Murine Bladder Cancer Model Research Articles

Abstract 4429: Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells.

Abstract Erlotinib is a first-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor and have well-established efficacy in non-small cell lung cancer patients with activating EGFR mutations. Afatinib, on the other hand, is a second-generation EGFR tyrosine kinase inhibitor with anti-HER2 activity and was found to be of benefit to patients with advanced lung adenocarcinoma who failed previous gefetinib or erlotinib. In previous study we have demonstrated the in vitro and in vivo radiosensitising activity of afatinib in a murine bladder cancer model. However, the radiosensitizing effect of afatinib and erlotinib, has never been compared in cancer cells including bladder cancer. We performed RTK (Receptor Tyrosine Kinase) antibody arrays to investigate the relative levels of phosphorylation in T24 human bladder cancer cell line. We found that EGFR and HER2 signals were activated after radiation10 Gy. Afatinib 100 nM suppressed both irradiation-induced EGFR and HER2 signals but erlotinib 100 nM only suppressed irradiation-induced EGFR signal. Clonogenic assay of T24 and NTUB1 human bladder cancer cell lines was then examined. Afatinib (100-500 nM) showed better radiosensitizing effect (radiation dose: 2-10 Gy) than erlotinib (300-1500 nM) in both T24 and NTUB1 cells by significantly decreasing the numbers of colonies 7 days after treatment. The effect is more prominent in high doses of afatinib. Flow cytometry was used to determine the distribution of cells among various cell cycle phases. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 200 nM, erlotinib 200 nM or the combination of radiation and afatinib or erlotinib. When compared with radiation or drug alone, treatment combining radiation and afatinib in T24 cells resulted in a significant increase of cells in sub-G1 phase but the phenomenon was not observed in the combination of erlotinib and radiation. A similar trend was found in NTUB1 cells. The results implicated that afatinib might enhance radiation effect by increasing apoptosis in bladder cancer cells. To study the DNA damage status after treatment, we checked intra-nuclear γH2AX foci by mmunofluorescence microscopy. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 100 nM, erlotinib 100 nM or the combination of radiation and afatinib or erlotinib. Radiation alone significantly increased γH2AX foci and combining radiation with afatinib further increased the foci in both T24 and NTUB1 cells. The enhancement was absent in the combination of radiation and erlotinib. Our data clearly show that afatinib, a new generation EGFR tyrosine kinase inhibitor with anti-HER2 activity, is superior to erlotinib as a radiosensitizer in the treatment of human bladder cancer cells. Citation Format: Yu-Chieh Tsai, Tsung-Fan Tuan, Pei-Yin Ho, Wei-Lin Liu, Liang-Yu Chang, Yeong-Shiau Pu, Ann-Lii Cheng, Jason Chia-Hsien Chen. Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2013-4429

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 ...

Cytogenetic characterization of the murine bladder cancer model MB49 and the derived invasive line MB49-I

Bladder cancer is frequently associated with chromosomal abnormalities, and the complexity of karyotypes increases with tumor progression. The murine model MB49 is one of the most widely studied models of bladder cancer. We developed the invasive cell line MB49-I by successive invivo passages of MB49 primary tumors. Because little is known about the chromosomal alterations of this model, our goal was to perform cytogenetic analyses of the MB49 and MB49-I lines. The karyotypes of both lines were analyzed by G-banding and fluorescence in situ hybridization techniques. Both lines were composed of two cell subpopulations, a diploid population, which was found mainly in the MB49 line, and the tetraploid population, which was found mainly in the MB49-I line. A translocation between chromosomes 5 and 9 and an isochromosome of chromosome 19 were observed in the subpopulations of both lines. New structural abnormalities and additional chromosomal imbalances were detected in the MB49-I line. Tumor progression in the MB49/MB49-I model was associated with a selection of polyploid cells with accompanying chromosomal abnormalities. This model may be advantageous for the study of the genetic changes associated with the progression of bladder cancer.

Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

Effect of lenalidomide on the response of bladder cancer to BCG immunotherapy in an in vivo murine model.

