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933 Establishment of a new orthotopic in vivo examinable model of non-muscle invasive bladder cancer using RT112 reporter cells
933
Establishment of a new orthotopic in vivo examinable model of non-muscle invasive bladder cancer using RT112 reporter cells Eur Urol Suppl 2016;15(3);e933
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Fragoulis A. 1 , Fera C.2 , Schemmert S. 2 , Strick K. 2 , Anton M. 3 , Möhring M. 4 , Steitz J. 4 , Tolba R.4 , Grosse J.O. 2 1 Uniklinik 3 TU
RWTH Aachen, Dept. of Orthopaedic Surgery, Aachen, Germany, 2 Uniklinik RWTH Aachen, Dept. of Urology, Aachen, Germany,
Munich, Institute of Molecular Immunology and Experimental Oncology, Munich, Germany, 4 Uniklinik RWTH Aachen, Institute for
Laboratory Animal Science, Aachen, Germany INTRODUCTION & OBJECTIVES: About 70% of bladder cancers are non-muscle invasive bladder cancers (NMIBC). Treatment strategies are still limited and include initial transurethral resection of the tumour with subsequent intravesical immuno- and/or chemotherapy (e.g. mitomycin C, MMC). Still, about 80% of patients with superficial bladder cancer develop a recurrence of tumours and in even 10% muscleinvasive carcinoma can be found. This demonstrates the extant requirement for new efficient treatment approaches. For this reason we established a new orthotopic NMIBC mouse model using urothelial RT112 reporter cells for non-invasive in vivo characterisation of tumour progression. MATERIAL & METHODS: The used RT112 cells carry a transgene that leads to a constitutive expression of a firefly luciferase/GFP fusion protein (RT112-luc/GFP). These cells were characterized in vitro regarding functional and stable luc expression, their proliferation, and vulnerability against MMC short- and long-term treatment. The benign urothelial cell line UROtsa was used as control. Subsequently, the RT112-luc/GFP cells were intravesically instilled in Swiss Webster nu/nu mice to induce tumour formation and test the NMIBC model. This was monitored and quantified for 28 days by non-invasive in vivo measurements of RT112-derived bioluminescence signals (BL) using the XENOGEN in vivo imager. After establishing the model we performed a MMC treatment approach (1mg/mL for 2 hrs) to underline the model’s efficacy to test pharmacological treatment strategies. The BL of the animals was quantified every 7 days after MMC treatment. After dissection the bladders were prepared for further histopathological analysis. RESULTS: The in vitro experiments revealed that the RT112-luc/GFP cells were suitable for the NMIBC mouse model. Luc expression was sufficient and stable; the proliferation was higher and they showed enhanced vulnerability against the chemotherapeutic MMC compared to normal growing benign UROtsa cells. After tumour induction in vivo, BL was restricted to the bladder for at least 21 days. Basically the signals increased during cancer development (day 4-28). MMC treatment starting 7 days after tumour induction resulted in almost complete tumour regression (ca. 90%). Histopathological evaluation further confirmed our findings, especially the non-muscle invasive growth of the tumour over 21 days. CONCLUSIONS: In this study we established a novel and robust NMIBC mouse model using RT112-luc/GFP cells. Due to the use of these cells local tumour growth and the time to progression can be easily monitored and quantified during cancer development or new treatment strategies respectively. The suitability for future treatment studies was also confirmed using a well-established MMC treatment that clearly inhibited tumour progression in our model.
Establishment of a new orthotopic in vivo examinable model of non-muscle invasive bladder cancer using RT112 reporter cells Eur Urol Suppl 2016;15(3);e933
Print! Print!
Fragoulis A. 1 , Fera C.2 , Schemmert S. 2 , Strick K. 2 , Anton M. 3 , Möhring M. 4 , Steitz J. 4 , Tolba R.4 , Grosse J.O. 2 1 Uniklinik 3 TU
RWTH Aachen, Dept. of Orthopaedic Surgery, Aachen, Germany, 2 Uniklinik RWTH Aachen, Dept. of Urology, Aachen, Germany,
Munich, Institute of Molecular Immunology and Experimental Oncology, Munich, Germany, 4 Uniklinik RWTH Aachen, Institute for
Laboratory Animal Science, Aachen, Germany INTRODUCTION & OBJECTIVES: About 70% of bladder cancers are non-muscle invasive bladder cancers (NMIBC). Treatment strategies are still limited and include initial transurethral resection of the tumour with subsequent intravesical immuno- and/or chemotherapy (e.g. mitomycin C, MMC). Still, about 80% of patients with superficial bladder cancer develop a recurrence of tumours and in even 10% muscleinvasive carcinoma can be found. This demonstrates the extant requirement for new efficient treatment approaches. For this reason we established a new orthotopic NMIBC mouse model using urothelial RT112 reporter cells for non-invasive in vivo characterisation of tumour progression. MATERIAL & METHODS: The used RT112 cells carry a transgene that leads to a constitutive expression of a firefly luciferase/GFP fusion protein (RT112-luc/GFP). These cells were characterized in vitro regarding functional and stable luc expression, their proliferation, and vulnerability against MMC short- and long-term treatment. The benign urothelial cell line UROtsa was used as control. Subsequently, the RT112-luc/GFP cells were intravesically instilled in Swiss Webster nu/nu mice to induce tumour formation and test the NMIBC model. This was monitored and quantified for 28 days by non-invasive in vivo measurements of RT112-derived bioluminescence signals (BL) using the XENOGEN in vivo imager. After establishing the model we performed a MMC treatment approach (1mg/mL for 2 hrs) to underline the model’s efficacy to test pharmacological treatment strategies. The BL of the animals was quantified every 7 days after MMC treatment. After dissection the bladders were prepared for further histopathological analysis. RESULTS: The in vitro experiments revealed that the RT112-luc/GFP cells were suitable for the NMIBC mouse model. Luc expression was sufficient and stable; the proliferation was higher and they showed enhanced vulnerability against the chemotherapeutic MMC compared to normal growing benign UROtsa cells. After tumour induction in vivo, BL was restricted to the bladder for at least 21 days. Basically the signals increased during cancer development (day 4-28). MMC treatment starting 7 days after tumour induction resulted in almost complete tumour regression (ca. 90%). Histopathological evaluation further confirmed our findings, especially the non-muscle invasive growth of the tumour over 21 days. CONCLUSIONS: In this study we established a novel and robust NMIBC mouse model using RT112-luc/GFP cells. Due to the use of these cells local tumour growth and the time to progression can be easily monitored and quantified during cancer development or new treatment strategies respectively. The suitability for future treatment studies was also confirmed using a well-established MMC treatment that clearly inhibited tumour progression in our model.