Orthotopic Bladder Cancer Model Research Articles

Abstract 1480: Systemic immunotherapeutic efficacy of an immunocytokine, NHS-muIL12, in a superficial murine orthotopic bladder cancer model

Abstract Interleukin-12 (IL-12) is one of the most powerful proinflammatory cytokines capable of supporting T and NK cell function, inducing interferon-gamma while driving a TH1 adaptive immune response. Yet, to date, its success as an antitumor agent in many preclinical models has yet to be realized in a clinical setting due to systemic toxicity. Investigators have developed IL-12 delivery systems to maximize deposition of the cytokine directly in the tumor microenvironment that may be the preferred site for IL-12 while mitigating the dose-limiting systemic effects. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of human (hu) or murine (mu) IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). NHS76 recognizes exposed chromatin-DNA often found in human/murine tumors that have outpaced their blood vessels and the inadequate perfusion quickly results in tumor necrosis. Indeed, previous studies have shown selective tumor uptake of NHS-IL12 in necrotic subcutaneous murine tumors. In the present study, we evaluated the use of NHS-muIL12 in a murine orthotopic bladder cancer model (MB49 Luc). MB49 luciferase positive cells, instilled into the bladder form superficial, multifocal tumors which can be monitored in real time with a luciferase-based intravital imaging system. Urothelial bladder cancer is known to respond favorably to immunotherapeutic agents due to the presence of multiple somatic mutations, a high number of TILs, and a response to the live bacterium Bacillus Calmette-Guerin (BCG). NHS-muIL12 was found to be a very potent anti-tumor agent in both subcutaneous and intravesical MB49 tumor models, reducing tumor volume in a dose-dependent manner. For example, in the intravesical bladder model, antitumor effects were initially seen at 2.5 ug/kg administrated as three separate systemic injections. Mice were completely cured of their bladder tumors when treated at 20 ug/kg x 3 NHS-muIL12 injections with durable tumor-free long-term survival. Immune analyses revealed potent p15E-specific CTLs and IFN-γ responses, indicating the development of a specific anti-tumor immune response in mice treated with NHS-muIL12. Underlying the durable tumor-free long-term survival was an immune memory response that protected mice following re-challenge with either subcutaneous or intravesical MB49 tumor cells. Anti-tumor efficacy required the presence of NK or CD8+ T cells as depletion of either abrogated the anti-tumor effects of this agent. NHS-huIL12, is currently being evaluated against solid tumors, in a Phase 1 clinical trial (NCT01417546). We acknowledge the kind contribution of NHS-muIL12 from EMD Serono, Billerica, MA. Citation Format: Amanda J. Vandeveer, Jeffrey Schlom, John W. Greiner. Systemic immunotherapeutic efficacy of an immunocytokine, NHS-muIL12, in a superficial murine orthotopic bladder cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1480.

Cancer-specific promoters for expression-targeted gene therapy: ran, brms1 and mcm5.

To expand the library of promoters that can be used for expression-targeted gene delivery to cancer cells, the specificity and strength of expression of three cancer-related gene promoters was evaluated: RAS-related nuclear protein ((P) ran), breast cancer metastasis suppressor 1 ((P) brms1) and minichromosome maintenance complex component 5 ((P) mcm5). The expression of reporter genes under the control of these promoters demonstrated selectivity in cancer cell lines of breast, prostate and ovarian origins versus a panel of normal cell types. The (P) ran was next used to regulate the expression of a bioactive exon (a constitutively active form of human caspase 3) to induce apoptosis in cancer cells. Further evaluation was performed in an orthotopic model of murine bladder cancer. The average strengths of reporter expression had relative intensities of 99.8% ((P) ran), 87.7% ((P) brms1) and 55.8% ((P) mcm5) versus the strong (P) cmv-driven positive control. Comparisons of expression-targeted reporter gene expression for these three promoters versus the clinically interesting promoter for the human telomerase reverse transcriptase gene ((P) hTERT) yielded an improvement of two- to 15-fold. Following transfection, cell death was evident from morphologic observations and viability assays performed on the cancer cells lines, with little (if any) effects seen when the same genes were delivered to normal cells. Cell viability was reduced by up to 60% after one treatment, with cell death via apoptosis implied by caspase 3 detection. During the in vivo preclinical study, reduced tumor burden, lack of mineralization and decreased inflammation were demonstrated after only three treatments. The ran, brms1, and mcm5 promoters have the specificity and strength needed for cancer-specific expression-targeted gene therapy. (p) ran in particular produced exciting results when coupled with a version of the caspase 3 exon to treat bladder cancer. Copyright © 2016 John Wiley & Sons, Ltd.