288 Background: Intravesical BCG is the gold standard for non-muscle-invasive bladder cancer (NMIBC). However, many patients do not respond to therapy while others relapse and/or progress. As a result, there remains a need for therapies that can enhance the efficacy of BCG. We explore the efficacy of lenalidomide, an immunomodulatory agent used in multiple myeloma and myelodysplastic syndrome, in combination with BCG in vitro and in an in vivo bladder cancer model. Methods: We studied the effects of lenalidomide in combination with BCG induced cytokines in MBT-2 cells using PI-FACS. For in vitro studies, we used 10 and 100 nM of lenalidomide in combination with TNF-a and FasL. We then performed Western blotting for cell cycle and apoptosis regulatory proteins. Subsequently, we tested the efficacy of this combination in an immunocompetent murine model of bladder cancer with MBT-2 cells in C3H mice using the flank injection method. Drug dosages were 30 mg/kg for lenalidomide and 105 CFU of BCG. Tumor growth curves were created for the control, lenalidomide, BCG and combination treatment mice groups. Immunohistochemistry (IHC) was then performed using antibodies against proteins related to cell cycle, apoptosis, angiogenesis and immune response. Results: PI-FACS identified increased DNA fragmentation in the combinations of lenalidomide and TNF-a and FasL compared to control and each agent alone. Using Western blotting, we demonstrated that the combination resulted in apoptosis via caspase-3 activation. In the murine model, combination therapy resulted in a statistically significant decreased tumor size compared to the control group. While the BCG alone and lenalidomide alone groups did show a trend toward smaller tumor, they did not reach statistical significance. Furthermore, the TUNEL assay showed a substantial increase in apoptosis only in the combination group. Immunohistochemistry demonstrated decreased angiogenesis in all treatment groups compared to control, as well as, decreased T-cell infiltration. Conclusions: Our study demonstrates a potential role for the immunomodulatory agent, lenalidomide, in combination with BCG for NMIBC. This in vivo model serves as a template for future clinical trials.

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.

Tumor Establishment Features of Orthotopic Murine Bladder Cancer Models

PurposeAnimal tumor models are important for the evaluation of novel therapeutic modalities. Since the initial report of an orthotopic bladder tumor model, several modifications have been proposed to improve the tumor take rate. Here we compared the HCl-pretreated and electrocauterization-pretreated orthotopic murine bladder tumor models.Materials and MethodsMBT-2 murine bladder cancer cells were transurethrally implanted in the bladder of syngeneic C3H/He mice. The mice were divided into three groups according to pretreatment methods (electrocautery, HCl, and control group) and were subjected to pretreatment before instillation of MBT-2 tumor cells into the bladder. Mice were sacrificed on day 21, and bladders were harvested, weighed, and examined histopathologically.ResultsThe tumor take rate of the control, electrocautery, and HCl groups was 0%, 54%, and 100%, respectively. The tumor take rate of the HCl group was significantly higher than that of the control group (p<0.01) and the electrocautery group (p=0.01). Pathologic reports revealed that all established bladder tumors were high-grade papillary urothelial carcinomas.ConclusionsThe HCl pretreatment model was a preferable murine bladder tumor model for evaluating further therapeutic interventions.

CD24 Offers a Therapeutic Target for Control of Bladder Cancer Metastasis Based on a Requirement for Lung Colonization

Metastasis is lethal in most bladder cancer patients. Expression of CD24, a glycosyl phosphatidylinositol (GPI)-linked sialoglycoprotein and cancer stem cell marker, is associated with metastatic progression in multiple cancer types, yet the role of CD24 in this process remains unclear. While developing a murine model of human metastatic bladder cancer, we observed that tumor cell CD24 expression correlated with a propensity to metastasize to the lung. Our immunohistochemical evaluation of 60 paired primary and metastatic human bladder cancer samples revealed increased intensity (P < 0.001) and frequency (P < 0.001) of CD24 expression in metastases. To directly evaluate the role of CD24 in metastatic colonization, we manipulated CD24 expression in human bladder cancer cell lines using short hairpin RNA depletion, cDNA overexpression, and fluorescence-activated cell sorting selection. Although suppression of CD24 reduced acute tumor cell retention in the lungs of mice inoculated intravenously with cancer cells, this differential retention was no longer apparent after 24 hours, prompting us to evaluate the role of CD24 in lung colonization. Here, CD24 was found necessary for subsequent development of lung metastases. We next treated clinically detectable lung metastases in mice with anti-CD24 antibody and observed reduced tumor growth and prolonged survival. These findings suggest that CD24 is a lynchpin of metastatic progression and a promising therapeutic target for antimetastatic therapy.

