Murine Bladder Cancer Cell Line Research Articles

Abstract 2037: Oral nitroxoline in combination with intravesical bacille calmette-guerin (BCG) shows synergistic antitumor efficacy in mouse bladder cancer orthotopic xenograft

Abstract Nitroxoline, an antibiotic used to treat urinary tract infection, had previously been identified as a MetAP2 inhibitor and found to show antitumor efficacy in mouse models of human breast cancer xenograft and bladder cancer orthotopic xenograft [1]. Our purpose in this study was to evaluate the feasibility of combining oral nitroxoline with intravesical BCG, the first-line therapy for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC), in mouse bladder cancer orthotopic xenograft. The luciferase-expressing murine bladder cancer cell line MBT-2-Luc cells was transurethrally implanted in the bladder of syngeneic female C3H/He mice. The animals were randomly divided into groups and treated with phosphate-buffered saline (PBS), oral nitroxoline, intravesical BCG alone, and a combination as described in methods. The IVIS spectrum images analyses of luciferin-stained tumor at 21 days after inoculation revealed that combining oral nitroxoline with intravesical BCG showed synergistically enhanced antitumor efficacy and prolonged long-term survival, implicating the potential of combining bladder cancer treatment using oral nitroxoline with intravesical BCG in the clinical setting. The mechanism behind this observation is currently under investigated. Citation Format: Qiang Li, Kevin Pan, Xiezhao Li, Naijin Xu, Peng Huang. Oral nitroxoline in combination with intravesical bacille calmette-guerin (BCG) shows synergistic antitumor efficacy in mouse bladder cancer orthotopic xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2037. doi:10.1158/1538-7445.AM2017-2037

A novel synthetic derivative of quercetin, 8-trifluoromethyl-3,5,7,3',4'-O-pentamethyl-quercetin, inhibits bladder cancer growth by targeting the AMPK/mTOR signaling pathway.

Quercetin is a naturally existing compound and shows attractive anticancer properties for a variety of solid tumors including glioma, bladder cancer, hepatocellular carcinoma, breast cancer, hematological malignancies and prostate carcinoma. However, these anticancer properties have not been clinically approved due to unclear mechanistic information and its low bioactivity. In our previous study, we elucidated that quercetin activates AMPK pathway which is the major mechanism for its unique anticancer effect in bladder cancer. In the present study, we are trying to enhance its bioactivity by chemical modification using fluorination approach to prepare novel chemical entities, based on the principle of intermediate derivative method (IDM). The compound we obtained is named 8-trifluoromethyl-3,5,7,3′,4′-O-pentamethyl- quercetin (TFQ), characterized by NMR spectra and mass spectrum (MS). The results from MTT and cologenic assay in two human and one murine bladder cancer cell lines showed that TFQ exhibits more potent inhibition on the three bladder cancer cell lines than quercetin (Que) although this enhanced effects is not very dramatic. Furthermore, we found that the survival of normal bladder cells PEBC was not significantly suppressed by TFQ compared with Que. Western blot analysis showed that TFQ possess more potent AMPK activation than Que. The downstream of AMPK was further examined by western blot. TFQ treatment is able to inactivate mTOR signaling pathway with the regulation of mTOR, 4EBP1 and P70S6K. These results demonstrated that the fluorinated quercetin derivative TFQ inhibits bladder cancer cell growth through the AMPK/mTOR pathway. Altogether, our findings suggest that TFQ could serve as a new potential therapeutic agent for bladder cancer more effective than Que.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

The establishment of a growth-controllable orthotopic bladder cancer model through the down-regulation of c-myc expression.

To properly evaluate the biological effects of immunotherapy, it is critical to utilize a model of cancer in immune-competent mice. Currently, MBT-2 is the most common murine bladder cancer cell line used in orthotopic bladder cancer models, even though this cell type often has an inappropriate genetic mutation landscape. In these models, after tumors are detected with in vivo imaging, the mouse usually dies within two to three weeks due to post-renal azotemia caused by the rapidly growing mass. This event prohibits the evaluation of tumor behavior upon intravesical drug treatment. We explored whether an shRNA-induced decrease in the expression of the c-myc oncogene in MBT-2 cells could slow down their in vitro proliferation and in vivo tumor growth. We transduced MBT-2 cells with shRNA lentiviruses that bound c-myc, established MBT2.cMYCshRNA and confirmed the retardation of the growth of tumors implanted in C3H/He mice. Accordingly, this study suggests that this novel orthotopic bladder cancer model in immune-competent mice may be more appropriate for the analysis of the effects of the intravesical instillation of immunotherapeutic agents.

