MB49 Cells Research Articles

EBAG9 modulates host immune defense against tumor formation and metastasis by regulating cytotoxic activity of T lymphocytes.

Estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is a primary estrogen-responsive gene that we previously identified in MCF-7 breast cancer cells using the CpG genomic binding-site cloning technique. The expression of EBAG9 protein is often upregulated in malignant tumors, suggesting that this protein is involved in cancer pathophysiology. In the present study, we investigated the role of EBAG9 in host defense against implanted tumors in Ebag9-knockout (Ebag9KO) mice. MB-49 mouse bladder cancer cells were subcutaneously implanted into Ebag9KO and control mice. We found that tumor formation and metastasis to the lung by MB-49 cells were substantially reduced in Ebag9KO mice compared with control mice. The infiltration of CD8+, CD3+ and CD4+ T cells into the generated tumors was enhanced in Ebag9KO mice compared with controls. Notably, CD8+ T cells isolated from tumors in Ebag9KO mice exhibited substantial upregulation of immunity- and chemoattraction-related genes, including interleukin-10 receptor, interferon gamma, granzyme A, granzyme B and chemokine (C-X-C motif) receptor 3 compared with CD8+ T cells from tumors in control mice. The CD8+ T cells isolated from tumors in Ebag9KO mice also exhibited enhanced degranulation and increased cytolytic activity. Furthermore, the adoptive transfer of CD8+ T cells isolated from tumors in Ebag9KO host could repress tumor growth by MB-49 cells implanted in wild-type host. These results suggest that EBAG9 modulates tumor growth and metastasis by negatively regulating the adaptive immune response in host defense. EBAG9 could be a potential target for tumor immunotherapy.

Abstract 3127: The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model

Abstract Bladder cancer is the second most common malignancy of the genitourinary tumors in the world and tenth most common cancer in Taiwan. The most common type of bladder cancer is urothelial carcinoma (UC, formerly known as transitional cell carcinoma, TCC). The incidence rate is particularly high in southwestern coast of Taiwan. Treatment for superficial non-invasive bladder carcinoma is currently using intravesical chemotherapy with mitomycin C and BCG after transurethral resection of bladder tumor (TUR-BT). However, this therapy usually causes adverse effects including urinary frequency, urinary urgency, hematuria and tuberculosis infection. Therefore, we intend to find a complementary and alternative medicine for bladder cancer treatment which can bring a good result with less side effects. In our previous study, we found that a traditional Chinese Medicine (TCM), Guizhi Fuling Wan (GFW) had an effect on growth inhibition of human bladder cancer cell lines as same as mitomycin-C, doxorubicin and cisplatin, but had mild toxicicity on normal human bladder epithelial cells. In this study we investigated whether GFW could be used as an intravesical chemotherapy agent in our orthotopic bladder cancer mouse model and compared its adverse effect with mitomycin-C and BCG. A mouse bladder cancer cell line, MB49, was selected and evaluated its response to GFW treatment. Cell Counting Kit-8 (CCK-8) assay was employed to analyze cell viability and flow cytometry was used to determine cell cycle and apoptosis. MB49 cells were implanted into female C57BL/6 mice to generate our orthotopic bladder tumor model which was confirmed under a microscope in histology by hematoxylin and eosin staining. Our data showed that GFW was a potent inhibitor of the proliferation of mouse bladder cancer cells in vitro as well as inhibited the tumor growth on MB49 bladder cancer orthotopic model. In addition, bladder hemorrhage which was observed in bladder cancer mice had completely disappeared 17 days after the intravesical GFW-treatment. These results support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer. Citation Format: Chi-Chen Lu, Ling-Huei Tseng, Syue-Yi Chen, Jiann-Der Wu, Shu-Fen Wu, Michael W.Y. Chan, Yu-Wei Leu, Cheng-Da Hsu. The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2014-3127

The natural flavonoid silybin improves the response to Photodynamic Therapy of bladder cancer cells

Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments.In the present work, we evaluated the cytotoxicity of the combination of ALA–PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA.Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou–Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells.Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment.Overall, our results suggest that the combination of silybin and ALA–PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.