Abstract 2697: Enhanced antitumor effect of combination intravesical BCG plus Docetaxel therapy in an orthotopic bladder cancer model

Abstract Introduction and Objective: Little progress in the management of non-muscle invasive bladder cancer (NMIBC) has been made. We explored the combination of intravesical BCG plus Doc in an orthotopic model. Methods: An orthotopic murine bladder cancer model was used. To assess the antitumoral effect of Doc, intravesical Doc therapy was administered at various dose-escalating concentrations: 0 μg (control: PBS), 25 μg, 50 μg and 100 μg (n = 12 for each group; total of 4 groups). Next, a comparative evaluation of tumor growth among the control group, the BCG-alone group, the Doc-alone group, and three combined BCG + Doc groups (a coincident group [CO] and two sequential groups) was performed (n = 12 for each group; total of 6 groups). The sequential combined groups consisted of SA group (SA; Doc administration followed by BCG administration on the next day) and an SB group (SB; BCG administration followed by Doc administration on the next day). Intravesical therapy was administered at 3-day intervals starting on day 5 and was repeated 5 times. To evaluate proliferation, T cell infiltration and apoptosis among the different groups, the tumor cells in the animal model were stained for Ki-67, CD8 and Cleaved caspase 3 after 4 instillations performed every 3 days. Results: In the Doc groups (12 mice each) treated with a dose of 0 μg (survival period: 32.3 ± 9.4 days), 25 μg (36.8 ± 11.4), 50 μg (38.1 ± 11.0) or 100 μg (40.0 ± 12.7), one, two, two and three mice failed to develop tumors and survived, respectively. The survival advantage for intravesical Doc in the 100 μg administration group was statistically significant when compared with that in the control group (p = 0.04). Out of the 12 mice in each group treated with control, Doc-alone, BCG-alone or combined Doc + BCG (CO, SA, SB), 1, 2, 1, 4, 4 and 1 mice failed to develop tumors and survived, respectively. Significant survival advantages were observed in the combined BCG + Doc group (CO: 50.7 ± 9.0, SA: 51.5 ± 6.9) compared with that in the BCG-alone group (33.1 ± 10.4) and the Doc-alone group (38.8 ± 11.5). The Ki-67 index of the cancer cells was significantly lower in the combined group, compared with that in the BCG-alone group (p &amp;lt; 0.0001), the Doc-alone group (p &amp;lt; 0.05) and the control group (p &amp;lt; 0.0001). BCG-alone significantly increased the number of CD8+ T cells (p &amp;lt; 0.05), while Doc-alone reduced the number of CD8+ T cells (p &amp;lt; 0.05), compared with that in the control. Apoptosis was significantly increased when Doc was administrated to the animals irrespective of the presence (p &amp;lt; 0.05) or absence (p &amp;lt; 0.05) of BCG instillation. Conclusions: Our results suggested that the combination of BCG and Doc treatment decreased the tumor appearance rate and improved the survival period. The enhancement in tumor growth inhibition enabled by combination regimens was presumably associated with a reduction in the tumor proliferation rate and might not depend on either CD8+ T cells or apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2697. doi:10.1158/1538-7445.AM2011-2697