Abstract 3068: Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model

Abstract Purpose: Paclitaxel is an effective anti-neoplastic agent in many solid tumors including bladder cancer. With the development of new drug delivery strategies, novel formulations of Cremophor EL-free paclitaxel have been developed. Here, we investigated the in vitro and in vivo anti-tumor effect of a mucoadhesive oral formulation of paclitaxel (DHP107) in comparison with iv paclitaxel in a human bladder cancer model. Methods: Four human bladder cancer cell lines (RT4, T24, J82, HT1376) and one murine bladder cancer cell line (MBT-2) were used in this study. In vitro antitumor activity and underlying mechanisms were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell-cycle analysis and apoptosis assay. In the in vivo study, RT4 cell line was implanted subcutaneously in the flank of nu/nu mice, and treated with iv paclitaxel and 2 doses of DHP107. The effects were assessed by the tumor growth, body weight and Ki-67 staining in tumor tissues. Results: The IC50 values of DHP107 ranged from 4.15 to 34.72 μg/ml with MTT assay. In RT4 cell line, both drugs induced similar G2-M arrest and apoptosis. The xenograft experiments showed that both DHP107 (per os, 50 mg/kg, equivalent of paclitaxel 10 mg/kg) and iv paclitaxel (intraperitoneal, 10 mg/kg) attenuated the growth of RT4 tumors in mice, equivalently. The higher dose (per os, 100 mg/kg) of DHP107 resulted in increased efficacy compared with the lower dose (50 mg/kg) without weight loss in mice. However, significant weight loss was observed in iv paclitaxel (-20% at Day 11) treated group. We observed significantly decreased proliferation with Ki-67 staining in DHP107 (100 mg/kg) treated group than in DHP107 (50 mg/kg) or iv paclitaxel (10 mg/kg) treated groups. Conclusion: Our findings provide the evidence that DHP107 showed comparable antitumor effect with iv paclitaxel and is a promising anti-tumor agent in bladder cancer. Clinical studies in patients with bladder cancer warrant further investigation. Citation Format: Se Hyun Kim, Jung Min Kim, Tae Soo Kim, Won Suk Lee, Woo Sun Kwon, Seung Tak Lim, Joong Bae Ahn, Se-Kyu Kim, Yeong Woo Jo, Sun Young Rha. Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in 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 3068.

The role of PD-L1 in the radiation response and clinical outcome for bladder cancer.

Identification of potential factors that can stratify a tumor’s response to specific therapies will aid in the selection of cancer therapy. The aim was to highlight the role of programmed cell death 1 ligand 1 (PD-L1) in bladder cancer. In this study, 92 of muscle-invasive bladder cancers and 28 of non- muscle invasive bladder cancers were selected for immunohistochemical staining analysis. Furthermore, human and murine bladder cancer cell lines were used to examine the correlation between PD-L1 and radiation response. Our data revealed that PD-L1 was overexpressed in the bladder tumor specimens compared with adjacent non-malignant specimens. Furthermore, the staining of PD-L1 was significantly linked to higher clinical stage, lower complete response rates and reduced disease-free survival rates. By in vitro and in vivo experiments, irradiation up-regulated the expression of PD-L1 in tumor cells, and its increase correlated with the irradiation dose. In immunocompetent mouse models, blocking PD-L1 induced a longer tumour growth delay following irradiation. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells was responsible to the effects of PD-L1 on radiation response. In conclusion, PD-L1 could be a significant clinical predictor for clinical stage and treatment response of bladder cancer.

High efficacy of intravesical treatment of metformin on bladder cancer in preclinical model.