Systemic Immunotherapy of Non-Muscle Invasive Mouse Bladder Cancer with Avelumab, an Anti-PD-L1 Immune Checkpoint Inhibitor.

Bacillus Calmette-Guerin (BCG) is the standard of care for intravesical therapy for carcinoma in situ and non-muscle invasive, nonmetastatic human urothelial carcinoma. Although the responsiveness to this immunotherapeutic is believed to be linked with (i) a high number of somatic mutations and (ii) a large number of tumor-infiltrating lymphocytes, recent findings of the roles that inhibitory immune receptors and their ligands play in tumor evasion may provide insights into the limitations of the effectiveness of BCG and offer new targets for immune-based therapy. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49(luc)), was used to study the antitumor effects of avelumab, an antibody to PD-L1. MB49(luc) murine tumor cells form multifocal tumors on the mucosal wall of the bladder reminiscent of non-muscle invasive, nonmetastatic urothelial carcinomas. MB49(luc) bladder tumors are highly positive for the expression of PD-L1, and avelumab administration induced significant (P < 0.05) antitumor effects. These antitumor effects were more dependent on the presence of CD4 than CD8 T cells, as determined by in vivo immune cell depletions. The findings suggest that in this bladder tumor model, interruption of the immune-suppressive PD-1/PD-L1 complex releases a local adaptive immune response that, in turn, reduces tumor growth. This bladder tumor model can be used to further identify host antitumor immune mechanisms and evaluate combinations of immune-based therapies for carcinoma in situ and non-muscle invasive, nonmetastatic urothelial carcinoma, to provide the rationale for subsequent clinical studies. Cancer Immunol Res; 4(5); 452-62. ©2016 AACR.

JNK2 downregulation promotes tumorigenesis and chemoresistance by decreasing p53 stability in bladder cancer.

Bladder cancer is one of the most common malignancies of the urinary system, and the 5-year survival rate remains low. A comprehensive understanding of the carcinogenesis and progression of bladder cancer is urgently needed to advance treatment. c-Jun N-terminal kinase-2 (JNK2) exhibits both tumor promoter and tumor suppressor actions, depending on tumor type. Here, we analyzed the JNK2 function in bladder cancer. Using gene expression microarrays, we demonstrated that JNK2 mRNA is downregulated in an orthotopic rat model of bladder cancer. JNK2 protein levels were lower in rat and human bladder cancer tissues than in normal tissues, and the levels correlated with those of p53. Moreover, JNK2 phosphorylated p53 at Thr-81, thus protecting p53 from MDM2-induced proteasome degradation. Decreased expression of JNK2 in T24 cells conferred resistance to cell death induced by mitomycin C. Furthermore, lower JNK2 expression was associated with poorer overall survival among patients who underwent radical cystectomy. These results indicate that JNK2 acts as a tumor suppressor in bladder cancer, and that decreased JNK2 expression promotes bladder cancer tumorigenesis.