521 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS DOCETAXEL THERAPY IN AN ORTHOTOPIC BLADDER CANCER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 2011521 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS DOCETAXEL THERAPY IN AN ORTHOTOPIC BLADDER CANCER MODEL Minoru Horinaga, Ryuichi Fukuyama, Masahiro Iida, Masashi Matsushima, Hitoshi Yanaihara, Yoko Nakahira, Munehisa Ueno, and Hirotaka Asakura Minoru HorinagaMinoru Horinaga Saitama, Japan More articles by this author , Ryuichi FukuyamaRyuichi Fukuyama Konan, Japan More articles by this author , Masahiro IidaMasahiro Iida Saitama, Japan More articles by this author , Masashi MatsushimaMasashi Matsushima Saitama, Japan More articles by this author , Hitoshi YanaiharaHitoshi Yanaihara Saitama, Japan More articles by this author , Yoko NakahiraYoko Nakahira Saitama, Japan More articles by this author , Munehisa UenoMunehisa Ueno Saitama, Japan More articles by this author , and Hirotaka AsakuraHirotaka Asakura Saitama, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1217AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical BCG therapy is currently the most successful adjuvant agent for patients with non-muscle invasive bladder cancer (NMIBC). However, little progress in the management of NMIBC has been made. We explored the combination of intravesical BCG + Docetaxel(Doc) in an orthotopic bladder cancer model. METHODS An orthotopic murine bladder cancer model was used. To assess the antitumoral effect of Doc, intravesical Doc therapy was administered at various dose-escalating concentrations: 0μg (control: PBS), 25μg, 50μg and 100μg (n = 12 for each group; total of 4 groups). Next, a comparative evaluation of tumor growth among the control, the BCG-alone group(100μg), the Doc-alone group, and three combined BCG (100μg) + Doc group (a coincident(CO) and two sequential groups) was performed (n= 12 ; total of 6 groups). The sequential combined groups consisted of SA group (SA; Doc administration followed by BCG administration on the next day) and SB group (SB; BCG administration followed by Doc administration on the next day). Therapy was administered at 3-day intervals starting on day 5 and was repeated 5 times. To evaluate proliferation, T cell infiltration and apoptosis among the different groups, the tumor cells in the animal model were stained for Ki-67, CD8 and Cleaved caspase 3 after 4 instillations performed every 3 days. (n = 5). RESULTS In the Doc groups treated with a dose of 0 μg(survival; 32.3 ± 9.4 days), 25μg (36.8 ± 11.4), 50μg (38.1 ± 11.0) or 100μg (40.0 ± 12.7) one, two, two and three of the mice survived, respectively. Significant survival advantages were observed in the combined BCG + Doc group (CO: 50.7 ± 9.0, SA: 51.5 ± 6.9) compared with that in the BCG-alone group (33.1 ± 10.4) and the Doc-alone group (38.8 ± 11.5). The Ki-67 index of the cancer cells was significantly lower in the combined group, compared with that in the BCG-alone group (p < 0.0001), the Doc-alone group (p < 0.05) and the control(p < 0.0001). BCG-alone significantly increased the number of CD8+ T cells (p < 0.05), while Doc-alone reduced the CD8+ T cells (p < 0.05), compared with that in the control. Apoptosis was significantly increased when Doc was administrated to the animals irrespective of the presence (p < 0.05) or absence (p < 0.05) of BCG instillation. CONCLUSIONS Our results suggested that the combination of BCG + Doc treatment decreased the tumor appearance rate and improved the survival period. The enhancement in tumor growth inhibition enabled by combination regimens was presumably associated with a reduction in the tumor proliferation rate and might not depend on either CD8+ T cells or apoptosis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e212 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Minoru Horinaga Saitama, Japan More articles by this author Ryuichi Fukuyama Konan, Japan More articles by this author Masahiro Iida Saitama, Japan More articles by this author Masashi Matsushima Saitama, Japan More articles by this author Hitoshi Yanaihara Saitama, Japan More articles by this author Yoko Nakahira Saitama, Japan More articles by this author Munehisa Ueno Saitama, Japan More articles by this author Hirotaka Asakura Saitama, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DAB389 EGF for the treatment of superficial urothelial cancer.

