Mouse Models of Human Bladder Cancer as a Tool for Drug Discovery

Catherine Seager1, Anna M. Puzio‐Kuter1, Carlos Cordon‐Cardo1, James McKiernan1, Cory Abate‐Shen1

1 Departments of Urology, Pathology, and Cell Biology, Columbia University Medical Center, Herbert Irving Comprehensive Cancer Center, New York, New York
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.14
DOI:  10.1002/0471141755.ph1414s49
Online Posting Date:  June, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Muscle‐invasive bladder cancer is a deadly condition in dire need of effective new treatments. This unit contains a description of mouse models suitable for the evaluation of potential new therapies. Included is a genetically engineered mouse model of bladder cancer generated by the delivery of an adenovirus expressing Cre recombinase into the bladder lumen. Also described is an orthotopic mouse model created by the instillation of human bladder tumor cells into the bladder lumen of immune deficient mice. Protocols are also provided on the use of these models for the preclinical evaluation of new chemical entities, with mTOR inhibitors shown as an example. Curr. Protoc. Pharmacol. 49:14.14.1‐14.14.18. © 2010 by John Wiley & Sons, Inc.

Keywords: bladder cancer; GEM mouse models; orthotopic models; mTOR inhibition; preclinical studies

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Genetically Engineered Mouse Models of Bladder Cancer
  • Basic Protocol 2: Orthotopic Murine Models of Bladder Cancer
  • Basic Protocol 3: Using Mouse Models to Evaluate Treatments for Bladder Cancer
  • Support Protocol 1: Analyses and Tissue Collection of Mice Following Bladder Cancer Treatment Evaluation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Genetically Engineered Mouse Models of Bladder Cancer

  Materials
  • Dulbecco's modified Eagle medium (D‐MEM; Invitrogen, cat. no. 11995)
  • Hexadimethrine bromide (Sigma, cat. no. 107689)
  • Adenovirus expressing Cre‐recombinase (University of Iowa Vector Core Facility pacAd5, high titer (4 × 1011 pfu/ml) (http://www.uiowa.edu/∼gene)
  • Mice (with floxed alleles)
  • Floxed mouse alleles (R26R reporter allele is used to monitor recombination efficacy and specificity):
    • Conditional alleles for Pten (Ptenflox/flox, C57Bl6;129SVJ) (Lesche et al., ) and p53 (p53flox/flox, FVB;129SJv) (Jonkers et al., ) obtained from the NCI Mouse Models of Human Cancer Consortium repository (http://mouse.ncifcrf.gov/)
    • R26R reporter allele (GT(ROSA)26Sortm1Sor) (C57Bl6;129SVJ) (Soriano, ) obtained from the Jackson Laboratory Induced Mutant Resource (Bar Harbor, Maine)
  • Isoflurane (or appropriate anesthetic)
  • 2% Chlorhexidine solution (Sigma)
  • Optimal cutting temperature (OCT) compound (Tissue‐Tek, cat. no. 4583)
  • 4% paraformaldehyde/phosphate‐buffered saline (PBS)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Staining solution (see recipe)
  • 10% formalin
  • PBS‐T (1 ml Tween 20 per 100 ml PBS)
  • Nuclear Fast Red (Vector Labs, cat. no. H3403)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylene
  • Clear Mount (American MasterTech, cat. no. MMCLEPT)
  • 100‐µl Hamilton glass syringe (Hamilton, cat. no. 81001)
  • 30‐G, ½‐in. needles (BD, cat. no. 305106)
  • 1‐ml disposable tuberculin syringes (BD, cat. no. 309602)
  • Sterile surgical area complete with operating board
  • Precision vaporizer and nose cone for administering isoflurane (available at institutional animal care facility; e.g., Kent Scientific)
  • Animal hair clippers
  • Dissecting tools (Fine Science Tools) including:
    • Surgical scissors
    • Serrated tissue forceps
    • Dressing forceps
  • Dissecting microscope
  • 5‐0 coated, braided silk sutures with attached needle (Seneture, cat. no. S1173)
  • Wound clip applicator (BD, cat. no. 427630)
  • Autoclip 9‐mm wound clips (BD, cat. no. 427631)
  • Animal warmer (such as model TR‐200 from Fine Science Tools)
  • VWR Superfrost slides
  • 37°C humidified chamber
NOTE: For optimal results, use high‐titer virus that has been divided into aliquots at the time of its initial preparation and not subject to freeze‐thawing.NOTE: The National Cancer Institute (NCI) at Frederick Web site lists general and safety precautions for adenovirus. See http://home.ncifcrf.gov/ehs/uploadedFiles/ISM‐193.pdf.

