User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

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

¹Departments 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

Are you the author of this protocol? Login or register and return to this page.

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

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: 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 (Pten^flox/flox, C57Bl6;129SVJ) (Lesche et al., 2002) and p53 (p53^flox/flox, FVB;129SJv) (Jonkers et al., 2001) obtained from the NCI Mouse Models of Human Cancer Consortium repository (http://mouse.ncifcrf.gov/)
- R26R reporter allele (GT(ROSA)26Sor^tm1Sor) (C57Bl6;129SVJ) (Soriano, 1999) 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, 2006)

Table 14.14.1 Commercially 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, 2006) and euthanizing the mice (Donovan and Brown, 2006)

Support Protocol: 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, 2006)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 14.14.1

Surgical procedure for instilling cells into the mouse bladder.

View Image
Figure 14.14.2

Placement of a superficial silk purse-string suture around the urethral meatus.

View Image
Figure 14.14.3

Modeling early-stage bladder cancer in mutant mice. (A to C) Representative H&E-stained sections from bladders of a control mouse (uninjected p53flox/flox; Ptenflox/flox) or two representative experimental mice (Adeno-Cre–injected p53flox/flox; Ptenflox/flox) 6 weeks subsequent to delivery of Adeno-Cre (or mock injection for the control). Shown are representative histologic sections from a total of 10 mice in each of the experimental and control groups. Note that the experimental, but not control, mice have a dysplastic and expanded epithelium (arrows) as well as abnormal stroma. Inset, high power view. (D to F) KI67-immunostained adjacent sections show elevated proliferation in the bladder epithelium of the experimental mice relative to the control mice. Inset, high power view. Scale bar, 100 µm.
(Source: Reprinted with permission from Seager et al. (2009).)

View Image
Figure 14.14.4

Rapamycin inhibits bladder tumor progression. Representative examples from the vehicle- or rapamycin-treated mutant mice show the following: (A to D) gross anatomy of bladder tissues/tumors; (E to H) H&E-stained sections; or (I to T) immunostained sections using the indicated antibodies. In all groups, gross analyses of bladder tissues and H&E analyses were done on all control and experimental mice in each group; immunohistochemistry was done on a minimum of four animals from each group; representative data are shown. Scale bars, 100 µm.
(Source: Reprinted with permission from Seager et al. (2009).)

View Image

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.