255 Background: Intravesical BCG has remained the standard treatment for superficial bladder cancer for decades, despite frequent recurrences after its use. Toxic fusion proteins are a novel class of agents containing both a targeting and a treatment component; correspondingly, DAB389EGF contains an EGF and a diphtheria element, thereby targeting any cell expressing EGFR. Notably, the normal urothelium has little or no luminal EGFR expression in contrast to urothelial cancers, which frequently overexpress EGFR. Methods: DAB389EGF was tested in vitro to determine the effect of DAB389EGF on cell viability in culture. Tests of clonogenicity were performed by treating plated cells for 2 hours and following colony counts over 14 days. DAB389EGF was tested in an orthotopic murine model of bladder cancer with implanted HTB9 cells modified to express luciferase. DAB389EGF was administered intravesically via catheterization of the murine bladder and held in place for 2 hours with a temporary suture to close the urethra. Treatments were continued twice weekly for 2 weeks, with DT-GMCSF used as a control. Results: Treatment with DAB389EGF yielded IC50's ranging from 0.5 to 15.7 ng/ml in 8 tested bladder cancer cell lines, but the IC50 was &gt; 500ng/ml in the EGFR- negative H520 control cell line. A single 2-hour exposure to DAB389EGF (50ng/ml) decreased colony formation in all of the bladder cancer cell lines, with complete suppression in HT1376 and T24 cells. In the animal model, luciferase activity was lost in 5 of 6 mice treated with DAB389EGF, versus 1 of 6 mice treated with control. After the 2 week treatment period, histological assessment of the treated mice was undertaken and was consistent with the luciferase findings. FISH was used to detect human or murine-originated cells in order to confirm the presence or absence of xenograft tissue histologically. Conclusions: Targeted-toxin fusion proteins represent a novel treatment approach for superficial bladder cancer. By capitalizing on the differential expression of EGFR between cancerous and normal urothelium, DAB389EGF selectively targets bladder cancer cells. Preliminary animal studies support the efficacy and tolerability of this approach with additionally animal work currently underway. No significant financial relationships to disclose.

Abstract 5614: Interleukin-10 inhibits the induction of macrophage cytotoxicity against bladder cancer cells by Mycobacterium bovis bacillus Calmette-Guérin (BCG)

Abstract Intravesical BCG therapy has been used for the treatment of superficial bladder cancer for three decades. However, the mechanisms underlying BCG therapy currently remain elusive. We have focused on BCG induction of macrophage cytotoxicity against bladder cancer cells. Previously, we observed that macrophages are cytotoxic to bladder cancer cells upon stimulation with BCG in vitro. However, we found that macrophages from C57BL/6 mice are less potent than those from C3H/HeN mice for the killing of both bladder cancer MBT-2 cells and MB49 cells. This study was to determine whether macrophage-derived interleukin (IL)-10 is responsible for this discrepancy between these two commonly used murine bladder cancer models. Thioglycollate-elicited peritoneal exudate cells were harvested from mice 3 days after intraperitoneal injection of 2 ml 3% thioglycollate. Cells were incubated for 3 hours and non-adherent cells were removed after incubation. The remaining cells were 96% F4/80-positive cells as determined by flow cytometry. The prepared macrophages were then used for the analysis of BCG induction of cytotoxicity, cytokines, and nitric oxide (NO) in vitro. Studies demonstrated that BCG-stimulated C57BL/6 macrophages produced a substantially high level of IL-10 compared to BCG-stimulated C3H/HeN macrophages. This high IL-10 production correlated with reduced production of tumor necrosis factor (TNF)-α, IL-6 and NO in BCG-stimulated C57BL/6 macrophages. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3.2-fold, along with increased production of TNF-α by 6.4-fold and NO by 3.6-fold, respectively. Macrophages from genetically modified C57BL/6 mice lacking IL-10 (IL-10−/−) also exhibited increased killing of MB49 cells and production of TNF-α and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. Taken together, our results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients, particularly for BCG non-responders who are often associated with high IL-10 production in response to BCG treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5614.

Abstract 1549: Oral inhibitors of macrophage migratory inhibitory factor (MIF) lead to reduced tumor burden in a murine model of invasive bladder cancer