MP45-04 PATERNALLY EXPRESSED GENE-10 PROMOTES CELL GROWTH AND INVASION OF BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-04 PATERNALLY EXPRESSED GENE-10 PROMOTES CELL GROWTH AND INVASION OF BLADDER CANCER Yoshihisa Kawai, Shusuke Akamatsu, Tetsutaro Hayashi, Eliana Beraldi, Fan Zhang, Roland Seiler, Jeffrey Leong, Htoo Oo, Igor Moskalev, Ladan Fazli, Hideyasu Matsuyama, Peter Black, Colin Collins, and Martin Gleave Yoshihisa KawaiYoshihisa Kawai More articles by this author , Shusuke AkamatsuShusuke Akamatsu More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Eliana BeraldiEliana Beraldi More articles by this author , Fan ZhangFan Zhang More articles by this author , Roland SeilerRoland Seiler More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Htoo OoHtoo Oo More articles by this author , Igor MoskalevIgor Moskalev More articles by this author , Ladan FazliLadan Fazli More articles by this author , Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author , Peter BlackPeter Black More articles by this author , Colin CollinsColin Collins More articles by this author , and Martin GleaveMartin Gleave More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.281AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We have recently identified that paternally expressed gene-10 (PEG10) promotes cell cycle progression in the context of TP53 and RB1 loss in neuroendocrine prostate cancer. Based on the evidence that TP53 mutations and RB1 inactivation are more prevalent in muscle invasive bladder cancer (MIBC), it is possible that PEG10 may contribute to the progression and prognosis of MIBC. To test this hypothesis, we characterized PEG10 function in bladder cancer and evaluated if PEG10 can be a novel therapeutic target for these cancers. METHODS PEG10 gene expression of tumor samples was analyzed using the cancer genome atlas (TCGA) cohort. The PEG10 expression in several bladder cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR (qRT-PCR). Silencing of PEG10 in vitro was achieved using siRNA. The in vivo effect of PEG10 antisense oligonucleotide (ASO) treatment was assessed in the T24 orthotopic bladder cancer model. RESULTS Higher mRNA level of PEG10 was significantly associated with poorer disease-free survival after cystectomy in 131 patients in the TCGA, Nature 2014 cohort. We examined PEG10 expression in a panel of 12 bladder cancer cell lines by Western blot. UM-UC14 showed the highest PEG10 expression, and T24 showed higher PEG10 expression than the other bladder cancer cell lines. Similar results were confirmed by qRT-PCR. PEG10 transient knockdown using two independent siRNAs resulted in significant growth suppression in UM-UC14 (RB1inactivatedTP53mut) and T24 (RB1wtTP53mut) cells. PEG10 knockdown induced higher expression of key cell cycle dependent kinase inhibitors p21 and p27 than observed in the control cells. Conversely, forced PEG10 RF1b/2 isoform over-expression induced cell growth in UC14 and T24 cells. Furthermore, UM-UC14 cell invasion was significantly decreased with PEG10 transient knockdown. Using BrdU incorporation assay after cell cycle synchronization by double-thymidine block, we found that PEG10 drove cell cycle progression from G0/G1. In the orthotopic bladder cancer model, systemic PEG10-ASO administration to athymic nude mice delayed tumor progression in T24 cells. CONCLUSIONS We have demonstrated that PEG10 promotes bladder cancer progression. Inhibition of PEG10 may be a novel treatment strategy for a subset of bladder cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e608 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yoshihisa Kawai More articles by this author Shusuke Akamatsu More articles by this author Tetsutaro Hayashi More articles by this author Eliana Beraldi More articles by this author Fan Zhang More articles by this author Roland Seiler More articles by this author Jeffrey Leong More articles by this author Htoo Oo More articles by this author Igor Moskalev More articles by this author Ladan Fazli More articles by this author Hideyasu Matsuyama More articles by this author Peter Black More articles by this author Colin Collins More articles by this author Martin Gleave More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Photo-thermal therapy of bladder cancer with Anti-EGFR antibody conjugated gold nanoparticles.

The aim of this study was to enhance the effectiveness of photo thermal therapy (PTT) in the targeting of superficial bladder cancers using a green light laser in conjunction with gold nanoparticles (GNPs) conjugated to antibody fragments (anti-EGFR). GNPs conjugated with anti-EGFR-antibody fragments were used as probes in the targeting of tumor cells and then exposed to a green laser (532nm), resulting in the production of sufficient thermal energy to kill urothelial carcinomas both in vitro and in vivo. Nanoparticles conjugated with antibody fragments are capable of damaging cancer cells even at relatively very low energy levels, while non-conjugated nanoparticles would require an energy level of 3 times under the same conditions. The lower energy required by the nanoparticles allows this method to destroy cancerous cells while preserving normal cells when applied in vivo. Nanoparticles conjugated with antibody fragments (anti-EGFR) require less than half the energy of non-conjugated nanoparticles to kill cancer cells. In an orthotopic bladder cancer model, the group treated using PTT presented significant differences in tumor development.