Anticancer potential of metformin has been extensively studied. However, its anticancer clinical use remains yet to be approved since sufficient concentration on target organs could not be achieved via conventional administration. To overcome this drawback, we aim to examine the efficiency of novel intravesical treatment of metformin on syngeneic orthotopic preclinical model. Three human and one murine bladder cancer cell lines were tested in vitro for inhibitory sensitivity by MTT and cologenic assays. AMPK pathway including AKT, Erk and S6K was examined by western blot and further explored by regulating activated levels using specific inhibitors. In vivo efficacy was determined by Kaplan-Meier survival curves and measurements of body and bladder weights plus tumor biomarkers. Lactic acid and metformin levels of plasma were measured by standard procedures. The results demonstrated that metformin activated AMPK and decreased phosphorylation of Akt and Erk. Furthermore, combinations of metformin with either Akt or Erk inhibitors synergistically diminished cancer proliferation, suggesting the involvement of Akt- and Erk- related pathways. Intravesical metformin 26 and 104 mg/kg, twice per week demonstrated a rapid elimination of the implanted tumor without any evidence of toxicity. In contrast, oral treatment at a dose of 800mg/kg/d exhibited little efficacy whereas severe toxicity existed if the dosage is higher. Collectively, intravesical metformin displays potent inhibition on bladder cancer in vitro and this preclinical study reveals the profound therapeutic application of metformin with durable tolerance via intravesical administration route.

The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines.

Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer- mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345-1356. © 2015 Wiley Periodicals, Inc.

Efficacy of intravesical Bacillus Calmette-Guérin therapy against tumor immune escape in an orthotopic model of bladder cancer.

The aim of this study was to evaluate the antitumor immune response of the Bacillus Calmette-Guérin (BCG) in an orthotopic bladder cancer model. The murine bladder cancer cell line MBT-2 was transurethrally implanted in the bladder of syngeneic female C3H/He mice. The animals were randomly divided into three treatment groups: Phosphate-buffered saline (PBS), low-dose BCG and high-dose BCG. The analyses of luciferin-stained tumor images 28 days after each treatment showed significant tumor growth inhibition in the high-dose group in comparison with that in the low-dose- or PBS-treated groups. In addition, the percentage of myeloid-derived suppressor cells in the high-dose group was significantly suppressed in comparison with that in the PBS and low-dose agent treatment groups. These findings are notable in terms of the clinical evaluations of this therapy for patients with bladder cancer. The outcomes of this study also provide important implications regarding antitumor immune responses in human cancer.

Parallel synthesis of poly(amino ether)-templated plasmonic nanoparticles for transgene delivery.

Plasmonic nanoparticles have been increasingly investigated for numerous applications in medicine, sensing, and catalysis. In particular, gold nanoparticles have been investigated for separations, sensing, drug/nucleic acid delivery, and bioimaging. In addition, silver nanoparticles demonstrate antibacterial activity, resulting in potential application in treatments against microbial infections, burns, diabetic skin ulcers, and medical devices. Here, we describe the facile, parallel synthesis of both gold and silver nanoparticles using a small set of poly(amino ethers), or PAEs, derived from linear polyamines, under ambient conditions and in absence of additional reagents. The kinetics of nanoparticle formation were dependent on PAE concentration and chemical composition. In addition, yields were significantly greater in case of PAEs when compared to 25 kDa poly(ethylene imine), which was used as a standard catonic polymer. Ultraviolet radiation enhanced the kinetics and the yield of both gold and silver nanoparticles, likely by means of a coreduction effect. PAE-templated gold nanoparticles demonstrated the ability to deliver plasmid DNA, resulting in transgene expression, in 22Rv1 human prostate cancer and MB49 murine bladder cancer cell lines. Taken together, our results indicate that chemically diverse poly(amino ethers) can be employed for rapidly templating the formation of metal nanoparticles under ambient conditions. The simplicity of synthesis and chemical diversity make PAE-templated nanoparticles useful tools for several applications in biotechnology, including nucleic acid delivery.

Licochalcone B inhibits growth of bladder cancer cells by arresting cell cycle progression and inducing apoptosis

To examine the mechanisms by which licochalcone B (LCB) inhibits the proliferation of human malignant bladder cancer cell lines (T24 and EJ) in vitro and antitumor activity in vivo in MB49 (murine bladder cancer cell line) tumor model. Exposure of T24 or EJ cells to LCB significantly inhibited cell lines proliferation in a concentration-dependent and time-dependent manner, and resulted in S phase arrest in T24 or EJ cells, respectively. LCB treatment decreased the expression of cyclin A, cyclin-dependent kinase (CDK1 and CDK2) mRNA, cell division cycle 25 (Cdc25A and Cdc25B) protein. In addition, LCB treatment down-regulated Bcl-2 and survivin expression, enhanced Bax expression, activated caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) protein. Consistently, the tumorigenicity of LCB-treated MB49 cells was limited significantly by using the colony formation assay in vitro and the MB49 tumor model performed in C57BL/6 mice in vivo. These findings provide support for the use of LCB in chemoprevention and bladder cancer therapy.