Abstract INTRODUCTION AND OBJECTIVES: MIF is a proinflammatory cytokine with regulatory properties over tumor suppressor proteins involved in bladder cancer. We have reported that absence of MIF in transgenic mice leads to decreased angiogenesis and invasion in the BBN model of bladder cancer. We evaluated the impact of 2 oral inhibitors of MIF (Cytokine PharmaSciences, King of Prussia, PA; CPSI) in the BBN model. METHODS: 30, 3 mnth old male C57Bl/6 mice were treated with BBN 0.05% in drinking water for 22 wks. Mice (n=10/grp) received vehicle (PEG 300, methylcelluose), oral inhibitor CPSI-2705 or CPSI-1306 daily (25 mg/kg) by gavage from wks 16-22, corresponding to time of progression from CIS to invasive disease as determined in previous experiments. Animals were inspected daily for general health with weights recorded weekly. Bladders were weighed, fixed and assessed for tumor stage, grade, burden and associated angiogenesis. RESULTS: In general, the group receiving CPSI-1306 appeared healthier during drug treatment. There was a 16% weight loss from the start of gavage in the control group vs 10-11% in the drug groups. Two animals each from the control and CPSI-2705 group died prior to study completion. The majority of control animals had evidence of upper tract obstruction with less noted in CPSI 2705 and none in the 1306 group. Average bladder weights were 0.5 ± 0.5 gm, 0.3 ± 0.3 gm and 0.2 ± 0.2 gm for control, 2705 and 1306 respectively which approached statistical significance for control vs 1306 (log transf p=0.06). There was a higher proportion of pT3 disease in the control (80%) vs inhibitor groups (60%). Tumor burden was markedly diminished in the drug groups (81% cntrl vs 62% 2705 vs 59% 1306, involvement invasive disease) with some bladders in these arms having only focal areas of invasion. Tumor grade trended lower in the drug groups as well. CONCLUSIONS: The use of MIF oral inhibitors CPSI 2705 and CPSI 1306 in the BBN bladder cancer model resulted in less weight loss, improved general health, decreased tumor burden with a trend toward lower stage, grade and tumor associated angiogenesis. The results of this experiment warrant larger studies to validate and expand these findings with a goal of development for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1549.

Abstract 1903: Enhanced antitumor effect of coincident combination intravesical MMC plus BCG therapy in an orthotopic bladder cancer model

Abstract Introduction and Objective: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of superficial bladder tumors. However, superficial bladder tumors recur in 60 to 70% of all cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties. We evaluated the combination of intravesical MMC plus BCG treatment against bladder tumors in a model of orthotopic murine bladder cancer. Methods: An orthotopic murine bladder cancer model was established using the simple instillation of 1 × 106 MBT-2 cells into the lumen of the bladder of a female C3H/HeN mouse through a urethral catheter. To assess the antitumoral effect of MMC, intravesical MMC therapy was administered at various dose-escalating concentrations: 0μg (control: PBS), 25μg, 50μg, 100μg, and 200μg, (n = 8 for each group; a total of 5 groups). Next, a comparative evaluation of tumor growth in a control group(PBS), a MCC-alone group, a BCG-alone group(100μg), and a combined MMC plus BCG (100μg) group were performed (n= 8 for each group; a total of 4 groups). Intravesical therapy was administered at 3-day intervals starting on day 5 and repeated 5 times. On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate macrophage infiltration in the tumor and the proliferative activity among the different groups, immunohistochemical staining analysis of the tumor cells utilizing CD68 and Ki-67 was performed in animal model. Results: In the MMC groups of 8 mice each treated with a dose of 0μg (survival; 26.4 ± 3.2 days), 25μg (34.8 ± 7.0 days) and 50μg (40.0 ± 13.2 days), 0, 0 and 2 of the mice failed to develop tumor and survived, respectively. In both the 100μg (17.3 ± 2.8 days) and 200μg (11.9 ± 3.0 days) groups, all eight mice died from adverse events. A significant survival advantage was observed in the combined MMC (50μg) plus BCG (100μg) group (51.5 ± 8.1 days), compared with that in the BCG-alone group (33.4 ± 16.8 days; p = 0.03) and the MMC-alone group (39.5 ± 13.5 days; p = 0.04). BCG significantly suppressed while MMC increased macrophage infiltration in the tumors, compared with in the control groups. The Ki-67 expression level, which is regarded as a proliferation index was significantly lower in the combined MMC (50μg) plus BCG (100μg) group (31.1 ± 5.2%) than in the BCG-alone group (50.9 ± 4.1%; p &amp;lt;0.001) and the MCC-alone group (39.2 ± 2.5%; p &amp;lt;0.05). Conclusion: Combining BCG plus MMC treatment decreased the tumor appearance rate, improved the survival period and reduced the proliferation rate in tumors, as compared with BCG-alone and MMC-alone treatment, possibly providing a greater therapeutic benefit over either agent alone. Immunochemotherapy with a combination of intravesical MMC plus BCG may be a promising alternative treatment for bladder tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1903.