Preclinical evaluation of VAX-IP, a novel bacterial minicell-based biopharmaceutical for nonmuscle invasive bladder cancer

The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder cancer patient populations where complete cures are achievable.

Systemic immunotherapy of superficial mouse bladder cancer with Avelumab (MSB0010718C), an anti-PD-L1 immune checkpoint inhibitor

Bladder cancer is considered a malignancy that is responsive to immunotherapy because of the presence of a (a) high number of somatic mutations, (b) large number of tumor-infiltrating lymphocytes and (c) clinical response to the immune stimulant, Bacillus Calmette-Guerin (BCG). Recent findings of the roles that inhibitory immune receptors and their ligands play in tumor evasion provide some possible explanation as to the limitations of BCG and may offer new therapeutic approaches to some patients with bladder cancer. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49luc), was used to study the antitumor effects of an anti-PD-L1 antibody. MB49luc murine tumor cells form multifocal, superficial tumors on the mucosal wall of the bladder reminiscent of superficial human transitional cell bladder carcinomas. MB49luc bladder tumors were shown to constitutively express high PD-L1 levels and the administration of Avelumab, an anti-PD-L1 antibody, induced significant tumor reduction. Antitumor effects subsequently improved overall survival, and both were abrogated by selective in vivo depletion of CD4 or CD8 T cells, but not with NK cell depletion. These findings suggest that in this murine bladder tumor model, interruption of the immune suppressive PD-1/PD-L1 complex releases a local adaptive immune response that, in turn, significantly reduces tumor growth. This experimental bladder tumor model can be used to (a) identify host immune mechanisms responsible for tumor resistance or destruction and (b) evaluate combinations of immune-based therapy for bladder cancer which could provide rationale for future clinical studies.

Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus.

Urothelial carcinoma of the bladder (UCB) is the most common malignancy of the urinary tract, nearly half of which contains a mutation in TP53 gene. Hence, therapeutic approach by restoring functional p53 protein in cancer cells will be beneficial. Recent studies have demonstrated the inhibition of cancer cell growth by p53 reactivation using a peptide derived from the p53 C-terminus (p53C). However, the outcome of reactivating p53 in controlling bladder cancer development is limited by its efficiency and specificity of peptide delivery, especially in metastatic animal models. Herein, we report that the cell penetrating peptide (polyarginine, R11)-conjugated p53C can exhibit a preferential uptake and growth inhibit of UCB cells expressing either mutant or wild-type TP53 by the activation of p53-dependent pathway. R11-p53C peptide treatment of preclinical orthotopic and metastatic bladder cancer models significantly decreased the tumor burden and increased the lifespan without a significant cytotoxicity. Based on these results, we believe that R11-p53C peptide has therapeutic potential for primary and metastatic bladder cancer, and R11-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to tumor cells in vitro and in vivo.

Metformin can block precancerous progression to invasive tumors of bladder through inhibiting STAT3-mediated signaling pathways.

BackgroundMetformin is the first line of oral antidiabetic drug in the biguanide class for treatment of type 2 diabetes. Increasing evidence has suggested that it is a potential anti-tumor drug. However, the mechanisms underlying inhibiting tumor development remain elusive, especially in bladder tumors.MethodsT24 and J82 cell lines were used as an in vitro model, and 24 female SD rats were used to build an N-methyl-N-nitrosourea (MNU)-induced orthotopic rat bladder cancer model. Transfection of lentivirus-based shRNA was used to construct the STAT3-KNOCKDOWN T24 cell line. After metformin treatment, the viability of bladde cancer cells was determined by CCK8. Cell cycle distribution and apoptosis were assessed by flow cytometry. The migration and invasion abilities of cells were evaluated by wound healing and transwell asssays. The inactivation of stat3 pahtway was examined by qRTPCR, western blot and Immunofluorescence.ResultsMetformin can effectively inhibit precancerous progression to invasive cancer in an MNU-induced rat orthotopic bladder tumor model, although it could not completely suppress normal cells transforming into tumor cells. While the MNU could induce 50 % rats (4/8) to develop invasive bladder cancers, the rats co-administrated with metformin failed to develop invasive tumors but retained at precancerous or non-invasive stages, exhibiting as dysplasia, papillary tumor and/or carcinoma in situ (CIS). Accordingly, phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is a well known oncogene, was significantly inhibited in the tumors of rats treated with metformin. In vitro experiments revealed that the metformin could efficiently inhibit STAT3 activation, which was associated with the cell cycle arrest, reduction of cell proliferation, migration and invasiveness, and increase in apoptotic cell death of bladder cancer cell lines.ConclusionsThese findings provide for the first time the evidence that metformin can block precancerous lesions progressing to invasive tumors through inhibiting the activation of STAT3 pathway, and may be used for treatment of the non-invasive bladder cancers to prevent them from progression to invasive tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0183-0) contains supplementary material, which is available to authorized users.