Basic Protocol 2: Orthotopic Murine Models of Bladder Cancer

  Materials
  • Trypsin/EDTA (Fisher Scientific, cat. no. BP2474‐100)
  • Bladder cancer cell line (from ATCC) in appropriate medium (see Table 14.14.1)
  • Appropriate medium (see Table 14.14.1)
  • Ice
  • 8‐week‐old female, athymic nude mice (Taconic)
  • Isoflurane
  • Ophthalmic ointment (Vetropolycin, Pharmaderm)
  • 2% chlorhexidine solution (Sigma)
  • Surgical lubricant (Fougera)
  • Commercial‐grade phosphate‐buffered saline (PBS; see recipe for 10×), 1×
  • Sterile surgical area with operating board
  • Precision vaporizer and nose cone for administering isoflurane (available at institutional animal care facility)
  • 5‐0 coated, braided silk sutures with attached needle (Seneture, cat. no. S1173)
  • Forceps (Dumoxel, cat. no. 11251‐35)
  • 24‐G angiocatheter (Jelco, cat. no. 4073)
  • Plastic syringes (BD, cat. no. 309602)
  • 100‐µl Hamilton glass syringe (Hamilton, cat. no. 81001)
  • Animal warmer (Gaymar T/Pump or similar)
  • Additional reagents and equipment for standard tissue culture procedures including trypsinization and counting cells using trypan blue (Phelan, )
    Table 4.4.1   MaterialsCommercially Available Bladder Cancer Cell Lines

    Malignancy characteristics Propagation Source Comments
    UM‐UC‐3 “High‐grade” IFN non‐responsive Hypertriploid (modal chromosome number = 80) McCoy's medium (Invitrogen) + 10% heat‐inactivated FBS Urinary bladder (epithelium), male patient
    KU‐7 “High‐grade” IFN non‐responsive DMEM + 5% FBS
    MGH‐U3 “Low‐grade” IFN responsive MEM +10% FBS
    RT4 “Low‐grade” Transitional cell papilloma IFN responsive McCoy's medium (Invitrogen) + 10% heat‐inactivated FBS Urinary bladder (epithelium), male patient
    T24 Transitional cell carcinoma McCoy's Medium + 10% FBS Urinary bladder (epithelium), female patient Contains H‐ras oncogene 19 hr generation time
    J82 Transitional cell carcinoma Eagle's Minimum Essential Medium + 10% FBS Urinary bladder (epithelium), male patient Contains H‐ras oncogene
    5637 Grade 2 carcinoma RPMI‐1640 medium +10% FBS Urinary bladder (epithelium), male patient pRB negative p16 positive
    HT‐1376 Grade 3 carcinoma Tumorigenic in mice Eagle's Minimum Essential Medium + 10% FBS Urinary bladder (epithelium) female patient
    TCCSUP Grade 4 transitional cell carcinoma Minimum essential medium (Eagle) in Earle's BSS with non‐essential amino acids and 1 mM sodium pyruvate +10% FBS Bladder neck (epithelium), female patient
    HT‐1197 Tumorigenic in mice Eagle's Minimum Essential Medium + 10% FBS Urinary bladder (epithelium), male patient
    SW 780 Transitional cell carcinoma Leibovitz's L‐15 medium +10% FBS Urinary bladder (epithelium), female patient Patient had pre‐op chemotherapy (Thiotepa) 41% plating efficiency