1150 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS MMC THERAPY IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101150 ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL BCG PLUS MMC THERAPY IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL Minoru Horinaga, Masashi Matsushima, Ryuichi Fukuyama, Hitoshi Yanaihara, Masahiro Iida, Yoko Nakahira, and Hirotaka Asakura Minoru HorinagaMinoru Horinaga Saitama, Japan More articles by this author , Masashi MatsushimaMasashi Matsushima Saitama, Japan More articles by this author , Ryuichi FukuyamaRyuichi Fukuyama Konan, Japan More articles by this author , Hitoshi YanaiharaHitoshi Yanaihara Saitama, Japan More articles by this author , Masahiro IidaMasahiro Iida Saitama, Japan More articles by this author , Yoko NakahiraYoko Nakahira Saitama, Japan More articles by this author , and Hirotaka AsakuraHirotaka Asakura Saitama, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.649AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of superficial bladder tumors. However, superficial bladder tumors recur in 60 to 70% of all cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties. We evaluated the combination of intravesical BCG plus MMC treatments against bladder tumors in a model of orthotopic murine bladder cancer. METHODS An orthotopic murine bladder cancer model was established using the simple instillation of one million MBT-2 cells into the lumen of the bladder of a female C3H/HeN mouse. To assess the antitumoral effect of MMC, intravesical MMC therapy was administered at various dose-escalating concentrations: 0ug(control: PBS), 25ug, 50ug, 100ug and 200ug (n = 8 for each group). Next, a comparative evaluation of tumor growth in a control group (PBS), a BCG-alone group(100ug), a MCC-alone group, and a combined BCG (100ug) plus MMC group was performed (n= 8 for each group; total 4 groups). Intravesical therapy was administered at 3-day intervals starting on day 5 and repeated 5 times. On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate the macrophage infiltration in the tumor and the proliferative activity among the different groups, CD-68 and Ki-67 staining of the tumor cells was performed in animal model. RESULTS In the MMC groups of 8 mice each treated with a dose of 0ug (survival; 26.4 days), 25ug (34.8 days) and 50ug (40.0 days), 0, 0 and 2 of the mice survived, respectively. In both the 100ug (survival;17.3 days) and 200ug (11.9 days) groups, all eight mice died from adverse events. A significant survival advantage was observed in the combined BCG (100ug) plus MMC (50ug) group (51.5 days), compared with that in the BCG-alone (100ug) group (33.4 days p = 0.03) and the MMC-alone (50ug) group (39.5 days p = 0.04). BCG significantly suppressed while MMC increased macrophage infiltration in the tumor, compared with in the control group. The Ki-67 expression level, which is regarded as a proliferation index, was significantly lower in the BCG plus MCC group (31.1 ± 5.2%) than in the BCG-alone group (50.9 ± 4.1%; p <0.001) and the MCC-alone group (39.2 ± 2.5%; p <0.05). CONCLUSIONS Combining BCG plus MMC treatment decreased the tumor appearance rate, improved the survival period, and reduced the proliferation rate in tumors, as compared with BCG-alone and MMC-alone treatment, possibly providing a greater therapeutic benefit over either agent alone. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Minoru Horinaga Saitama, Japan More articles by this author Masashi Matsushima Saitama, Japan More articles by this author Ryuichi Fukuyama Konan, Japan More articles by this author Hitoshi Yanaihara Saitama, Japan More articles by this author Masahiro Iida Saitama, Japan More articles by this author Yoko Nakahira Saitama, Japan More articles by this author Hirotaka Asakura Saitama, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors

The present gold standard for bladder cancer is Mycobacterium bovis, Bacillus Calmette Guerin (BCG) immunotherapy. But it has a non-responder rate of 30-50% and side effects are common. Lactobacillus casei strain Shirota has been reported to reduce the incidence of recurrence in bladder cancer patients and to cure tumor-bearing mice. Our aim was to determine if Lactobacillus rhamnosus GG (LGG) could be as efficacious as BCG in a murine model of bladder cancer. MB49 bladder cancer cells secreting human prostate-specific antigen were implanted orthotopically in female C57BL/6 mice and urinary prostate-specific antigen levels were used as a marker of tumor growth. Mice were treated with either live or lyophilized LGG given via intravesical instillation, or both oral and intravesical LGG given once a week for a period of 6 weeks starting at day 4 after tumor implantation. A comparison of LGG and BCG immunotherapy was also carried out. LGG therapy (live or lyophilized) significantly (P = 0.006) increased the number of cured mice. Cytokine arrays and immune cell recruitment analysis revealed differences between untreated, treated, cured, and tumor-bearing mice. LGG therapy restored XCL1 levels to those in healthy bladders. LGG also recruited large numbers of neutrophils and macrophages to the tumor site. Intravesical LGG and BCG immunotherapy had cure rates of 89 and 77%, respectively, compared with 20% in untreated mice. LGG has the potential to replace BCG immunotherapy for the treatment of bladder cancer.