596 A NOVEL AND EFFICIENT GENE THERAPY FOR THE TREATMENT OF UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013596 A NOVEL AND EFFICIENT GENE THERAPY FOR THE TREATMENT OF UROTHELIAL CARCINOMA Eduardo Landerer, Maximiliano Bendek, Lorena Lobos, Miguel Ávila, Alexis Rivas, Vincenzo Borgna, Luis O. Burzio, Carlos González, and Jaime Villegas Eduardo LandererEduardo Landerer Santiago, Chile More articles by this author , Maximiliano BendekMaximiliano Bendek Santiago, Chile More articles by this author , Lorena LobosLorena Lobos Santiago, Chile More articles by this author , Miguel ÁvilaMiguel Ávila Santiago, Chile More articles by this author , Alexis RivasAlexis Rivas Santiago, Chile More articles by this author , Vincenzo BorgnaVincenzo Borgna Santiago, Chile More articles by this author , Luis O. BurzioLuis O. Burzio Santiago, Chile More articles by this author , Carlos GonzálezCarlos González Santiago, Chile More articles by this author , and Jaime VillegasJaime Villegas Santiago, Chile More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1992AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The epidemiological data related to bladder cancer recurrence and mortality, support the need of novel therapeutic approaches in order to avoid recurrence, invasion and improve the survival rate and the quality of life of patients diagnosed as having this neoplasm. In the early 2000, our laboratory described the existence of a novel family of non-coding mitochondrial RNAs (ncmtRNAs), named Sense (S-ncmtRNA) and Antisense (AS-ncmtRNA). Lipofection of antisense oligodeoxynucleotides (AS-ODN) full phosphorotioate (PS), complementary to the human AS-ncmtRNA on HeLa cells, in vitro, induces cell death. Interestingly, similar treatment does not affect the viability of cultures of normal cell lines such us human keratinocytes. Aim To demonstrate that the transfection of AS-ODN complementary to the AS-ncmtRNA on bladder cancer cell lines represent a novel and efficient gene therapy for the treatment of urothelial carcinoma. METHODS Murine cell lines: The murine bladder cancer cell line MB49 were harvested at 60% of confluence, and lipofected in optimem (serum an antibiotic free) for 6 hours with 200 nM full PS AS-ODN complementary to the murine AS-ncmtRNA. Afterwards, the original culture medium was re-established, and the cells were incubated at 37° C, 5% CO2 in humidity chamber, for 48 hrs. We analysed cell viability; migration, invasion and wound healing capabilities. We used normal urothelial cells obtained as primary culture from wild type C57/Bl 6 mice like control, carrying out the same protocol described above. Human cell lines: The human bladder cancer cells line RT-4, T24 and UM-UC3 were harvested at 60% of confluence, and lipofected in optimem (serum an antibiotic free) for 6 hours with 200 nM full PS AS-ODN complementary to the murine AS-ncmtRNA. Afterwards, the original culture medium was re-established, and the cells were incubated at 37° C, 5% CO2 in humidity chamber, for 48 hrs. We analysed cell viability; migration, invasion and wound healing capabilities. RESULTS The treatment of all the bladder cancer cell lines described with AS ODN complementary to the ASncmtRNAs (human or murine) caused massive cell death, decreased proliferative capacity and diminished wound healing closure compared with those treated with a control AS-ODN and non-treated cells. Furthermore, in normal cells, the same treatment did not affect their viability. CONCLUSIONS Taken together, these results demonstrate that: 1 The AS-ncmtRNA is a suitable target for a new therapeutic strategy for bladder cancer. 2 The use of this approach can be evaluated in a murine (in vivo) bladder cancer model. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e243-e244 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eduardo Landerer Santiago, Chile More articles by this author Maximiliano Bendek Santiago, Chile More articles by this author Lorena Lobos Santiago, Chile More articles by this author Miguel Ávila Santiago, Chile More articles by this author Alexis Rivas Santiago, Chile More articles by this author Vincenzo Borgna Santiago, Chile More articles by this author Luis O. Burzio Santiago, Chile More articles by this author Carlos González Santiago, Chile More articles by this author Jaime Villegas Santiago, Chile More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 1577: Lactobacilli secreting a tumor antigen and IL15 activates neutrophils and dendritic cells and generates cytotoxic T lymphocytes against cancer cells