CD40L gene therapy tilts the myeloid cell profile and promotes infiltration of activated T lymphocytes

CD40 ligand (CD40L) is a potent stimulator of tumor immunity via its activation of dendritic cells, which in turn initiate T-cell activation. However, T cells are inhibited by suppressive myeloid cells, which constitute an important part of immune evasion. We hypothesized that CD40L may revert the function of suppressive myeloid cells to generate a T-cell stimulatory environment, and this was investigated in the murine bladder cancer model MB49/C57BL/6. Upon intratumoral adenoviral CD40L (AdCD40L) gene therapy, the infiltration of CD11b(+)Gr-1(+) cells was significantly reduced, whereas activated T cells were increased. In vitro, CD40L-expressing MB49 cells tilted the myeloid subpopulations in favor of granulocytic CD11b(+)Gr-1(high) myeloid cells instead of monocytic CD11b(+)Gr-1(int/low) myeloid cells. Further, the level of macrophages in splenocyte co-cultures with MB49 cells was evaluated. In cultures with MB49 cells expressing CD40L, the overall level of macrophages was reduced and the remaining cells were differentiated into M1-like cells. Hence, these data support that CD40L tilts myeloid immune cell populations in favor of anti-tumor immunity (M1) instead of immunosuppression (CD11b(+)Gr-1(int/low) and M2), and this was accompanied by an increased level of activated T cells in the tumor tissue.

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.

Enhanced therapeutic anti-tumor immunity induced by co-administration of 5-fluorouracil and adenovirus expressing CD40 ligand.

Bystander immune activation by chemotherapy has recently gained extensive interest and provided support for the clinical use of chemotherapeutic agents in combination with immune enhancers. The CD40 ligand (CD40L; CD154) is a potent regulator of the anti-tumor immune response and recombinant adenovirus (RAd)-mediated CD40L gene therapy has been effective in various cancer models and in man. In this study we have assessed the combined effect of local RAd-CD40L and 5-fluorouracil (5-FU) administration on a syngeneic MB49 mouse bladder tumor model. Whereas MB49 cells implanted into immunocompetent mice responded poorly to RAd-CD40L or 5-FU alone, administration of both agents dramatically decreased tumor growth, increased survival of the mice and induced systemic MB49-specific immunity. This combination treatment was ineffective in athymic nude mice, highlighting an important role for T cell mediated anti-tumor immunity for full efficacy. 5-FU up-regulated the expression of Fas and immunogenic cell death markers in MB49 cells and cytotoxic T lymphocytes from mice receiving RAd-CD40L immunotherapy efficiently lysed 5-FU treated MB49 cells in a Fas ligand-dependent manner. Furthermore, local RAd-CD40L and 5-FU administration induced a shift of myeloid-derived suppressor cell phenotype into a less suppressive population. Collectively, these data suggest that RAd-CD40L gene therapy is a promising adjuvant treatment to 5-FU for the management of bladder cancer.

A chorioallantoic membrane model for the determination of anti-angiogenic effects of imatinib