Transabdominal Micro-ultrasound Imaging of Bladder Cancer in a Mouse Model: A Validation Study

To validate the use of transabdominal micro-ultrasound imaging (MUI) in an orthotopic murine bladder cancer model. The current in vivo imaging systems for murine bladder cancer include magnetic resonance imaging, bioluminescent and fluorescent imaging, and intravesical ultrasound. We implanted murine bladder tumor-2 tumor cells into C3H/He female mice. Mice underwent MUI before, and every 3 days after instillation of tumor cells. Three mice were killed at every MUI session. Bladder tumors were measured and tumor volumes were calculated during MUI and gross stereomicroscopy. Bladders were harvested and examined under gross stereomicroscopy to confirm the presence, location, and size of bladder tumors, and were prepared for histology review. Overall, 15 of 33 (45%) mice were confirmed to have tumors, using MUI, gross stereomicroscopy, and histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). MUI identified all tumors that were present on final histology. The smallest confirmed tumor on MUI was detected at 0.52 mm(3), and mean tumor volume was 0.95 mm(3). No tumors that were not detected first using MUI were found on final histology. Transabdominal MUI is a valuable tool to use for translational studies involving orthotopic mouse bladder cancer models. MUI provides real-time, high resolution in vivo images of bladder tumors. Tumor presence can be confirmed with a high degree of accuracy pertaining to tumor volume before initiation of treatment. In addition, tumor growth or regression can be followed up in vivo longitudinally.

Bladder cancer: modeling and translation: Table 1.

Transitional cell carcinoma of the bladder is a common malignancy worldwide that is associated with significant morbidity and mortality. Although superficial tumors can often be treated effectively, invasive cancers not only require invasive surgery, but are also refractory to aggressive chemotherapy and radiotherapy. In this issue of Genes & Development, Puzio-Kuter and colleagues (pp. 675-680) describe an elegant genetically engineered murine model of bladder cancer that recapitulates many of the cardinal features of the human disease. The development of such models together with the application of new approaches to enumerate the complement of genetic alterations in bladder will provide new insights into the molecular nature of this disease. Moreover, the anatomy of this urinary malignancy provides a unique opportunity for innovative translational studies.

Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti-invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC 50 0.2 –> 10 μM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibition-resistant and -sensitive xenografts. The activity of AZD0530 in NBT-II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell–cell adhesion. These data suggest a dominant anti-invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.

Preventing Bladder Tumor Implantation With Photodynamic Therapy in a Rat Model Mimicking Post-Fluorescence Guided Transurethral Resection

Fluorescence guided transurethral resection has gained acknowledgment from the urological community and it is progressively becoming more applied. It has been shown to decrease the recurrence rate of nonmuscle invasive bladder cancer due to incomplete resection due to lack of visualization. The implantation of viable tumor cells seeded during transurethral resection is another reason for recurrence. We investigated whether applying photodynamic therapy on sensitized tumor cells would decrease the amount of viable intraluminal cells and tumor cell implantation. Two models were designed to mimic the situation after fluorescence guided transurethral resection, including partly or fully de-epithelialized bladders and circulating tumor cells loaded with protoporphyrin IX. Photodynamic therapy was performed. Controls consisted of no drug with no light, light only and drug only. Immediately after photodynamic therapy the intravesical contents were retrieved and clonogenic assays were performed on cells. Bladders were harvested 10 days after cell administration and subjected to pathological analysis. In the photodynamic therapy and control groups tumor volume was proportional to the instilled cell load. Clonogenic assays showed that viable cells were decreased a tenth of the initial administered amount. Tumor implantation decreased to less than a fifth of control values. Photodynamic therapy can effectively decrease the amount of viable tumor cells in the bladder lumen. This results in a significant decrease in tumor implantation. This technique could possibly be used to further decrease the recurrence rate of nonmuscle invasive bladder cancer.