Abstract 5146: Establishment of an orthotopic bladder cancer model to evaluate continuous intravesical delivery of small molecule inhibitors in the nude rat

Abstract Bladder cancer is a disease with high unmet need, with no new treatments being registered in the last two decades. Intravesical delivery is an attractive option for organ-confined bladder cancer; indeed, non-muscle invasive disease is currently treated by intravesical administration of agents such as bacillus Calmette-Guerin (BCG) and mitomycin C, and neoadjuvant intravesical therapy has the potential to preserve the bladder and improve survival in M0 muscle invasive disease. Intravesical delivery has the potential to increase the therapeutic index compared with systemic dosing routes, particularly if the duration of exposure can be extended. In order to explore this concept, we have developed an orthotopic model of bladder cancer and a drug delivery method to administer gemcitabine into the nude rat bladder for one week. An orthotopic model of bladder cancer was established by serial passaging of MGH-U3 human bladder cancer cells into the muscle wall. Tumor take-rate and growth was monitored by ultrasound imaging. Serial ex vivo - in vivo passaging increased growth and take rate of the tumors in both orthotopic and subcutaneous locations. Orthotopic implantation of cells following two previous passages in vivo resulted in reproducible tumor growth and take rates of &amp;gt;90%. Immunohistochemistry analysis showed that the orthotopic tumors were stably epithelial in phenotype after three serial in vivo passages. Principal component analysis of gene expression (GE) data from RNA sequencing of the parental cell line, sequentially passaged cells growing ex vivo, and both subcutaneous and orthotopically implanted tumours after various numbers of passages revealed three clusters; two distinct but closely related clusters representing the orthotopic and subcutaneously growing tumors, and another distinct cluster representing the various cell lines. This analysis showed that in vitro/in vivo location of growth is a more important determinant of global GE than the process of serial passaging in vivo. We have used the iPRECIO programmable infusion pump system to continuously infuse gemcitabine into the bladders of rats with orthotopic bladder tumors. Infusion of 2.5 mg/day at a flow rate of 5 ul/h for 7 days resulted in urine concentrations in the range of 1- 10 mM (∼ 1000-fold greater than systemic plasma drug concentrations), a 4-fold increase of γH2AX and induction of both necrosis and apoptosis (10-fold induction of cleaved casapse 3) in the orthotopic tumors. This model system holds considerable potential for evaluation of intravesical small molecule therapies that are potentially translatable to patients. Citation Format: Leigh J. Williams, Stephanie K. Klein, Danielle Greenawalt, Dawn Trueman, Helen Musgrove, Helen Jones, Linette Ruston, Barry R. Davies. Establishment of an orthotopic bladder cancer model to evaluate continuous intravesical delivery of small molecule inhibitors in the nude rat. [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 5146. doi:10.1158/1538-7445.AM2015-5146

γ Irradiated Mycobacteria Enhance Survival in Bladder Tumor Bearing Mice Although Less Efficaciously than Live Mycobacteria

γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent. The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer. Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin. Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.

Local Salmonella immunostimulation recruits vaccine-specific CD8 T cells and increases regression of bladder tumor

The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in complement component 5a, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

Recombinant hIFN-α2b-BCG inhibits tumor growth in a mouse model of bladder cancer.