Abstract Introduction and Objective Lactobacillus rhamnosus GG (LGG) are lactic acid bacteria, which were found to be better than BCG at inducing cell death in human bladder cancer cell lines. This study compares both LGG and BCG in vivo in an orthopic model and further evaluates the potential of recombinant LGG to induce a directed anti-tumor response. Methods MB49-PSA (murine bladder cancer cells secreting human PSA) cells were implanted orthotopically in C57BL/6 mice and either LGG or BCG was instilled weekly for 6 weeks. Tumor cure rates were noted and an immnohistochemical examination was performed on the tumors. The human prostate specific antigen (PSA) PSA was cloned into pLP500-slpAp vector under the control of S-layer promoter of L. acidophilus. The murine IL-15 cDNA minus its termination codon was cloned upstream of the PSA protein sequence. Lactobacilli were transformed with the plasmids to generate LGG-IL15-PSA, LGG-PSA and LGG-S (pLP500-slpAp) respectively. PSA and IL15 secretion was 16-19 ng/2x109 CFU/ml. Bone marrow cells were isolated from the tibia and femur of 8-10 weeks old female C57BL/6 mice. Neutrophils were isolated by positive selection with anti-Ly6G microbead kit (Miltenyi Biotech, Germany). To generate DCs, bone marrow derived cells were cultured for 9 days in media with 40ng/ml of GM-CSF (BD Bioscience) which was replaced every other day. Neutrophils and DC were exposed to the recombinant LGG for 2h and used to stimulate T cells to generate CTL against the murine bladder cancer cell lines expressing PSA (MB49-PSA). Results LGG was as effective as BCG in the mice. Analysis of tumors after repeated weekly LGG instillations showed increased infiltration of neutrophils and macrophages into the tumor mass after therapy. In vitro, recombinant LGG activated neutrophils (elevated MHC class I expression) induced DC maturation (increased expression of CD86, CD80, CD40, MHC II and CD83), T cell proliferation and PSA specific cytotoxic T lymphocytes (CTL) activity. Activated DC were more efficient than activated neutrophils in inducing T cell IL2 and IFNα production. DC activated with LGG for 2 h or with LGG treated neutrophils were re-isolated and plated with naïve T cells for 5 days to generate PSA specific T cells. Then PSA secreting or control MB49 cells were added to the wells. IL15 enhanced DC generation of CTL. Conclusions Intravesical instillation of LGG cured mice of tumors. LGG secreting tumor antigens and cytokines could be used to activate DC in vitro for DC therapy in patients or used as intravesical therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1577. doi:1538-7445.AM2012-1577

