our group demonstrated comparable efficacy and overall survival with intravesical anti-PD-1 treatments in NMIBC in mice. Additionally, oncolytic viral therapy with reovirus has shown pre-clinical efficacy in the bladder & other cancers. We hypothesized that intravesical instilment with both anti-PD-1 inhibitor & oncolytic reovirus would demonstrate a greater synergistic effect than either treatment as monotherapy. METHODS: We investigated an orthotopic mouse model of urothelial bladder cancer using MBT2 cells implanted into the bladders of (n=40) syngeneic, wild-type C3H mice. Groups of 10 mice were treated 14 days after engraftment at the point of palpable tumor for comparison between no treatment, intravesical anti-PD-1, intravesical oncolytic reovirus, & intravesical oncolytic reovirus + anti-PD1. The primary outcome was overall survival; secondary outcomes include long-term immunity & immune milieu changes indicated by mass cytometric evaluation. RESULTS: 40 total mice were engrafted with tumor & then treated. At time of submission, all 10 mice receiving no treatment had died, with median survival of 41 days. Median overall survival for the mice receiving reovirus, anti-PD-1 or anti-PD-1 + reovirus had not been reached. Mice receiving reovirus experienced longer survival than control (HR 14.4; 95% CI 3.9-32.6, p<0.0001), as did mice receiving anti-PD-1 alone (HR 28.4; 95% CI 7.0-115.9, p<0.0001). When compared to control alone, combination therapy with anti-PD-1 & reovirus also demonstrated superior survival (HR 28.4; 95% CI 7.0-115.9, p<0.0001). Comparison between anti-PD-1 and reovirus demonstrated no significant differences in survival (p=0.15). See Figure 1 for Kaplan-Meier analysis comparing survival. CONCLUSIONS: Treatment of mice engrafted with MBT2 orthotopic bladder tumors with intravesical therapy with reovirus, anti-PD-1 antibody, or combination anti-PD-1 antibody & reovirus confer superior survival to intravesical controls. Immune profile analysis examining tumor microenvironment differences in immune response are ongoing with mass cytometry and will further bolster our mechanistic understanding of each therapy. Source of Funding: T32 Grant (WS) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e78 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Woodson Smelser More articles by this author Jian Wang More articles by this author Sam Chang More articles by this author Austin Kirschner More articles by this author Expand All Advertisement PDF DownloadLoading ...

A Novel RGD-4C-Saporin Conjugate Inhibits Tumor Growth in Mouse Models of Bladder Cancer.

Although toxin may have some advantages compared to chemotherapeutic drugs in cancer therapy, e.g. a potent cytotoxic activity and a reduced risk of resistance, their successful application in the treatments to solid tumors still remains to be fully demonstrated. In this study, we genetically modified the structure of the plant-derived single-chain ribosome inactivating protein saporin (SAP) by fusing its N-terminus to the ACDCRGDCFCG peptide (RGD-4C), an αv-integrin ligand, and explored the anti-tumor activity of the resulting protein (called RGD-SAP) in vitro and in vivo, using a model of muscle invasive bladder cancer. We found that the RGD-4C targeting domain enhances the cytotoxic activity of SAP against various tumor cell lines, in a manner dependent on αv-integrin expression levels. In a subcutaneous syngeneic model of bladder cancer, RGD-SAP significantly reduced tumor growth in a dose-dependent manner. Furthermore, systemic administration of RGD-SAP in combination with mitomycin C, a chemotherapeutic drug currently used to treat patients with bladder cancer, increased the survival of mice bearing orthotopic bladder cancer with no evidence of systemic toxicity. Overall, the results suggest that RGD-SAP represents an efficient drug that could be exploited, either alone or in combination with the state-of-the-art therapies, for the treatment of bladder cancer and, potentially, of other solid tumors.

Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors

Chemoimmunotherapy has recently failed to demonstrate significant clinical benefit in advanced bladder cancer patients; and the mechanism(s) underlying such suboptimal response remain elusive. To date, most studies have focused on tumor-intrinsic properties that render them “immune-excluded”. Here, we explore an alternative, drug-induced mechanism that impedes therapeutic response via disrupting the onset of immunogenic cell death. Using two immune-excluded syngeneic mouse models of muscle-invasive bladder cancer (MIBC), we show that platinum-based chemotherapy diminishes CD8+ T cell tumor infiltration and constraines their antitumoral activity, despite expression of activation markers IFNγ and granzyme B. Mechanistically, chemotherapy induces the release of prostaglandin E2 (PGE2) from dying cancer cells, which is an inhibitory damage-associated molecular pattern (iDAMP) that hinderes dendritic cell maturation. Upon pharmaceutical blockade of PGE2 release, CD8+ T cells become tumoricidal and display an intraepithelial-infiltrating (or inflamed) pattern. This “iDAMP blockade” approach synergizes with chemotherapy and sensitizes bladder tumors towards anti-PD1 immune checkpoint inhibitor therapy. These findings provide a compelling rationale to evaluate this drug combination in future clinical trials.

Blockade of integrin signaling reduces chemotherapy-induced premature senescence in collagen cultured bladder cancer cells.

BackgroundDiminished sensitivity towards chemotherapy remains the major impediment to the clinical treatment of bladder cancer. However, the critical elements in control of chemotherapy resistance remain obscure.MethodsWe adopted improved collagen gels and performed cytotoxicity analysis of doxorubicin (DOX) and mitomycin C (MMC) of bladder cancer cells in a 3D culture system. We then detected the expression of multidrug resistant gene ABCB1, dormancy-associated functional protein chicken ovalbumin upstream-transcription factor 1 (COUPTF1), cell proliferation marker Ki-67, and cellular senescence marker senescence-associated β-galactosidase (SA-β-Gal) in these cells. We further tested the effects of integrin blockade or protein kinase B (AKT) inhibitor on the senescent state of bladder cancer. Also, we examined the tumor growth and survival time of bladder cancer mouse models given the combination treatment of chemotherapeutic agents and integrin α2β1 ligand peptide TFA (TFA).ResultsCollagen gels played a repressive role in bladder cancer cell apoptosis induced by DOX and MMC. In mechanism, collagen activated the integrin β1/AKT cascade to drive bladder cancer cells into a premature senescence state via the p21/p53 pathway, thus attenuating chemotherapy-induced apoptosis. In addition, TFA had the ability to mediate the switch from senescence to apoptosis of bladder cancer cells in xenograft mice. Meanwhile, TFA combined with chemotherapeutic drugs produced a substantial suppression of tumor growth as well as an extension of survival time in vivo.ConclusionsBased on our finding that integrin β1/AKT acted primarily to impart premature senescence to bladder cancer cells cultured in collagen gel, we suggest that integrin β1 might be a feasible target for bladder cancer eradication.

FimH confers mannose-targeting ability to Bacillus Calmette-Guerin for improved immunotherapy in bladder cancer

BackgroundBladder cancer is a common disease worldwide with most patients presenting with the non-muscle-invasive form (NMIBC) at initial diagnosis. Postoperational intravesical instillation of BCG is carried out for patients with...

The combination therapy of Everolimus and anti-PD-1 improves the antitumor effect by regulating CD8+ T cells in bladder cancer.

This study aimed to investigate the efficacy of Everolimus (EVE) in combination with immune checkpoint inhibitors (ICIs) in bladder cancer treatment and the underlying mechanisms. In vitro, MB49 cells were exposed to gradient concentrations (0nM-100nM) of EVE for 48h, to investigate the cell viability and cell proliferative potential. In vivo, we applied a subcutaneous tumor mouse model of bladder cancer and the mice were treated with EVE monotherapy (different doses) or in combination with anti-programmed cell death protein 1 (PD-1) agents to study the impacts on tumor growth and explore the immune mechanism. The influences of treatments on peripheral immune profiles and tumor immune microenvironment were also discussed. EVE could inhibit the growth of MB49 cells in vitro. Though high-dose EVE monotherapy could induce tumor regression in vivo, it also contributed to immunosuppression. High-dose EVE inhibited the expression of PD-L1 by inhibiting Th1 cytokine secretion, while combined therapy with PD-1 inhibitors showed no extra profit. Low-dose EVE in combination with PD-1 inhibitors could effectively suppress tumor growth by increasing periphery CD8+ T cell frequency and GZMB+ CD8+ T cell frequency in the tumor microenvironment. High-dose EVE monotherapy induced tumor regression, but with immunosuppression to some content. Combination therapy with low-dose EVE and PD-1 inhibitor could effectively inhibit the growth of bladder tumors by enhancing the antitumor immunity of CD8+ T cells in both periphery and tumor microenvironment.