Tumor angiogenesis is of major importance in the growth and metastasis of solid tumors, and the development of anti-angiogenic treatment strategies is thus a relevant option in oncology. The chorioallantoic membrane (CAM) model is a rapid and simple alternative to in vivo studies for the evaluation of anti-angiogenic compounds, thus allowing to reduce animal experiments and, upon establishment of robust and reproducible procedures, to more efficiently and objectively assess the anti-angiogenic efficacy of a given drug.In this paper, we compare two different methods for tumor establishment on a CAM model: (i) a Murine Urothelial Carcinoma (MB49) cell suspension mixed with Matrigel and (ii) an MB49 cell suspension absorbed in Gelfoam gelatin sponges. Based on the applicability of both methods for implant formation, we identify Gelfoam gelatin sponges as superior due to better attachment of the tumors on the membrane surface. For the precise quantitation of tumor xenograft growth and angiogenesis, we furthermore establish in this paper the electronic capturing of the xenografts and the computer-based analysis of the microscopic CAM images in order to determine the number of intersecting vessels and to measure vessel diameters.Beyond its direct effect on tumor cells by inhibiting the tyrosine kinase domain of the abl gene, imatinib has been reported to reduce the Bcr-Abl-mediated secretion of the angiogenesis factor VEGF and hence to interfere with angiogenesis. To test our CAM model for its ability to monitor anti-angiogenic effects, Gelfoam gelatin sponge-based tumor implants were treated by topical application of imatinib at various concentrations. Besides anti-tumor effects, we observed an inhibition of angiogenesis as determined by the number or total diameter of intersecting vessels. We also demonstrate that the calculation of the “blood vessel index” (vessel total diameter/tumor circumference) in our model allows to assess anti-angiogenic effects of imatinib independently of tumor growth inhibition. We conclude that our CAM assay and computer-based analysis represent a useful in vitro technique for the rapid assessment of anti-angiogenic effects of various agents.

Target expression of Staphylococcus enterotoxin A from an oncolytic adenovirus suppresses mouse bladder tumor growth and recruits CD3+ T cell

We recently engineered an oncolytic adenovirus (PPE3-SEA) that expresses the superantigen, Staphylococcus enterotoxin A (SEA), and that has enhanced tumor specificity because the telomerase reverse transcriptase and hypoxia-inducible factor promoters regulate expression of E1A and E1B genes, respectively. Here, we evaluated the PPE3-SEA adenovirus anti-tumor activity against MB49 mouse bladder cancer cell proliferation in vitro and in vivo. PPE3-SEA infection of murine MB49 cells in vitro induced cytopathic effects, and significant expression of SEA mRNA and protein, as measured by RT-PCR and western blot, respectively. Subcutaneous MB29 bladder tumors were established in syngeneic C57BL/6 mice. After 10 days, tumors were injected with either oncolytic virus or PBS. Tumor dimensions were measured on days 1, 3, 5, 7, 9, and 11 post-treatment and tumor volumes were calculated. One of eight PPE3-SEA-treated mice had no tumor by day 9. PPE3-SEA treated group had significantly lower mean tumor volume beginning on day 5 post-treatment (p < 0.01), more fibrous tissue in the tumor, and increased presence of infiltrating CD3+ T cells than those of the control group. Gross appearance and histologic sections from the livers and kidneys of the PPE3-SEA-treated group were similar to those of the control group. In conclusion, oncolytic adenoviruses can provide a novel delivery vehicle for SEA to tumor sites, and PPE3-SEA warrants further study as a potential anti-tumor agent for bladder cancer.

584 THERAPEUTIC EFFECT OF INTRAVESICAL ADMINISTRATION OF A NOVEL NANOTRANSPORTER FOR PACLITAXEL IN AN ORTHOTOPIC BLADDER CANCER MODEL

was to examine the potential of intravesical chitosan/IL-12 immunotherapy to eliminate established orthotopic bladder tumors and elicit adaptive, tumor-specific immunity. METHODS: C57BL/6 mice bearing orthotopic MB49 bladder tumors were treated intravesically with saline, IL-12 or chitosan/IL-12 on days 6, 9, and 12 after tumor implantation. Mice eliminating orthotopic MB49 tumors following intravesical chitosan/IL-12 treatment were rechallenged subcutaneously on the flank with MB49 cells or an irrelevant tumor (B16 melanoma). Lymphocytes isolated from cured mice were evaluated for cytotoxic T lymphocyte activity against MB49 or B16 targets. RESULTS: Intravesical chitosan/IL-12 immunotherapy was found to eliminate established tumors in 100% of mice. In addition, all cured mice were protected from a distant MB49 rechallenge, but not B16 challenge. Similarly, lymphocytes from cured mice were found to lyse MB49, but not B16 cells in vitro. Protective immunity was found to be dependent on CD4 and CD8 subsets as their depletion abrogated protection. CONCLUSIONS: These data are the first to show that an intravesical IL-12-based immunotherapy for bladder cancer can induce a systemic tumor-specific immune response. As such, this treatment may be suitable for patients with recurrent superficial disease as well as invasive and metastatic bladder cancers for which there are no effective treatment options.