568 LACTOBACILLI SECRETING A TUMOR ANTIGEN AND IL15 ACTIVATES NEUTROPHILS AND DENDRITIC CELLS AND GENERATES CYTOTOXIC T LYMPHOCYTES AGAINST CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2012568 LACTOBACILLI SECRETING A TUMOR ANTIGEN AND IL15 ACTIVATES NEUTROPHILS AND DENDRITIC CELLS AND GENERATES CYTOTOXIC T LYMPHOCYTES AGAINST CANCER CELLS Kesavan Esuvaranathan, Matheswaran Kandasamy, Boon Huat Bay, Yuan Kun Lee, and Ratha Mahendran Kesavan EsuvaranathanKesavan Esuvaranathan Singapore, Singapore More articles by this author , Matheswaran KandasamyMatheswaran Kandasamy Singapore, Singapore More articles by this author , Boon Huat BayBoon Huat Bay Singapore, Singapore More articles by this author , Yuan Kun LeeYuan Kun Lee Singapore, Singapore More articles by this author , and Ratha MahendranRatha Mahendran Singapore, Singapore More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.643AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Lactobacillus rhamnosus GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. Analysis of tumors after repeated weekly LGG instillations showed increased infiltration of neutrophils and macrophages into the tumor mass after therapy. This study evaluates the potential of LGG to induce a directed anti-tumor response. METHODS The human prostate specific antigen (PSA) PSA was cloned into pLP500-slpAp vector under the control of S-layer promoter of L. acidophilus. The murine IL-15 cDNA minus its termination codon was cloned upstream of the PSA protein sequence. Lactobacilli were transformed with the plasmids to generate LGG-IL15-PSA, LGG-PSA and LGG-S (pLP500-slpAp) respectively. PSA and IL15 secretion was 16-19 ng/2x109 CFU/ml. Bone marrow cells were isolated from the tibia and femur of 8-10 weeks old female C57BL/6 mice. Neutrophils were isolated by positive selection with anti-Ly6G microbead kit (Miltenyi Biotech, Germany). To generate DCs, bone marrow derived cells were cultured for 9 days in media with 40ng/ml of GM-CSF (BD Bioscience) which was replaced every other day. Neutrophils and DC were exposed to the recombinant LGG for 2h and used to stimulate T cells to generate CTL against the murine bladder cancer cell lines expressing PSA (MB49-PSA). RESULTS Recombinant LGG activated neutrophils (elevated MHC class I expression) induced DC maturation (increased expression of CD86, CD80, CD40, MHC II and CD83), T cell proliferation and PSA specific cytotoxic T lymphocytes (CTL) activity. Activated DC were more efficient than activated neutrophils in inducing T cell IL2 and IFNγ production. DC activated with LGG for 2 h or with LGG treated neutrophils were re-isolated and plated with naïve T cells for 5 days to generate PSA specific T cells. Then PSA secreting or control MB49 cells were added to the wells. IL15 enhanced DC generation of CTL and increased specific cytotoxicity. CONCLUSIONS LGG secreting tumor antigens activate antigen specific immune responses and IL15 enhances this response. This approach could be possibly be used for ex-vivo or intravesical immunotherapy. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e232 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kesavan Esuvaranathan Singapore, Singapore More articles by this author Matheswaran Kandasamy Singapore, Singapore More articles by this author Boon Huat Bay Singapore, Singapore More articles by this author Yuan Kun Lee Singapore, Singapore More articles by this author Ratha Mahendran Singapore, Singapore More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

563 IL-17 PRODUCTION BY γδ T CELLS IS IMPORTANT FOR THE ANTITUMOR EFFECT OF MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUERIN TREATMENT AGAINST BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2012563 IL-17 PRODUCTION BY γδ T CELLS IS IMPORTANT FOR THE ANTITUMOR EFFECT OF MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUERIN TREATMENT AGAINST BLADDER CANCER Takashi Dejima, Ario Takeuchi, Masatoshi Eto, Tatsuya Nakatani, Yasunobu Yoshikai, and Seiji Naito Takashi DejimaTakashi Dejima Fukuoka, Japan More articles by this author , Ario TakeuchiArio Takeuchi Fukuoka, Japan More articles by this author , Masatoshi EtoMasatoshi Eto Kumamoto, Japan More articles by this author , Tatsuya NakataniTatsuya Nakatani Osaka, Japan More articles by this author , Yasunobu YoshikaiYasunobu Yoshikai Fukuoka, Japan More articles by this author , and Seiji NaitoSeiji Naito Fukuoka, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.638AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical inoculation of Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used for the treatment of bladder cancer. However the antitumor effector mechanisms remain elusive. Recent studies demonstrated that neutrophils infiltrated in the bladder after BCG treatment played a key role in the antitumor effect. Interleukin (IL)-17 (also known as IL-17A) is a T cell-derived proinflammatory cytokine, which is involved in various pathogenesis where neutrophils are involved. However, at present, the mechanism of neutrophil infiltration after BCG treatment is not fully understood. METHODS γδ T cell-deficient (Cδ knock out (KO) and IL-17KO mice were kindly provided by Dr. S. Itohara and Dr. Y. Iwakura, respectively. The murine bladder cancer cell line, MB49, was kindly provided by Dr T. L. Ratliff. Mice were catheterized to receive an intravesical inoculate of 1 x 105 MB49 tumor cells on day 0. On days 1, 8, 15, and 22, mice were treated intravesically with either 3 x 106 CFU of BCG Connaught strain or PBS. Cytokines in the bladder were measured by ELISA. Cellular infiltrations in the bladder were analyzed by flow cytometry. For identification of IL-17-producing cells, intracellular cytokine staining method was used. RESULTS Significant infiltration of neutrophils was observed from one week after starting BCG treatment, and it gradually increased during the observation period. IL-17 production was induced as early as 1 day after BCG injection. During the course of repeated BCG administration, similar level of IL-17 production was induced after each injection. Infiltration of neutrophils was significantly reduced in IL-17KO mice. The control wild type (WT) mice treated with BCG exhibited significantly longer survival compared to PBS-treated mice. On the other hand, there was no difference in the survival between BCG- and PBS-treated IL-17KO mice. The major source of IL-17A was γδT cell population in the bladder. BCG-treated CδKO mice showed significant reduction of IL-17 production and neutrophil infiltration compared with BCG-treated control mice. On the other hand, there was no difference in either IL-17 production or neutrophil count between CD4 or NK cell-depleted mice and the control mice. Survival of CδKO mice was not improved by BCG treatment. CONCLUSIONS γδ T cells in the bladder have the anti tumor effect of intravesical BCG treatment via IL-17 production. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e230 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Dejima Fukuoka, Japan More articles by this author Ario Takeuchi Fukuoka, Japan More articles by this author Masatoshi Eto Kumamoto, Japan More articles by this author Tatsuya Nakatani Osaka, Japan More articles by this author Yasunobu Yoshikai Fukuoka, Japan More articles by this author Seiji Naito Fukuoka, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In Vivo Targeted Contrast Enhanced Micro-Ultrasound to Measure Intratumor Perfusion and Vascular Endothelial Growth Factor Receptor 2 Expression in a Mouse Orthotopic Bladder Cancer Model