Exosome-Mediated miR-4792 Transfer Promotes Bladder Cancer Cell Proliferation via Enhanced FOXC1/c-Myc Signaling and Warburg Effect.

Bladder cancer is the second-most common malignancy in the urogenital system and the most common in men. However, our understanding of the driving mechanisms of bladder cancer remains incomplete. The forkhead box (FOX) family of transcription factors is implicated in urogenital development and bladder malignancies. Many exosomal microRNAs have been identified as regulators and mediators of the expression of FOX, including the expression of FOXC1. miR-4792 has been known as a tumor miRNA suppressor. However, the function of miR-4792/FOXC1 signaling in bladder cancer development remains unknown. Here, we studied the role of miR-4792/FOXC1 signaling in bladder cancer by using multiple bladder cancer cell lines and bladder cancer mouse models through in vitro and in vivo approaches. We showed that FOXC1 is highly expressed in multiple bladder cancer cell lines and bladder tumor tissues. The knockdown of FOXC1 expression in bladder cancer cell lines decreases c-Myc expression levels, retards cell growth, and reduces aerobic glycolysis (also known as the Warburg effect) and lactic acid content. By contrast, the overexpression of FOXC1 elicits the opposite effects. FOXC1-downregulated bladder cancer cells form significantly smaller tumors in vivo. The inhibition of c-Myc reverses the effects of FOXC1 overexpression and leads to reduced cell proliferation, aerobic glycolysis, and lactic acid content. miR-4792 expression is downregulated in bladder tumor tissues. miR-4792 exposure to bladder cancer cells reduces the expression levels of FOXC1 and c-Myc, slows down cell growth, and decreases aerobic glycolysis and lactic acid content. However, the enhanced miR-4792 expression elicits opposite effects. These findings provided the first evidence that the exosome-mediated delivery of miR-4792 could play an important role in bladder cancer development through the downregulation of FOXC1 and c-Myc, which further inhibited aerobic glycolysis and lactic acid content.

Abstract P008: Effect of route of Bacillus Calmette Guérin administration on the tumor immune microenvironment in a mouse model of non-muscle invasive bladder cancer

Abstract Background: Bladder cancer is the fifth most common cancer in North America, with up to 80% of cases being non-muscle invasive bladder cancer (NMIBC). The standard of care for high-risk NMIBC involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG), a live attenuated bacterium. Unfortunately, most patients do not respond fully to this therapy, resulting in recurrences that sometimes progress to invasive disease. Moreover, our understanding of how BCG exerts its immunotherapeutic effect is incomplete. Using a mouse model of NMIBC, we compared the bladder tumor immune microenvironment (TiME) following intravesical versus intravenous (IV) administration of BCG. Methods: Female C57Bl/6 mice (6-8 weeks old) were catheterized and 2.5 × 105 MB49 bladder cancer cells were instilled into the bladders after poly-L-lysine treatment. On day 7 post cancer cell instillation, mice with equal tumor volumes were randomized to receive three weekly intravesical or IV administrations of saline or BCG (2 mg/50 μl). Similarly, following catheterization, 50 μl of poly-L-lysine was administered into the bladders of control non-tumor bearing mice, which was then followed by three weekly intravesical instillations of BCG (2 mg/50 μl). In both cohorts, mice were sacrificed on day 23 and bladders were harvested and enzymatically dispersed to generate single-cell suspensions for analysis by polychromatic flow cytometry. Results: Compared with intravesical BCG treatment, the TiME of mice treated with BCG intravenously was associated with a significantly higher proportion of CD11b− CD3+, CD3−, and CD4− lymphoid cells, as well as a significantly lower proportion of immature myeloid cells. Furthermore, compared with saline IV treatment, BCG administered IV resulted in a significantly larger CD4− T cell population. There were no significant differences in the TiME of intravesically BCG-treated vs intravesically saline-treated tumor-bearing mice. However, compared with control non-tumor-bearing mice treated with saline intravesically, bladders of non-tumor-bearing mice treated intravesically with BCG had a significantly higher proportion of leukocytes, which were predominantly immature myeloid cells. Conclusion/Significance: These results provide evidence that the route of BCG administration is an important determinant of the composition of the TiME in bladder cancer. Understanding the link between the TiME and BCG therapy may facilitate the development of new approaches to improve outcomes and reduce recurrence rates in patients with NMIBC. This knowledge may also help optimize BCG treatment regimen to avoid unnecessary therapy and induce an optimal anti-tumor immune response. Citation Format: Aline Atallah, Arielle Grossman, William Tran, Jean-Francois Paré, Tiziana Cotechini, Charles H. Graham. Effect of route of Bacillus Calmette Guérin administration on the tumor immune microenvironment in a mouse model of non-muscle invasive bladder cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P008.