Role of Peroxisome Proliferator Activated Receptor-Gamma in Bacillus Calmette-Guérin Bladder Cancer Therapy

We evaluated the effects of combined PPARg agonist with bacillus Calmette-Guérin in bladder cancer growth in vitro and in vivo, focusing on the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. PPARs are a superfamily of nuclear receptors that are transcription factors activated by ligands. Activation of PPARg, the γ subtype, causes proliferation inhibition or differentiation of tumor cells. Previously, we reported that the inhibition of murine bladder tumor growth induced by bacillus Calmette-Guérin, which is the standard treatment for patients with nonmuscle invasive, high grade bladder cancer, increased PPARg expression in vitro and in vivo. In vitro the cell growth inhibition induced by bacillus Calmette-Guérin was enhanced by the PPARg agonist 15-d-PGJ2, raising the possibility that PPARg activation may be a therapeutic modality for this disease. In MB49 cells bacillus Calmette-Guérin and 15-d-PGJ2 induced PPARg expression, nuclear translocation and transcriptional activity. In vivo bacillus Calmette-Guérin reduced tumor size, an effect that was partially reversed when bacillus Calmette-Guérin was combined with the PPARg agonist rosiglitazone. The same result was found when we analyzed the effect of the PPARg antagonist BADGE (Fluka Chemical, Buchs, Switzerland) combined with bacillus Calmette-Guérin. Analysis of the activation of macrophages and fibroblasts demonstrated that rosiglitazone inhibited the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. Results suggest that PPARg is involved in the antitumor action of bacillus Calmette-Guérin. However, exogenous PPARg agonists would not be a favorable therapeutic modality because they can inhibit the tissue remodeling needed for an overall satisfactory bacillus Calmette-Guérin response.

HMGB1 Release by Urothelial Carcinoma Cells is Required for the In Vivo Antitumor Response to Bacillus Calmette-Guérin

Prior series showed that a portion of urothelial carcinoma cells exposed to bacillus Calmette-Guérin undergoes nonapoptotic cell death and release of the chemokine HMGB1. We evaluated the role of tumor cell derived HMGB1 in mediating the in vivo antitumor effect of bacillus Calmette-Guérin. The murine urothelial carcinoma cell line MB49 was engineered to express a shRNA construct targeting HMGB1. The shRNA expressing cell line underwent characterization to ensure its comparability to the parental MB49 cell line. An orthotopic tumor model was used to compare the in vivo antitumor efficacy of bacillus Calmette-Guérin in the parental cell line (24 control and 24 bacillus Calmette-Guérin treated) vs the HMGB1 knockdown line (23 control and 21 treated). Expression of the shRNA construct decreased HMGB1 expression and its release in response to bacillus Calmette-Guérin. The parental and shRNA cell lines showed similar in vitro doubling time and cytotoxicity in response to bacillus Calmette-Guérin. Treatment significantly decreased tumor volume vs controls in parental MB49 tumor bearing mice (p = 0.036). Tumor volume in treated mice inoculated with the shRNA cell line was higher than that in sham treated shRNA controls (p = 0.12). Of the bacillus Calmette-Guérin treated mice tumor volume was significantly lower in parental tumor bearing mice vs the shRNA group (p <0.00001). ANOVA revealed a significant interaction between the cell line (shRNA vs parental) and the bacillus Calmette-Guérin effect (p = 0.0076). The direct tumor response to bacillus Calmette-Guérin, culminating in HMGB1 release, may be an important contributor to the clinical efficacy of bacillus Calmette-Guérin.