We evaluated the feasibility of using targeted contrast enhanced micro-ultrasound imaging to assess intratumor perfusion and vascular endothelial growth factor receptor 2 expressions in a mouse orthotopic bladder cancer model. We created an orthotopic mouse model by implanting MBT-2 murine bladder cancer cell lines in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Successful tumor implantation was confirmed by transabdominal micro-ultrasound imaging on post-implantation day 11. Contrast enhanced micro-ultrasound imaging was done on days 14 and 21. Vascular endothelial growth factor receptor 2 targeted contrast agent was prepared by adding biotinylated anti-vascular endothelial growth factor receptor 2 monoclonal antibodies to streptavidin coated microbubbles. The targeted contrast agents were injected via the retro-orbital route. We quantified intratumor perfusion, vascular endothelial growth factor receptor 2 endothelial expression and blood volume in real time. In the initial study intratumor perfusion data and vascular endothelial growth factor receptor 2 expression could only be measured in 10 of 14 mice (71%) due to motion artifact. We modified our technique by applying an elastic band over the lower abdomen to minimize body wall movement. After the modification complete images were acquired in all mice at 2 consecutive imaging sessions. Measurements were made of intratumor perfusion and in vivo vascular endothelial growth factor receptor 2 expression. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. Targeted contrast enhanced micro-ultrasound imaging enables investigators to detect and monitor vascular changes in orthotopic bladder tumors. It may be useful for direct, noninvasive, in vivo evaluation of novel anti-angiogenesis therapeutic agents. With the modified technique target enhanced contrast ultrasound can be applied in an orthotopic bladder cancer model.

Tumour growth inhibition by an imidazoquinoline is associated with c‐Myc down‐regulation in urothelial cell carcinoma