Immunotherapeutic effects of recombinant Bacillus Calmette-Guérin containing sic gene in ex vivo and in vivo bladder cancer models.

PurposeThe recombinant Bacillus Calmette–Guérin (BCG) containing the streptococcal inhibitor of the complement gene (rBCG-sic) may be more resistant to antimicrobial peptides and improve internalization; therefore, it can enhance the immunotherapeutic effect of the BCG. Here we determined the optimal dose of rBCG-sic and compared its effectiveness with that of BCG.Materials and MethodsWe fabricated a high-throughput 3D-bioprinted bladder cancer-on-a-chip (BCOC) and used it to evaluate the effectiveness of the rBCG-sic in terms of cell viability, cell migration, and cytokine concentrations. Using an orthotopic mouse model, we evaluated its anticancer effect and toxicity via bioluminescence imaging.ResultsT24 cell viability was decreased after treatment with rBCG-sic 30 multiplicities of infection (MOI) versus the same dosage of mock BCG (42.8%±6.4% vs. 75.7%±6.6%, p<0.05). THP-1 cell migration was positively correlated with rBCG-sic concentration (2.42-fold at 30MOI, p<0.01). The interleukin-6 concentration of rBCG-sic 30MOI was significantly higher than that of mock BCG 30MOI (11.2±1.3 pg/mL vs. 6.7±0.6 pg/mL, p<0.05). In the orthotopic bladder cancer mouse model, lower tumor volume was observed in the rBCG-sic 30MOI group than in the BCG 30MOI group after 10 days of treatment (p<0.05).ConclusionsWe concluded that rBCG-sic is a useful tool for overcoming BCG unresponsiveness in non-muscle invasive bladder cancer. Additionally, high-throughput BCOC with a microfluidic system can successfully reflect the bladder cancer microenvironment.

Animal Models in Bladder Cancer.

Background: Bladder cancer (urothelial cancer of the bladder) is the most common malignancy affecting the urinary system with an increasing incidence and mortality. Mouse models of bladder cancer should possess a high value of reproducibility, predictability, and translatability to allow mechanistic, chemo-preventive, and therapeutic studies that can be furthered into human clinical trials. Objectives: To provide an overview and resources on the origin, molecular and pathological characteristics of commonly used animal models in bladder cancer. Methods: A PubMed and Web of Science search was performed for relevant articles published between 1980 and 2021 using words such as: “bladder” and/or “urothelial carcinoma” and animal models. Animal models of bladder cancer can be categorized as autochthonous (spontaneous) and non-autochthonous (transplantable). The first are either chemically induced models or genetically engineered models. The transplantable models can be further subclassified as syngeneic (murine bladder cancer cells implanted into immunocompetent or transgenic mice) and xenografts (human bladder cancer cells implanted into immune-deficient mice). These models can be further divided—based on the site of the tumor—as orthotopic (tumor growth occurs within the bladder) and heterotopic (tumor growth occurs outside of the bladder).

Intravesical Pseudomonas aeruginosa mannose-sensitive Hemagglutinin vaccine triggers a tumor-preventing immune environment in an orthotopic mouse bladder cancer model.