Inducible Nitric Oxide Synthase and PPARγ are Involved in Bladder Cancer Progression

We evaluated the role of inducible nitric oxide synthase and PPARγ as prognostic factors for bladder cancer. Inducible nitric oxide synthase and PPARγ were evaluated by Western blot and immunohistochemistry in a mouse bladder cancer model of nonmuscle invasive and invasive MB49-I tumor cells, and in human bladder cancer samples. Inducible nitric oxide synthase expression was negative in mouse normal urothelium and higher in invasive than in noninvasive MB49 tumors. In vitro inducible nitric oxide synthase activity, determined as nitrite, was higher in MB49-I than in MB49 cells (p <0.001). In human samples expression was also associated with tumor invasion. Nuclear PPARγ expression was negative in normal mouse urothelium but higher in nonmuscle invasive MB49 than in MB49-I tumors. Similarly in human tumors low PPARγ was associated with poor prognosis factors, such as high histological grade (p = 0.0160) and invasion status (p = 0.0352). A positive correlation was noted between inducible nitric oxide synthase and PPARγ in low histological grade and nonmuscle invasive tumors (Pearson correlation index 0.6368, p = 0.0351, 0.4438 and 0.0168, respectively). As determined by gene reporter assay, PPARγ activity was induced by nitric oxide only in nonmuscle invasive MB49 cells (p <0.001). Results suggest that increased PPARγ controls inducible nitric oxide synthase expression at early tumor stages. However, regulation is lost at advanced tumor stages, when the increase in inducible nitric oxide synthase and the decrease in PPARγ seem to be associated with bladder cancer progression.

Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1‐phosphate signalling to regulate tumour metastasis

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP+/+SK1−/− mice, or lung metastasis of multiple cancer cells in SK1−/− animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2−/− MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

The preclinical activity of lenalidomide in urothelial carcinoma (UC).

e15002 Background: Lenalidomide is approved for multiple myeloma and deletion 5q myelodysplastic syndromes (MDS) and demonstrates immune modulation, anti-angiogenic activity and direct anti-tumor cytotoxicity. A rationale can be made to evaluate the preclinical activity of lenalidomide in urothelial carcinoma (UC) based on the importance of these pathways. Methods: The in vitro anti-tumor activity of lenalidomide was evaluated in 4 human (5637, TCC-SUP, RT4, RT112) and 1 murine (MB49) cell line. Anti-proliferative activity activity (MTT assay), apoptosis (Annexin-FITC immunohistochemistry [IHC], flow cytometry) and cell viability by colony forming assay were measured. In vivo examination of activity of daily oral lenalidomide 10 mg/kg orally once daily or placebo for 4 weeks is examined in a syngeneic immunocompetent mouse model employing MB49-Luc25 cells injected subcutaneously in C57BL/6 mice. Murine tumors will be studied for anti-tumor activity. Results: In vitro activity of lenalidomide was detected at low ~1 µM concentrations (attainable in human subjects) against a non-invasive human UC cell line (RT4). Long-term cultures of RT4 cells for 10 days with daily repletion of lenalidomide reduced cell viability and colony forming ability to 75.6% of controls. Induction of apoptosis in RT4 was demonstrated by Annexin-FITC IHC and flow cytometry compared to control (30.11 vs. 14.74%). Invasive human UC cells and murine MB49 cells did not demonstrate apoptosis with lenalidomide exposure in vitro. Futher, lenalidomide did not inhibit overall tumor growth in the syngeneic immunocompetent murine model; immune activity and stem cell directed activity will be presented. Conclusions: Lenalidomide demonstrated preclinical anti-tumor activity against non-invasive human UC cells. Given its favorable toxicity profile compared to cytotoxic chemotherapy, clinical evaluation in patients with non-muscle-invasive bladder cancer and recurrence after BCG therapy may be warranted.