Bacillus Calmette-Guérin (BCG) reduces the recurrence and progression of non-muscle invasive bladder cancer. The present study aimed to investigate the impact of a recombinant hIFN-α2b-secreting BCG (rBCG) on the mouse bladder MB49 cell line and an orthotopic mouse model of bladder cancer. MB49 cells were cultivated in the presence or absence of rBCG, BCG or BCG+hIFN-α2b. Cellular morphology and viability were assessed by microscopy and CCK-8 assay, respectively. Apoptosis was assessed by acridine orange, Hoechst 33258 staining and flow cytometry. MHC-I expression was assessed by flow cytometry. MB49 cells were transplanted into the bladders of C57BL/6 mice administered BCG, rBCG or BCG+hIFN-α2b. Local tissue Fas expression and T cell subsets were assessed by immunohistochemistry. Peripheral blood TNF-α and IL-12 levels were measured by ELISA, and circulating T lymphocyte subsets by flow cytometry. BCG, rBCG and BCG+hIFN-α2b increased the distortion and death of MB49 cells, yet rBCG reduced the proliferation and enhanced apoptosis most substantially. Apoptosis was increased after a 24-h co-culture with rBCG or BCG+hIFN-α2b. Mice administered rBCG survived longer than mice administered BCG (p<0.001), yet this result was not significantly different from mice administered BCG+hIFN-α2b. The average bladder weight was reduced by administration of rBCG (p<0.001). Fas expression and peripheral blood mTNF-α and mIL-12, cell counts of polymorphonuclear leukocytes, monocytes, T lymphocytes and CD4+/CD8+ ratios were significantly increased by all BCG treatments (p≤0.05), yet monocyte and T lymphocyte counts were higher in mice administered rBCG than in mice treated with BCG or BCG+hIFN-α2b (p=0.000). These results indicate that in an orthotopic murine bladder cancer model rBCG possesses superior antitumor activity to BCG+hIFN-α2b.

Intravesical administration of exogenous microRNA-145 as a therapy for mouse orthotopic human bladder cancer xenograft.

We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.

Intravesical dual PI3K/mTOR complex 1/2 inhibitor NVP-BEZ235 therapy in an orthotopic bladder cancer model.

NVP-BEZ235 is an inhibitor of both phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1/2 (mTORC1/2), and its antitumor activity is expected to be higher than that of mTORC1 inhibitors because it inhibits the upregulation of pAkt through mTORC2. We examined the efficacy of intravesical NVP-BEZ235 therapy in the treatment of bladder cancer using an orthotopic bladder cancer model. The cytotoxic effects of various concentrations of NVP-BEZ235 in MBT-2 cells were examined using a WST assay. The expression of pAkt, pS6 and p4EBP1 was evaluated in MBT-2 cells treated with NVP-BEZ235 using western blotting. Orthotopic models were established by implanting MBT-2 cells into the bladders of female C3H/He mice. We assigned C3H/He mice to 2 groups: a control group treated with vehicle control (n=15), and a group intravesically administered 40 µM (18.78 mg/l) of NVP-BEZ235 (n=15). NVP-BEZ235 inhibited the viability of MBT-2 cells in a dose-dependent manner. Furthermore, the expression of pAkt, pS6, and p4EBP1 was inhibited in NVP-BEZ235-treated MBT-2 cells. Bladder weights were significantly lower in the NVP-BEZ235-treated group than in the control group (P<0.05). An analysis of the tumor tissues revealed that the NVP-BEZ235 treatment strongly reduced pAkt, pS6 and p4EBP1 levels. An immunohistochemical analysis showed that NVP-BEZ235 significantly inhibited the expression of pS6. Intravesically administered NVP-BEZ235 exerted significant antitumor effects in the orthotopic bladder cancer model by inhibiting the PI3K/Akt/mTOR pathway. The intravesical instillation of a dual PI3K/mTORC1/2 inhibitor may represent a novel therapy for the treatment of bladder cancer.

Therapeutic effect of intravesical administration of paclitaxel solubilized with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) in an orthotopic bladder cancer model.