Bacillus Calmette-Guerin (BCG) immunotherapy can prevent recurrence and progression in selected patients with non-muscle-invasive bladder cancer (NMIBC); however, significant adverse events and treatment failure suggest the need for alternative agents. A commercial anti-infection vaccine comprises a genetically engineered heat-killed Pseudomonas aeruginosa (PA) expressing many mannose-sensitive hemagglutination (MSHA) fimbriae, termed PA-MSHA, which could be a candidate for bladder cancer intravesical therapy. In an immunocompetent orthotopic MB49 bladder cancer model, we characterized the antitumor effects and mechanisms of PA-MSHA compared with those of BCG. Three weekly intravesical PA-MSHA or BCG treatments reduced tumor involvement; however, only PA-MSHA prolonged survival against MB49 implantation significantly. In non-tumor-bearing mice after treatment, flow-cytometry analysis showed PA-MSHA and BCG induced an increased CD4/CD8 ratio, the levels of effector memory T cell phenotypes (CD44, CXCR-3, and IFN-γ), and the proportion of CD11b+Ly6G-Ly6C-IA/IE+ mature macrophages, but a decrease in the proportion of CD11b+Ly6G-Ly6C+IA/IE- monocytic myeloid-derived suppressor cells (Mo-MDSCs) and the expression of suppressive molecules on immune cells (PD-L1, PD-1, TIM-3, and LAG-3). Notably, PA-MSHA, but not BCG, significantly reduced PD-1 and TIM-3 expression on CD4+ T cells, which might account for the better effects of PA-MSHA than BCG. However, in tumor-bearing mice after treatment, the increased proportion of Mo-MDSCs and high expression of PD-L1 might be involved in treatment failure. Thus, modulating the balance among adaptive and innate immune responses was identified as a key process underlying PA-MSHA-mediated treatment efficacy. The results demonstrated mechanisms underlying intravesical PA-MSHA therapy, pointing at its potential as an alternative effective treatment for NMIBC.

Immunotherapy Strategy Targeting Programmed Cell Death Ligand 1 and CD73 with Macrophage-Derived Mimetic Nanovesicles to Treat Bladder Cancer.

Combination immunotherapy is a promising strategy to remove the inhibitory effect of the tumor microenvironment on immune effector cells, improving the efficacy of immune checkpoint inhibitor treatment in bladder cancer. However, it is challenging to deliver multiple drugs to the tumor tissue effectively and simultaneously to ensure optimal therapeutic effects. Macrophage-derived exosome-mimetic nanovesicles (EMVs) were designed and validated as a nanoplatform for coloading and delivery of the CD73 inhibitor (AB680) and the monoclonal antibody to programmed cell death ligand 1 (aPDL1). The tumor-targeting, biosafety, and therapeutic effects of these nanocomplexes (AB680@EMVs-aPDL1), as a combined immunotherapy strategy for bladder cancer, were assessed in vitro and in vivo. Our results indicate that the nanodrug system was highly stable, provided adequate biosafety, and enhanced tumor targeting in a mouse model of bladder cancer. Moreover, the CD73 inhibitor reduced extracellular adenosine production, and the combination therapy significantly promoted the activation and infiltration of cytotoxic T-lymphocytes, which helped to optimally suppress tumor growth and extend median survival in vivo. Therefore, using EMVs to deliver a combination of aPDL1 and the CD73 inhibitor may be a useful combined immunotherapy strategy for treating bladder cancer.

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.

PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (PD42)1 Sep 2021PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL Jae Hun Shim, Joonhee Gook, Se Young Choi, Byung Hoon Chi, Jin Wook Kim, Tae-Hyoung Kim, Soon Chul Myung, and In Ho Chang Jae Hun ShimJae Hun Shim More articles by this author , Joonhee GookJoonhee Gook More articles by this author , Se Young ChoiSe Young Choi More articles by this author , Byung Hoon ChiByung Hoon Chi More articles by this author , Jin Wook KimJin Wook Kim More articles by this author , Tae-Hyoung KimTae-Hyoung Kim More articles by this author , Soon Chul MyungSoon Chul Myung More articles by this author , and In Ho ChangIn Ho Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002056.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is the most widely used bladder cancer immunotherapy, innate immune responses involving antimicrobial peptides (AMPs) cause BCG failure. Here, we developed genetically modified recombinant BCG (rBCG) strains which escape AMPs and evaluate the efficacy and effects of rBCG. METHODS: We constructed rBCG strains expressing Streptococcal inhibitor of complement (Sic), which confers resistance to human α-defensin-1 and cathelicidin, and d-alanyl carrier protein ligase (dltA), which confers resistance to cationic AMPs. Sic and dltA were separately cloned into the pMV306 plasmid and introduced into BCG via electroporation. The efficacy of the Sic and dltA gene electroporation into BCG was evaluated by quantitative real-time polymerase chane reaction (qRT-PCR). The internalization rates and anti-cancer effects of the rBCG strains containing Sic (rBCG-Sic) and dltA (rBCG-dltA) was evaluated by the orthotopic bladder cancer mouse model. RESULTS: The cycle quantification (Cq) values of rBCG-Sic (y=-4.8823x+13.645, R2=0.9996) and rBCG-dltA (y=-5.438x+11.641, R2=0.9995) were inverse correlations to the amount of Sic and dltA genes dose dependently. The mean introduction proportions of Sic and dltA genes into BCG by electroporation were 22.2%, 27.5% and showed constant efficacy. In the orthotopic bladder cancer mouse model, the relative internalization number of rBCG-Sic, and rBCG-dltA into bladder cell in mouse bladder were higher than that of BCG and the tumor volume at rBCG-Sic were lower than at BCG and rBCG-dltA at 11, 14 m and 18 days. CONCLUSIONS: Our results showed that constructed rBCG-Sic and rBCG-dltA by electroporation and the rBCG-Sic and rBCG-dltA can effectively escape BCG-stimulated AMPs, and significantly improved immunotherapeutic tools to treat bladder cancer in orthotopic bladder cancer mouse model. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e724-e724 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jae Hun Shim More articles by this author Joonhee Gook More articles by this author Se Young Choi More articles by this author Byung Hoon Chi More articles by this author Jin Wook Kim More articles by this author Tae-Hyoung Kim More articles by this author Soon Chul Myung More articles by this author In Ho Chang More articles by this author Expand All Advertisement Loading ...

An oral cancer vaccine using a Bifidobacterium vector suppresses tumor growth in a syngeneic mouse bladder cancer model

Cancer immunotherapy using immune-checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors has been well established for various types of cancer. Monotherapy with ICIs, however, can achieve a durable response in only a subset of patients. There is a great unmet need for the ICI-resistant-tumors. Since patients who respond to ICIs should have preexisting antitumor T cell response, combining ICIs with cancer vaccines that forcibly induce an antitumor T cell response is a reasonable strategy. However, the preferred administration sequence of the combination of ICIs and cancer vaccines is unknown. In this study, we demonstrated that combining an oral WT1 cancer vaccine using a Bifidobacterium vector and following anti-PD-1 antibody treatment eliminated tumor growth in a syngeneic mouse model of bladder cancer. This vaccine induced T cell responses specific to multiple WT1 epitopes through the gut immune system. Moreover, in a tumor model poorly responsive to an initial anti-PD-1 antibody, this vaccine alone significantly inhibited the tumor growth, whereas combination with continuous anti-PD-1 antibody could not inhibit the tumor growth. These results suggest that this oral cancer vaccine alone or as an adjunct to anti-PD-1 antibody could provide a novel treatment option for patients with advanced urothelial cancer including bladder cancer.

Entinostat induces antitumor immune responses through immune editing of tumor neoantigens

Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

LncRNA HAND2-AS1 exerts anti-oncogenic effects on bladder cancer via restoration of RARB as a sponge of microRNA-146