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

Abstract 5386: Immunotherapy of bladder cancer using bacillus calmette guérin (BCG): Generation of cytotoxic T-cells is BCG strain dependent

Abstract Introduction: Forty years ago Zbar and colleagues demonstrated that bacillus calmette guérin (BCG) is capable of provoking an immune response against experimentally induced tumors. The conditions for successful tumor immunity were defined by a i) limited number of tumor cells in ii) close contact with a iii) sufficient number of bacteria and iv) the ability of the organism to mount a BCG specific immune response. These findings led to the development of what is currently the standard of care for the treatment of patients with non-muscle invasive bladder cancer. Whether the genetic drift between BCG strains that are routinely used in treatment protocols for bladder cancer affects the generation of successful tumor immunity is unknown. Here we compared two of the commonly used BCG strains (Connaught and Tice) in the treatment of bladder cancer in an animal model. Methods: Naïve or bladder cancer bearing (orthotopic implantation of syngeneic MB49 cells) Bl/6 mice received either a single s.c. injection or weekly intravesical instillation of 107 CFUs of BCG Tice or Connaught. Twelve days after the 4th intravesical treatment or after the single s.c. immunization, the bladders, spleens and draining lymph nodes (DLN) were removed, enzymatically digested and the resulting cell suspensions analyzed by flow cytometry, including assessment of BCG specific CD8+ T-cells. Lymph node extracts were cultivated for the enumeration of BCG CFUs. Tumor bearing animals were assessed for their overall survival. Results: In non-tumor bearing animals, BCG Connaught lead to a more robust influx of adaptive immune cells into the bladder and primed significantly more BCG specific cytotoxic CD8+ T-cells. Compared to BCG Connaught, BCG Tice was less capable to disseminate into the draining lymph nodes (DLN), and induced less Th1 polarized CD4+ T-cells in the bladder, DLN and spleens, as assed by intracellular T-bet staining. These findings were independent of BCG preparation (clinical grade versus laboratory grade) and were seen for identical treatment doses. Finally, mice, orthotopically challenged with MB49 bladder cancer cells, showed significantly better survival if treated with BCG Connaught as compared to BCG Tice. Conclusion: Here we show that different BCG strains significantly impact the immunological response of adaptive immunity. Along with a stronger Th1 immune response, favoring the generation of cytotoxic T-cells, BCG Connaught improves survival as compared to BCG Tice in vivo. These findings implicate potential consequences for clinical practice and corroborate testing of different BCG strains in prospective clinical trials. 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 5386. doi:1538-7445.AM2012-5386

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 ...

A Novel Therapeutic Vaccine of Mouse GM-CSF Surface Modified MB49 Cells Against Metastatic Bladder Cancer

Immunotherapy is considered effective for muscle invasive bladder cancer mini metastasis. We developed what is to our knowledge a novel technology by which streptavidin tagged mouse GM-CSF was displayed on the surface of biotinylated bladder cancer cells to induce antitumor immunity. Mouse subcutaneous and lung metastasis bladder cancer models were established. Mice were injected subcutaneously with 1 × 10(6) mouse GM-CSF surface modified MB49 bladder cancer cells and monitored for tumor growth and survival. Immunohistochemical and flow cytometric assay were done to assess the proportion of T lymphocytes. The T-lymphocyte cytotoxicity assay was performed to assess MB49 specific cytotoxicity. On day 60 after MB49 implantation the vaccine cured mice were injected subcutaneously with MB49 or RM-1 cells in the left or right hind leg, respectively. They were monitored for survival and T-lymphocyte cytotoxicity. Mouse GM-CSF surface modified vaccine significantly inhibited tumor growth in the subcutaneous model and extended survival in the lung model. More CD4 and CD8 T cells appeared at tumor sites and in peripheral blood in the vaccine treated group than in other control groups. Splenocytes from the vaccine treated group showed the most potent cytotoxicity on MB49 cells. Cured mice in the vaccine treated group resisted the second injection of MB49 bladder cancer cells but not the RM-1 prostate cancer cell challenge. Mouse GM-CSF surface modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient for metastatic bladder cancer.

Abstract A220: The efficacy of varying concentrations of intravesical apaziquone on murine bladder cancer in an animal model.