Basic Protocol 3: Using Mouse Models to Evaluate Treatments for Bladder Cancer

  Materials
  • Rapamycin (see recipe)
  • Animal balance
  • 1‐ml plastic syringes, (BD, cat. no. 309602)
  • 26‐G, 3/8‐inch needles (BD, cat. no. 305110)
  • Additional reagents and equipment for in vivo imaging using bioluminescence, ultrasound, or MRI imaging (Olive and Tuveson, ) and euthanizing the mice (Donovan and Brown, )

Support Protocol 1: Analyses and Tissue Collection of Mice Following Bladder Cancer Treatment Evaluation

  Materials
  • Phosphate‐buffered saline (PBS; see recipe for 10× stock), 1×
  • 10% buffered formalin (Fisher, cat. no. SF93‐4)
  • 4% paraformaldehyde (PFA; Sigma, cat. no. 158127)
  • Optimal cutting temperature (OCT) compound (Tissue‐Tek, cat. no. 4583)
  • Liquid nitrogen
  • Processing cassettes (Fisher, cat. no. 15‐182‐705)
  • Cryogenic vials (VWR, cat. no. 479‐081)
  • EDTA precoated tubes (Greiner Bio‐One, cat. no. 450403)
  • 22‐G 1‐in. needle (BD, cat. no. 305768)
  • 3‐ml disposable syringe (BD, cat. no. 309585)
  • 1.5‐ml tubes
  • Dissecting tools including: (Fine Science Tools)
    • Fine forceps
    • Surgical scissors
    • Disposable scalpel (Bard‐Parker, cat. no. 371611)
  • Petri dishes
  • Dissecting microscope
  • Additional reagents and equipment for euthanizing the mice (Donovan and Brown, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ahlering, T.E., Dubeau, L., and Jones, P.A. 1987. A new in vivo model to study invasion and metastasis of human bladder carcinoma. Cancer Res. 15:6660–6665.
   Branda, C.S. and Dymecki, S.M. 2004. Talking about a revolution: The impact of site‐specific recombinases on genetic analyses in mice. Dev. Cell 6:7‐28.
   Chin, J., Kadhim, S., Garcia, B., Kim, Y.S., and Karlik, S. 1991. Magnetic resonance imaging for detecting and treatment monitoring of orthotopic murine bladder tumor implants. J. Urol. 145:1297‐1301.
   Chong, L., Ruping, Y., Jiancheng, B., Guohong, Y., Yougang, F., Jiansong, W., Xiang, G., Jie, H., and Shusheng, X. 2006. Characterization of a novel transplantable orthotopic murine xenograft model of a human bladder transitional cell tumor (BIU‐87). Cancer Biol. Ther. 4:394–398.
   Cordon‐Cardo, C. 2008. Molecular alterations associated with bladder cancer initiation and progression. Scand. J. Urol. Nephrol. Suppl. 42:154‐165.
   Dalbagni, G., Presti, J., Reuter, V., Fair, W.R., and Cordon‐Cardo, C. 1993. Genetic alterations in bladder cancer. Lancet 342:469‐471.
   Dinney, C.P., McConkey, D.J., Millikan, R.E., Wu, X., Bar‐Eli, M., Adam, L., Kamat, A.M., Siefker‐Radtke, A.O., Tuziak, T., Sabichi, A.L., Grossman, H.B., Benedict, W.F., and Czerniak, B. 2004. Focus on bladder cancer. Cancer Cell 6:111‐116.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Eble, J., Sauter, G., Epstein, J., and Sesterhenn, I. 2004. Tumors of the Urinary System—Pathology and Genetics. IARC Press, Lyon, France.
   Fomchenko, E.I. and Holland, E.C. 2006. Mouse models of brain tumors and their applications in preclinical trials. Clin. Cancer Res. 12:5288‐5297.
   Hadaschik, B.A., Black, P.C., Sea, J.C., Metwalli, A.R., Fazli, L., Dinney, C.P., Gleave, M.E., and So, A.I. 2007. A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging. BJU Int. 100:1377‐1384.
   Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R.C., Ghafoor, A., Feuer, E.J., and Thun, M.J. 2005. Cancer statistics, 2005. CA Cancer J. Clin. 55:10‐30.
   Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van der Valk, M., and Berns, A. 2001. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29:418‐425.
   Kinkade, C.W., Castillo‐Martin, M., Puzio‐Kuter, A., Yan, J., Foster, T.H., Gao, H., Sun, Y., Ouyang, X., Gerald, W.L., Cordon‐Cardo, C., and Abate‐Shen, C. 2008. Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone‐refractory prostate cancer in a preclinical mouse model. J. Clin. Invest. 118:3051‐3064.
   Knowles, M.A. 2008. Bladder cancer subtypes defined by genomic alterations. Scand. J. Urol. Nephrol. Suppl. 2008 Sep;(218):116‐130.
   Lesche, R., Groszer, M., Gao, J., Wang, Y., Messing, A., Sun, H., Liu, X., and Wu, H. 2002. Cre/loxP‐mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32:148‐149.
   Mitra, A.P. and Cote, R.J. 2009. Molecular Pathogenesis and Diagnostics of Bladder Cancer. Annu. Rev. Pathol. 4:251‐285.
   Olive, K.P. and Tuveson, D.A. 2006. A “mouse hospital” for preclinical testing of novel cancer therapeutics. Clin. Cancer Res. 12:5277‐5287.
   Ouyang, X., Jessen, W.J., Al‐Ahmadie, H., Serio, A.M., Lin, Y., Shih, W.J., Reuter, V.E., Scardino, P.T., Shen, M.M., Aronow, B.J., Vickers, A.J., Gerald, W.L., and Abate‐Shen, C. 2008. Activator protein‐1 transcription factors are associated with progression and recurrence of prostate cancer. Cancer Res. 68:2132‐2144.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Puzio‐Kuter, A.M., Castillo‐Martin, M., Kinkade, C.W., Wang, X., Shen, T.H., Matos, T., Shen, M.M., Cordon‐Cardo, C., and Abate‐Shen, C. 2009. Inactivation of p53 and Pten promotes invasive bladder cancer. Genes Dev. 23:675‐680.
   Rangarajan, A. and Weinberg, R.A. 2003. Opinion: Comparative biology of mouse versus human cells: Modelling human cancer in mice. Nat. Rev. Cancer 3:952‐959.
   Seager, C.M., Puzio‐Kuter, A.M., Trushar Patel, T., Jain, S., Carlos Cordon‐Cardo, C., McKiernan, J., and Abate‐Shen, C. 2009. Intravesical delivery of rapamycin suppresses tumorigenesis in a mouse model of progressive bladder cancer. Cancer Prev. Res. 2:1008‐1014.
   Singh, M. and Johnson, L. 2006. Using genetically engineered mouse models of cancer to aid drug development: An industry perspective. Clin. Cancer Res. 12:5312‐5328.
   Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21:70‐71.
   St. Clair, M.B., Sowers, A.L., Davis, J.A., and Rhodes, L.L. 1999. Urinary bladder catheterization of female mice and rats. Contemp. Top. Lab. Anim. Sci. 38:78‐79.
   Watanabe, T., Shinohara, N., Sazawa, A., Harabayashi, T., Ogiso, Y., Koyanagi, T., Takiguchi, M., Hashimoto, A., Kuzumaki, N., Yamashita, M., Tanaka, M., Grossman, H.B., and Benedict, W.F. 2000. An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies. Cancer Gene Ther. 7:1575‐1580.
   Wu, X.R. 2005. Urothelial tumorigenesis: A tale of divergent pathways. Nat. Rev. Cancer 5:713‐725.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library