BackgroundGrowing evidence has shown that long noncoding RNA: microRNA: mRNA is implicated in tumor initiation, development, and progression. Long noncoding RNA HAND2-AS1 exhibits anti-cancer effects in diverse cancers. However, the knowledge of HAND-AS1 in bladder cancer development remains unknown.MethodsLncRNA and miRNA microarray was conducted to explore different expressed RNA in primary bladder cancer specimens. RNA-RNA interaction prediction tools miRcode (http://www.mircode.org/), DIANA-lncBase v2 (https://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-experimental), DIANA-TarBase v.8 (https://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8%2Findex) and miRDB (http://www.mirdb.org/) were employed to predict the interactions between RNA. Bladder cancer cell lines were used to perform cell proliferation and apoptosis assays. Western blot and quantitative Real-time Polymerase Chain Reaction were used to determine the expression of protein and RNA separately. Dual-luciferase assay was conducted to determine the activity of three prime untranslated region of retinoic acid receptor beta (RARB). Furthermore, 5637 human bladder cancer mouse models were established to investigate the interactions of lncRNA: miRNA: mRNA in vivo.ResultsBased on the RT2 lncRNA PCR Arrays analysis, we validated HAND2-AS1 declined in bladder cancer and negatively correlated with the depth of invasion and grades. The overexpression of HAND2-AS1 in human bladder cancer cells 5637 and RT4 hampered cell proliferation by provoking Caspase 3-triggered cell apoptosis. Besides, one of the HAND2-AS1 sponges, miR-146, elevated in bladder cancer and targeted the tumor suppressor, retinoic acid receptor beta (RARB). We further demonstrated that the HAND2-AS1: miR-146: RARB complex promoted Caspase 3-mediated apoptosis by suppressing COX-2 expression. Finally, the results gained in mouse xenografts suggested that HAND2-AS1 diminished miR-146 expression, thereby reversing the suppression of miR-146 on RARB-mediated apoptosis and contributing to bladder cancer regression.ConclusionThe present study sheds light on the fact that lncRNA HAND2-AS1 exerted as a tumor suppressor by releasing RARB from miR-146, leading to tumor proliferation and invasion inhibition. The findings expanded HAND2-AS-mediated regulatory networks' knowledge and provided novel insights to improve the RARB-targeted regimens against bladder cancer.

Abstract 2911: Intersection of aging, the microbiome and inflammation in a mouse model of bladder cancer

Abstract Bladder cancer (BCa) is a disease strongly related to aging and remains a major source of morbidity and mortality in the elderly. Older patients are at greater risk of developing BCa and treatment outcomes remain poor. Interventions aimed at delaying recurrence and enhancing therapy would be highly beneficial given the aging US population and association with BCa. Aging is associated with baseline immune dysfunction, increasing risk for chronic diseases such as cancers. Compounding this issue, dysregulated inflammation in cancer results in a switch in the inflammatory milieu toward a pro-tumorigenic phenotype. Recent studies have indicated immune dysregulation in multiple cancers is also likely related to the changes in the underlying microbiome. The impact of age-related changes in the microbiome and inflammatory status, and how this relates to progression of tumorigenesis in the bladder have not yet been defined though. We hypothesized aging would enhance inflammation and carcinogenesis in a mouse model of carcinogen induced BCa. To determine how aging affects BCa, we exposed middle aged mice (14 months old at study start), and young mice (2 months old at study start) to the urothelial carcinogen N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) 0.05% in drinking water for 6-20 weeks. Pathological analysis indicated older mice had higher indices of cellular inflammation at baseline and after BBN. Similarly, older mice had significantly more upregulation of pro-inflammatory mediators after 6 weeks exposure to BBN. While inflammatory metrics were increased, this did not extend to tumor size or stage. Middle aged mice (14 months) do not express prototypical biomarkers of aging yet though, and represent a very mild phenotype. To test the idea that truly older mice would be more susceptible to carcinogenesis, we treated young mice (8 weeks at start) or truly old mice (18 months at start) with 0.05% BBN for 16 weeks. We found older animals had larger tumors indicative of increased tumorigenesis after 16 weeks BBN. Moreover, multiple older mice muscle invasive tumors which were absent in the young group and increased recruitment of neutrophils indicative of increased inflammation. We subsequently analyzed the fecal microbiome using 16S sequencing of bacterial DNA. ß-diversity indices indicated substantial differences in microbiota constituency between young and older mice with cancer, although these differences were not observed at baseline. Future goals are to determine if aging-related alterations in the microbiome are responsible for the observed changes in tumor formation and inflammation. In conclusion, aged animals experience more inflammation, major changes in gut microbial content, and develop larger and more aggressive tumors after exposure to carcinogens implicating a pathological role for aging in BCa. Citation Format: Benjamin L. Woolbright, Erika Abbott, Ganeshkumar Rajendran, Cuncong Zhong, Hao Xuan, Shahid Umar, John Arthur Taylor. Intersection of aging, the microbiome and inflammation in a mouse model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2911.