J Korean Med Sci.  2014 Mar;29(3):343-350. 10.3346/jkms.2014.29.3.343.

Establishment of an Orthotopic Mouse Non-Muscle Invasive Bladder Cancer Model Expressing the Mammalian Target of Rapamycin Signaling Pathway

• Kim SJ
• Seo HK
• Seo HH
• Lee SJ
• Kwon JK
• Lee TJ
• Chi BH
• Chang IH

• KMID: 1734920
• DOI: http://doi.org/10.3346/jkms.2014.29.3.343

Abstract

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r2 = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r2 = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.

Keyword

Urinary Bladder Neoplasms; Mouse Orthotopic Model; mTOR

MeSH Terms

Animals
Blotting, Western
Cell Line, Tumor
Disease Models, Animal
Female
Genes, Reporter
Green Fluorescent Proteins/genetics/metabolism
Humans
Immunohistochemistry
Luciferases, Firefly/genetics
Luminescent Measurements
Mice
Mice, Nude
Neoplasm Staging
*Signal Transduction
TOR Serine-Threonine Kinases/*metabolism
Transplantation, Heterologous
Urinary Bladder Neoplasms/*metabolism/pathology/veterinary
Green Fluorescent Proteins
Luciferases, Firefly
TOR Serine-Threonine Kinases

Figure

• 

  Fig. 1 Serial bioluminescence imaging and histopathological findings in the mice bladder after intravesical instillation of Ku-7-luc cells. (A) Schematic summary of the investigation. (B) Microscopic appearance of the KU-7-luc bladder tumor after hematoxylin and eosin staining of each stage groups taken randomly on days 4, 7, 14, and 21.

• 

  Fig. 2 ×40 and ×100 magnification. Bioluminescence imaging of KU-7-luc tumors in vitro and in vivo. (A) Correlation between KU-7-luc cells and in vitro bioluminescence.Cells were serially diluted in a 96-well plate starting with 1×104 cell/well. Luciferin (0.15 mg/mL) was added to the wells, and the plate was imaged for 30 sec in an IVIS 2000 systemafter 15 min. Experiments were done in quadruplicate. (B) In vivo imaging of tumor growth over time. After intravesical instillation of 2×106 Ku-7-Luc cells on day zero, mice were imaged at 4, 7, 14, and 21 days. (C) Comparison of bioluminescence according to histological tumor stage. Error bars indicated standard error. (D) Correlation between bioluminescence and histological tumor volume. Bioluminescence is quantified in photons/s, n = 19. †P < 0.05, ‡P < 0.01.

• 

  Fig. 3 mTOR signaling pathway related protein expression in human bladder cancer cell lines and the orthotopic mouse non-muscle invasive bladder cancer model using KU-7-luc cells. (A) Western blot analysis to evaluate the expression of phosphorylated-mTOR (p-mTOR), mTOR, p-70S6K, p70S6K, p-4EBP1, 4EBP1, p-eIF4E, and eIF4E in the human bladder cancer cell lines. (B) Immunohistochemical staining for p-mTOR, mTOR, p70S6K, p-p70S6K, 4E-BP1, p-4E-BP1, eIF4E, p-eIF4E in the orthotopic mouse non-muscle invasive bladder cancer model using KU-7-luc cells. mTOR, mammalian target of the rapamycin; p70S6K, p70S6 kinase; 4E-BP1, eukaryotic initiation factor 4E binding protein 1; eIF4E, eukaryotic initiation factor 4E.

Reference

1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010; 60:277–300.

2. Skinner E. Will a new intravesical chemotherapy agent improve the treatment of non-muscle-invasive bladder cancer? Nat Clin Pract Urol. 2007; 4:248–249.

3. Gasión JP, Cruz JF. Improving efficacy of intravesical chemotherapy. Eur Urol. 2006; 50:225–234.

4. Davis JW, Sheth SI, Doviak MJ, Schellhammer PF. Superficial bladder carcinoma treated with bacillus Calmette-Guerin: progression-free and disease specific survival with minimum 10-year followup. J Urol. 2002; 167:494–500.

5. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999; 13:2905–2927.

6. Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, Yonezawa K. Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem. 1999; 274:34493–34498.

7. Seager CM, Puzio-Kuter AM, Patel T, Jain S, Cordon-Cardo C, Mc Kiernan J, Abate-Shen C. Intravesical delivery of rapamycin suppresses tumorigenesis in a mouse model of progressive bladder cancer. Cancer Prev Res (Phila). 2009; 2:1008–1014.