BackgroundTo evaluate the effects of intravesical administration of paclitaxel (PTX-30W), which was prepared by solubilization with a water-soluble amphiphilic polymer composed of PMB30W, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate, in an orthotopic bladder cancer model.MethodsThe cytotoxicities of PMB30W were examined in MBT-2 cell cultures and the results were compared with those of the conventional paclitaxel solubilizer Cremophor. In an orthotopic MBT-2 bladder cancer model, the effect of intravesical administration of PTX-30W was compared with that of paclitaxel solubilized with Cremophor (PTX-CrEL). The paclitaxel concentration in bladder tumors after the intravesical treatment was also evaluated using liquid chromatography tandem mass spectrometry (LC-MS/MS) system.ResultsIn vitro, Cremophor exhibited dose-dependent cytotoxicity towards MBT-2 cells, whereas no cytotoxicity was observed with PMB30W. In the orthotopic bladder cancer model, intravesical administration of PTX-30W resulted in a significant reduction of bladder wet weight compared with that of PTX-CrEL. The paclitaxel concentration in bladder tumors after the intravesical treatment was significantly higher in PTX-30W treated mice than in PTX-CrEL treated mice.ConclusionsIntravesically administered PTX-30W can elicit stronger antitumor effects on bladder tumors than conventional paclitaxel formulated in Cremophor, presumably because of its better penetration into tumor cells. PTX-30W might be a promising antitumor agent for intravesical treatment of non-muscle invasive bladder cancer.

MP49-19 EMP2 AS A NOVEL TARGET OF BLADDER CANCER IMMUNOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-19 EMP2 AS A NOVEL TARGET OF BLADDER CANCER IMMUNOTHERAPY Wujiang Liu, Jie Jin, liqun Zhou, and Yinglu Guo Wujiang LiuWujiang Liu More articles by this author , Jie JinJie Jin More articles by this author , liqun Zhouliqun Zhou More articles by this author , and Yinglu GuoYinglu Guo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2814AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES EMP2, epithelial membrane protein 2, is supposed to be upregulated in cancer (Clin Cancer Res. 2008: 7367). Antibody targeting EMP2 shows benefit on cancer growth arrest (Mol Cancer Ther. 2014: 902). Here we investigated EMP2 expression in bladder cancer tissue and the potential for using as a target for cancer immunotherapy. METHODS 31 muscle invasive bladder cancer tissue and adjacent normal bladder tissue were collected from patient underwent cystectomy with informed consent. Protein expression of EMP2 was assayed by immmunohistochemical stain. T24, 5637 and SV-HUC-1 were used as cell lines for bladder cancer cells and normal bladder epithelium. Real-time PCR and Western-blot were performed to measure the mRNA and protein expression of EMP2. Chimeric antigen receptors of EMP2 (CARs-EMP2) were successfully constructed from multiple murine antibodies using second generation (CD28.CD3£a) signaling domains. Gamma-retroviral vector were used for carrying CAR and CD28.CD3£a sequences and subsequently for peripheral blood lymphocytes (PBL) transduction. Anti-tumor effect of T cells expressing CARs-EMP2 was assayed by cytolysis and cytokine release (IL-6, IFN-ƒ× and TNF-ƒÑ)ƒw following co-culture with target cell lines T24 and 5637. In vivo anti-tumor effect of T cells expressing anti-EMP2 CARs was tested on orthotopic mouse bladder cancer model. Tumor growth curve and tumor cell apoptosis were used to test the anti-tumor effect. RESULTS EMP2 was significantly increased in bladder tumor tissue compared to adjacent normal bladder tissue (P<0.01). The location of EMP2 is bladder cancer cell membrane. RNA and protein expression of EMP2 were more intensive in T24 and 5637 cells than that in SV-HUC-1 (P<0.05). T cells expressing CARs-EMP2 were able to lysis T24 and 5637 cells in a dose dependent manner and increase the cytokine release especially IL-6 and IFN-ƒ×ƒ|ƒnƒnCompared with T cells transfected with empty vector, adoptive transfer therapy with T cells transfected with CARs-EMP2 dramatically delay the tumor growth and increase the cancer cell apoptosis (P<0.01). CONCLUSIONS EMP2 is a potential target of bladder cancer immunological therapy. Adoptive transferring T cells expressing anti-EMP2 chimeric antigen receptors could be an alternative option to treat bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e610 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wujiang Liu More articles by this author Jie Jin More articles by this author liqun Zhou More articles by this author Yinglu Guo More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

MP49-13 LIM-SH3 DOMAIN PROTEIN 1 KNOCKDOWN INHIBITS CELL GROWTH AND ENHANCES ACTIVITY OF CISPLATIN IN BLADDER CANCER.