To detect and characterize the potential role of c-Myc in the inhibition of proliferation and induction of cell death of urothelial cell carcinoma by imidazoquinolines, Toll-like receptor-7 (TLR7) agonists, that are thought to exert their immunogenic effects through the MyD88/NF-kappaB pathway. Human (T24) and murine (MBT-2) bladder cancer cell lines were cultured in normal culture medium or medium supplemented with imidazoquinoline. The effects of imidazoquinoline on gene expression, transcription and tumorigenesis were then evaluated. Effects of imidazoquinoline on in vivo bladder tumour growth and gene expression were investigated using a mouse model of orthotopic bladder cancer. There was a dose-dependent decrease in c-Myc expression in bladder cancer cells treated with imidazoquinoline; the transcriptional activity of c-Myc was also significantly reduced. Furthermore, the in vitro proliferation and tumorigenesis of MBT-2 cells were suppressed in a dose-dependent manner. For in vivo experiments, a third of mice with bladder cancer treated with intravesical imidazoquinoline showed evidence of residual bladder tumour, vs all the placebo-treated mice. In vivo expression of c-Myc, cyclin D2 and proliferating cell nuclear antigen in the bladder tumour tissue were also down-regulated. Imidazoquinolines can inhibit c-Myc expression and directly affect cell growth and tumorigenesis of bladder cancer cells, independent of an immune response. These direct effects might be synergistic with previously described immunogenic actions of imidazoquinolines. Our findings could broaden the potential application of imidazoquinoline therapy beyond dermatological malignancies, and further clinical studies are warranted.

Antitumor Effects of Imidazoquinolines in Urothelial Cell Carcinoma of the Bladder

Imidazoquinolines (Toll-like receptor-7 agonists) are a class of synthetic immune modulating agents. Imiquimod, a member of this drug family, is currently used as first line topical therapy for genital condyloma. It recently showed clinical efficacy against several benign and malignant skin lesions, including actinic keratosis and basal cell carcinoma. Working primarily through the stimulation of a proinflammatory immune response, the mechanism of action of imiquimod may be similar to that through which bacillus Calmette-Guerin is thought to act. We hypothesized that imidazoquinolines have therapeutic potential against bladder cancer. We determined the in vitro and in vivo effects of imidazoquinolines against bladder cancer cells. The human and murine J82, T24, TCC-SUP (American Tissue Culture Collection, Manassas, Virginia) and MBT-2 bladder cancer cell lines were cultured in normal culture medium or medium supplemented with imidazoquinoline. Effects on cell viability, apoptosis induction and cytokine production were evaluated. In addition, the effects of imidazoquinoline on in vivo bladder tumor growth were determined via intravesical instillation in an orthotopic bladder tumor model in the mouse. A dose dependent decrease in cell viability was observed in all tumor cell lines treated with imidazoquinoline. In addition, imidazoquinoline significantly induced apoptosis and cytokine production. In in vivo experiments most mice treated with imidazoquinoline showed only an intense inflammatory response with no evidence of tumor, while control mice showed tumor growth. Imidazoquinolines have potent direct activity against bladder cancer cells by decreasing cell viability and inducing apoptosis and cytokine production. In addition, in vivo data suggest antitumor effects in an orthotopic bladder cancer mouse model. Therefore, imidazoquinolines may have therapeutic potential as a synthetic intravesical agent against bladder cancer.

Non-viral tumor necrosis factor-alpha gene transfer decreases the incidence of orthotopic bladder tumors.

Our aim was to evaluate the feasibility and efficacy of tumor necrosis factor-alpha gene therapy in preventing bladder tumor recurrence using an orthotopic model of bladder cancer. We transiently transfected a murine bladder cancer cell line MB49 with pBud-TNF-alpha using a transfection system consisting of the cationic liposome N-(1-(2,3-dioleoyloxyl)propyl)-N,N,N-trimethylammoniummethyl sulfate (DOTAP) plus methyl-beta-cyclodextrin solubilized cholesterol (MBC). MB49 cells produced 893.7+/-24.0 pg/ml of TNF-alpha 2 days after transfection. Cell growth was inhibited, apoptosis was induced and MHC class I, B7.1 and Fas expression on the MB49 cells were increased. In vivo, an orthotopic murine bladder cancer model was established by intravesical instillation of bladder cancer cells after transurethral cauterization of the mouse bladder mucosa. TNF-alpha gene transfer was initiated 2 days after the tumor inoculation, when the tumor burden was small, and given twice per week for 3 weeks. RT-PCR showed TNF-alpha mRNA was observed to increase after the first instillation and then return to basal level 1 month after the sixth instillation. Histology revealed that TNF-alpha gene transfer decreases the bladder tumor incidence from 75% for the control group to 25% for the treated group. Increased level of T lymphocytes and NK cells was found in the TNF-alpha transfected bladders. In situ cytokine gene transfer provides significant protection against tumor growth. This approach may be useful to reduce the incidence of a subsequent tumor after endoscopic resection when used for prophylaxis.