8. Park SJ, Lee TJ, Chang IH. Role of the mTOR pathway in the progression and recurrence of bladder cancer: an immunohistochemical tissue microarray study. Korean J Urol. 2011; 52:466–473.

9. Soloway MS. Intravesical and systemic chemotherapy of murine bladder cancer. Cancer Res. 1977; 37:2918–2929.

10. Hadaschik BA, Black PC, Sea JC, Metwalli AR, Fazli L, Dinney CP, Gleave ME, So AI. A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging. BJU Int. 2007; 100:1377–1384.

11. Chang SG, Kim JI, Jung JC, Rho YS, Lee KT, An Z, Wang X, Hoffman RM. Antimetastatic activity of the new platinum analog [Pt(cis-dach) (DPPE). 2NO3] in a metastatic model of human bladder cancer. Anticancer Res. 1997; 17:3239–3242.

12. Jiang F, Zhou XM. A model of orthotopic murine bladder (MBT-2) tumor implants. Urol Res. 1997; 25:179–182.

13. Kleinerman DI, Dinney CP, Zhang WW, Lin SH, Van NT, Hsieh JT. Suppression of human bladder cancer growth by increased expression of C-CAM1 gene in an orthotopic model. Cancer Res. 1996; 56:3431–3435.

14. Bonfil RD, Russo DM, Schmilovich AJ, Garcia-Palazzo IB. Intravesical therapy with vinorelbine tartrate: antitumor activity in orthotopic murine cell carcinoma of the bladder. J Urol. 1997; 158:912–915.

15. Günther JH, Jurczok A, Wulf T, Brandau S, Deinert I, Jocham D, Böhle A. Optimizing syngeneic orthotopic murine bladder cancer (MB49). Cancer Res. 1999; 59:2834–2837.

16. Werthman PE, Drazan KE, Rosenthal JT, Khalili R, Shaked A. Adenoviral-p53 gene transfer to orthotopic and peritoneal murine bladder cancer. J Urol. 1996; 155:753–756.

17. Watanabe T, Shinohara N, Sazawa A, Harabayashi T, Ogiso Y, Koyanagi T, Takiguchi M, Hashimoto A, Kuzumaki N, Yamashita M, et al. An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies. Cancer Gene Ther. 2000; 7:1575–1580.

18. Chan ES, Patel AR, Smith AK, Klein JB, Thomas AA, Heston WD, Larchian WA. Optimizing orthotopic bladder tumor implantation in a syngeneic mouse model. J Urol. 2009; 182:2926–2931.

19. Mangsbo SM, Ninalga C, Essand M, Loskog A, Tötterman TH. CpG therapy is superior to BCG in an orthotopic bladder cancer model and generates CD4+ T-cell immunity. J Immunother. 2008; 31:34–42.

20. Chin J, Kadhim S, Garcia B, Kim YS, Karlik S. Magnetic resonance imaging for detecting and treatment monitoring of orthotopic murine bladder tumor implants. J Urol. 1991; 145:1297–1301.

21. Kikuchi E, Xu S, Ohori M, Matei C, Lupu M, Menendez S, Koutcher JA, Bochner BH. Detection and quantitative analysis of early stage orthotopic murine bladder tumor using in vivo magnetic resonance imaging. J Urol. 2003; 170:1375–1378.

22. Satoh H, Morimoto Y, Arai T, Asanuma H, Kawauchi S, Seguchi K, Kikuchi M, Murai M. Intravesical ultrasonography for tumor staging in an orthotopically implanted rat model of bladder cancer. J Urol. 2007; 177:1169–1173.

23. Arentsen HC, Hendricksen K, Oosterwijk E, Witjes JA. Experimental rat bladder urothelial cell carcinoma models. World J Urol. 2009; 27:313–317.

24. Van der Horst G, van Asten JJ, Figdor A, van den Hoogen C, Cheung H, Bevers RF, Pelger RC, van der Pluijm G. Real-time cancer cell tracking by bioluminescence in a preclinical model of human bladder cancer growth and metastasis. Eur Urol. 2011; 60:337–343.

25. Jurczok A, Fornara P, Soling A. Bioluminescence imaging to monitor bladder cancer cell adhesion in vivo: a new approach to optimize a syngeneic, orthotopic, murine bladder cancer model. BJU Int. 2008; 101:120–124.

26. Xiao Z, McCallum TJ, Brown KM, Miller GG, Halls SB, Parney I, Moore RB. Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model. Br J Cancer. 1999; 81:638–646.