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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Published: December 1st, 2013

DOI:

10.3791/50181

1Department of Microbiology & Immunology, Medical University of South Carolina

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

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.

Bladder cancer is the second most common cancer of the urogenital tract with nearly 75,000 new cases and 15,000 deaths expected in 20121. High rates of recurrence require lifelong follow-up, which makes bladder cancer one of the costliest cancers to treat. Bladder cancer that has invaded the muscle layer may metastasize to liver, lung or bone via the lymphatic system. Multimodal therapy of advanced tumors results in only 20-40% survival after 5 years. Therefore, effective treatment strategies aimed at reducing the recurrence and progression of superficial bladder cancer as well as improving therapeutic outcome in patients with advanced disease are urgently ....

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All procedures involving animals have been reviewed and were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.  The protocol was approved under USDA category D for pain. 

1. Cell Implantation

  1. Two days before performing the procedure, plate 1 x 106 MB49 cells into T-25 flasks. Use high glucose DMEM supplemented with 10% FBS (and antibiotics, if desired). One flask is sufficient for each group of up to f.......

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Hematuria is observed in nearly all mice within 8 days after implantation of 200,000 MB49 cells. As shown in Figure 1, bladder weight more than doubles from 34.7±3.3 mg (range 31-37 mg, n=4) in nontumor bearing mice to 87.5±19.2 mg (range 77-120 mg, n=10) in mice that have been implanted with MB49 cells. In terms of gene delivery, we found that imaging mice 24 hr after viral instillation yields a stronger signal than after 48 hr (Figure 2). Adenoviral delivery is highly va.......

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The primary methodology described in this protocol is catheterization of mouse bladders, which has broad applications for instillation of cells or any agent intended for local delivery to the bladder epithelium. The specific protocol outlined above has been optimized for short-term studies (~10 days). Implanting the accurate number of cells is critical, since a higher cell number will result in more rapid tumor growth and possibly loss of animals due to large tumor burden. Using 200,000 MB49 cells for instillation may re.......

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This work was supported by NIH R21 CA143505 to Christina Voelkel-Johnson.

....

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Name Company Catalog Number Comments

Name of reagent

Company

Catalog number

Comments

6-8 week old female mice

Jackson Laboratories

Strain Name: C57BL/6J

Stock Number: 000664

Trypsin*

MediaTech

MT25-053-CI

Obtained through Fisher

DMEM*

MediaTech

MT10-017-CV

Obtained through Fisher

FBS

Hyclone

SH30071.03

Heat-inactivated

T25 flasks*

Corning Costar

Corning No.:3056

Fisher: 07-200-63

Obtained through Fisher

MB49 cells

N/A

N/A

Obtained from Dr. Boehle (see reference11)

Puralube Vet Ointment*

Pharmaderm

Henry Schein Company

No.:036090-6050059

Fisher: NC9676869

Obtained through Fisher

Depilatory cream: Veet

local pharmacy

Lubricant:

K-Y Jelly

local pharmacy

Catheters*

Exel International

Exel International

No.:26751;

Fisher: 14-841-21

Obtained through Fisher

Isoflurane

Terrell

NDC 66794-011-25

Obtained though hospital pharmacy

1 ml slip tip TB syringes

Becton Dickinson

BD309659

Fisher:14-823-434

D-Luciferin

Gold Biotechnologies

L-123-1

Ad-CMV-Luc

VectorBiolabs

1000; Request large scale amplification and CsCl purification for in vivo use

Infectious agent that requires BSL2 containment

Steady-Glo Luciferase Assay System

Promega

E2510 (10 ml), E2520 (100 ml), or E2550 (10 x 100 ml)

*available through multiple vendors

EQUIPMENT

Material name

Company

Catalog number

Comments

Anesthesia system

E-Z Systems, Euthanex Corporation

Anesthesia system: EZ7000

5-port mouse rebreathing device: EZ109

Obtained through Fisher

Xenogen IVIS 200

Caliper Life Sciences

http://www.caliperls.com/products/preclinical-imaging/ivis-imaging-system-200-series.htm

FLUOstar Optima

BMG Labtech

http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2

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