An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies (original) (raw)
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An Orthotopic Bladder Cancer Model for Gene Delivery Studies
Journal of Visualized Experiments, 2013
Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.
Advances in Gene Therapy for Bladder Cancer
Current Gene Therapy, 2003
The efficacy of various currently available therapeutic strategies for bladder cancer is not always sufficient, especially for the advanced disease, recurrent superficial cancer, and treatment-resistant carcinoma in situ. Advances in genetic and molecular biology have led to novel approaches for cancer treatment. Gene therapy is currently one of the most promising strategies against various malignancies, and several clinical trials have been approved worldwide. Various strategies for modulating the genetic state have been applied in bladder cancer treatment, and encouraging results have been demonstrated both in vitro and in vivo. Although the therapeutic genes work dramatically when the transgenes are effectively expressed in the targeted cells, however, a sufficient rate of transduction cannot always be achieved. The most significant obstacle for clinical application of cancer gene therapy might be the method for sufficient delivery and expression of the therapeutic genes. Bladder is an easily accessible organ because of its anatomy; however, a glycosaminoglycan (GAG) layer on the bladder mucosa may protect integration of exo-delivered genetic vectors. Various strategies are applied for improving the transduction efficacy of the therapeutic genes into the bladder cancer cells. These strategies include the modification of adenoviral fibers, cotransduction of the materials for enhancing the viral infectivity, and disruption of the GAG layer. Recent advances in the field of gene therapy for bladder cancer are briefly summarized in this review.
Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy
Laboratory Animals, 2005
Bladder cancer is regarded as a promising candidate for innovative therapies in the field of immune and gene therapy. In this paper, we present the subcutaneous, metastatic and a novel orthotopic model of murine MB49 bladder cancer in C57BL/6 mice. We further show the potential of using adenoviral vectors together with different transduction enhancers to augment in vivo gene delivery. Finally, we present candidate genes for tumour detection, therapy or targeting.
European Urology, 2005
Objectives: Superficial bladder tumours are at high risk for recurrence, relapse after resection, escape to intravesical immunotherapy and they may become invasive. New therapeutics are therefore needed to achieve cure. Thus, gene therapy is an attractive new treatment modality for malignant bladder tumours. The purpose of this study was to evaluate the feasibility and the efficiency of retroviral mediated reporter gene transfer into malignant urothelial cells both in vitro and in vivo. Methods: We evaluated the feasibility of the transfection of bladder tumour with direct intravesical instillation of a defective retrovirus. The vector was derived from LXSN. The efficiency of transduction with the Moloney Leukaemia Murine virus-based vector, amphotrophic retroviral vector, was monitored through the expression of two marker genes (nls-LacZ and NeoR). The canine animal was chosen since it can present with spontaneous bladder carcinomas mimicking human pathology. Primary cultures of two normal canine bladder urothelium and two canine primary bladder tumours were first studied. We then investigated in vivo, in two normal and two spontaneous tumour bearing dogs, the transduction of urothelial cells following direct intravesical instillation of 2Á10 4 to 3Á10 6 of the retroviral vector. Results: Transduced cells were evidenced in all primary cultures of canine normal urothelium and transitional cell carcinoma. Bladder biopsies from sound dogs instilled with the viral solution showed long lasting transduction up to 60 days long. Bladder cryosections from tumour-bearing dogs displayed transduction of superficial layers of urothelial cancer cells without passing through lamina propria. In vivo transduction was evidenced in 1 to 15% (mean 5%) of the cells in the tumours and preferentially addressed malignant cells. Normal epithelium either originating from sound or tumour-bearing animals was not transduced. Conclusion: These results demonstrate for the first time the feasibility of in vivo retroviral transduction of bladder cancer using a clinically relevant procedure. #
Cancer Science, 2001
The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV-tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short-term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apoptosis. Following a long-term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.
Intravesical liposome-mediated interleukin-2 gene therapy in orthotopic murine bladder cancer model
Gene Therapy, 2000
Using a novel orthotopic MBT-2 murine bladder tumor model, we evaluated the feasibility of intravesical gene therapy utilizing a cationic liposome, DMRIE/DOPE. Superficial bladder tumors were consistently established by intravesical instillation of 5 × 10 5 MBT-2 cells in syngeneic C3H female mice. In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/DMRIE/DOPE lipoplex. -Galactosidase (-gal) gene expression was preferentially evident in bladder tumors and was present for at least 7 days after a single 30 min in situ transfection. Murine interleukin-2 (IL-2) gene was used for treatment of 3-day-old pre-established bladder tumors. Forty percent of animals treated with IL-2 gene were completely
Molecular targeting and gene delivery in bladder cancer therapy
Journal of B.U.ON.: official journal of the Balkan Union of Oncology
Urothelial carcinoma of the bladder is the second most common genitourinary malignancy and the second most common cause of genitourinary cancer-related deaths with a worldwide estimate of about 300,000 new cases diagnosed every year. A signifi cant problem in this type of cancer is the high recurrence rate of noninvasive primary tumors, leading to a high percentage of tumor progression and to a very poor 5-year survival rate. Targeted and gene therapy are currently the two major efforts in cancer treatment. Targeted therapy refers to strategies against specifi c cellular molecules deregulated in tumors, whereas gene therapy focuses on the genetic modifi cation of tumor cells, mainly for correcting gene defects, inducing selective tumor cell death or modulating host's immune response. Recent advances in our understanding of the pathogenesis of bladder cancer at the molecular level have provided a signifi cant number of cellular targets for therapy and have shown the importance of individualized therapy according to the molecular profi le exhibited by the tumor cells. While the major problems of both targeted and gene therapy are far from being solved yet, both lines of cancer therapy hold promising results. This article aims at providing a brief general overview of this broad subject.
Adenovirus-mediated suicide-gene therapy in an orthotopic murine bladder tumor model
International Journal of Urology, 2002
Background : Patients with high-grade transitional-cell carcinoma (TCC) of the bladder frequently experience recurrence and progress and have a low response rate to chemotherapy in metastatic TCC. In this study, we evaluated the feasibility and long-term efficacy of suicide-gene therapy using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase gene (HSV-TK) and prodrug ganciclovir (GCV) as a potential therapeutic approach in murine-orthotopic models of TCC. Methods : A replication defective adenoviral vectors containing toxic HSV-TK gene under the transcriptional control of RSV (Rous sarcoma virus) promoter (Ad-RSV-TK) was used. Orthotopic bladder TCC was established with 1 × 10 6 murine (MBT-2) TCC cells in syngenic C3H/He female mice. Intratumoral injection of Ad-RSV-TK in combination with GCV (20 mg/kg body weight/day i.p. b.i.d. × 7 days) was administered in vivo for the determination of treatment efficacy and long-term host survival in separate controlled experiments. Results : In vivo experiments demonstrated greater than three-fold reductions in MBT-2 tumor growth for the animals treated with Ad-RSV-TK (5 × 10 8 plaque forming units (pfu)/GCV therapy ( P < 0.01)). Central tumor necrosis and apoptosis were revealed by histomorphology and immunohistochemistry compared with other control animals (non-treated, GCV alone, Ad-RSV-TK alone). Direct intratumoral injection with Ad-RSV-TK/GCV also resulted in significantly improved survival over the control groups in separated experiment (log-rank test, P < 0.05). Conclusions : Suicide-gene therapy using Ad-RSV-TK/GCV provides an effective therapy in an experimental murine orthotopic bladder cancer by significantly inhibiting tumor growth and improving long-term host survival.