Abstract Introduction and Objectives: Apaziquone is a novel bioreductive alkylating agent in development for intravesical therapy of nonmuscle invasive bladder cancer. The standard dose of apaziquone in all ongoing clinical studies is 4 mg/ 40 mL (0.1mg/mL). The objective of this study was to test the efficacy of varying concentrations of apaziquone versus placebo in an animal model of bladder cancer. Methods: A total of 1×105 MB49 cells were instilled into the bladder of C57BL/6 mice after electrocauterization to establish the tumor model. Mice bearing orthotopic tumors were administered varying concentrations of apaziquone (0.05, 0.1, 0.4, and 0.8 mg/mL) or placebo (0.85% saline) via intravesical instillation (0.1 mL) one week after tumor implantation. Instillations were continued once a week for 4 weeks. Tumor incidence, overall and median survival is reported. Results: At the end of the observation period (80 days), all animals in the control (0.85% saline) and 0.05 mg/mL groups had died (fig. 1). The median survival of mice receiving apaziquone at concentrations of 0.1 mg/mL (42.5 days); 0.4 mg/mL (40.5 days) and 0.8 mg/mL (49 days) was longer than that of mice treated with 0.05 mg/mL (30 days) and 0.85% saline (27 days). Statistically significant differences in survival were observed in control vs apaziquone-treated groups [0.1 mg/ml(P &amp;lt; 0.05); 0.4 mg/mL(P &amp;lt; 0.01); 0.8 mg/mL (P &amp;lt; 0.01)], and in the low concentration (0.05 mg/mL) vs 0.1 mg/mL (P &amp;lt; 0.05), 0.4 mg/mL (P &amp;lt; 0.01) and 0.8 mg/mL (P &amp;lt; 0.01), respectively. The median survival of the 0.1, 0.4 and 0.8 mg/mL treated groups did not differ, but 33.3% in the 0.1 mg/mL cohort, and 14.3% in the 0.4 mg/mL cohort were long-term survivors (&amp;gt;80 days). All other animals (n = 37) had died of cancer by the end of the observation period. Three mice did not survive anesthesia. At necropsy all mice had advanced local disease with deep invasion of the muscularis propria, and usually with multiple lung metastases. Conclusion: Apaziquone at the 0.1 mg/mL concentration demonstrates excellent antitumor activity compared to placebo or 0.05 mg/mL solutions. In this model there was no significant difference in the median survival between 0.1 mg/mL and higher concentrations of apaziquone (0.4 and 0.8 mg/mL) supporting continued development with the current clinical dose (0.1mg/mL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A220.

Sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines against the metastatic bladder cancer

ObjectivesMany strategies are pursued to enhance tumor vaccine immune response, including the utilization of cytokines. We have developed a novel protein-anchor technology to immobilize cytokines on tumor cell surface. Here we reported the preparation of tumor cell vaccines by immobilizing GM-CSF or IL-2 on MB49 bladder cancer cells and evaluated their antitumor efficacy (administrated alone or sequentially) in a metastatic mouse model. Materials and methodsSA-mGM-CSF or SA-hIL-2 surface-modified MB49 cells were prepared as vaccine. Mice were treated with MB49 cell vaccines (administrated alone or sequentially). Survival time, tumor growth, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and cytotoxic T lymphocytes (CTL) assay were used to evaluate the antitumor efficiency of the vaccines in the pulmonary metastatic model of bladder cancer. ResultsGM-CSF vaccine induced more mature dendritic cells in the mice spleen. Combination with subsequent IL-2 vaccine significantly increased CD4+, CD8+, and IFN-γ+CD8+ T but not CD4+Foxp3+ T cell population and induced the highest production of IFN-γ, IL-12, but not IL-10. Furthermore, the splenocytes from the sequentially combined vaccines group showed the most potent cytotoxicity on MB49 cells. Finally, the sequentially combined vaccines evidently extended the survival time of mice (the median survival time of PBS, ethanol-fixed, anchored GM-CSF, anchored IL-2, and anchored GM-CSF + anchored IL-2 groups were 34, 37, 45, 47, and 59 days, respectively) and effectively protected the mice against a second MB49 cells but not RM-1 cells challenge. ConclusionsThis study demonstrated that sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines could effectively induce specific antitumor immune response.