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-13 LIM-SH3 DOMAIN PROTEIN 1 KNOCKDOWN INHIBITS CELL GROWTH AND ENHANCES ACTIVITY OF CISPLATIN IN BLADDER CANCER. Takashi Dejima, Ario Takeuchi, Tetsutaro Hayashi, Jeffrey Leong, Tabitha Tombe, Kevin Tam, Htoo Oo, Peter Black, Seiji Naito, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Kevin TamKevin Tam More articles by this author , Htoo OoHtoo Oo More articles by this author , Peter BlackPeter Black More articles by this author , Seiji NaitoSeiji Naito More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.517AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is the 6th most common cancer in the USA. The current standard therapy for the first line of metastatic or local advanced bladder cancer is combination therapy with cisplatin (CDDP) and gemcitabine (GEM). However 5-years survival is still below 50%; therefore additional therapy is needed. LIM-SH3 domain protein 1 (LASP1), identified from cDNA library of metastatic axillary lymph nodes of breast cancer, has been shown to promote cancer progression and invasion in several malignancies. In bladder cancer, LASP1 expression associates with cell invasion and can be used for detecting bladder cancer. However, the anti-tumor effects of LASP1 knockdown in vivo as well as combination therapy with chemotherapy remain unclear. In this study, we investigated the anti-cancer activity of LASP1 knockdown in bladder cancer. METHODS LASP1 gene expression of tumor samples is analyzed using the Affymetrix Exon Array. The LASP1 expression in several bladder cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR. Silencing of LASP1 in vitro was achieved using siRNA. The In vivo effect of LASP1 antisense oligonucleotide (ASO) treatment was assessed in the T24 CDDP-R (cisplatin-resistant) orthotopic bladder cancer model. RESULTS High LASP1 expression correlated with recurrence rate between patients receiving adjuvant chemotherapy or not. This difference is increased in patients after adjuvant chemotherapy. The LASP1 expression is higher in UC1 and UC15 cells than in UC13 and UC6 cells. Knockdown of LASP1 using siRNA inhibited cell growth, and was accompanied by an increase in p21 and p27. Conversely, stable LASP1 overexpression drove cell growth with increase of cyclinD1 in UC13 and UC6 cells. The treatment of CDDP and GEM induced LASP1 expression in vitro. Combination treatment with LASP1 knockdown using siRNA and CDDP inhibited cell growth in UC1 cells. Furthermore, compared with parental cell line, LASP1 is higher in T24 CDDP-R and RT112 CDDP-R cells than in parental cells. In the orthotopic bladder cancer model, systemic LASP1 ASO administration to athymic nude mice delayed tumor progression in T24 CDDP-R cells. CONCLUSIONS These data revealed that LASP1 inhibition might be as a promising novel therapeutics modality in the treatment of bladder cancer, as well as a possible synergy with chemotherapy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e607-e608 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Tetsutaro Hayashi More articles by this author Jeffrey Leong More articles by this author Tabitha Tombe More articles by this author Kevin Tam More articles by this author Htoo Oo More articles by this author Peter Black More articles by this author Seiji Naito More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2015MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE Ryuji Matsumoto, Masumi Tsuda, Takashige Abe, Satoru Maruyama, Kunihiko Tsuchiya, Naoto Miyajima, Nobuo Shinohara, and Shinya Tanaka Ryuji MatsumotoRyuji Matsumoto More articles by this author , Masumi TsudaMasumi Tsuda More articles by this author , Takashige AbeTakashige Abe More articles by this author , Satoru MaruyamaSatoru Maruyama More articles by this author , Kunihiko TsuchiyaKunihiko Tsuchiya More articles by this author , Naoto MiyajimaNaoto Miyajima More articles by this author , Nobuo ShinoharaNobuo Shinohara More articles by this author , and Shinya TanakaShinya Tanaka More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e535 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuji Matsumoto More articles by this author Masumi Tsuda More articles by this author Takashige Abe More articles by this author Satoru Maruyama More articles by this author Kunihiko Tsuchiya More articles by this author Naoto Miyajima More articles by this author Nobuo Shinohara More articles by this author Shinya Tanaka More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...