An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Summary

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.

Abstract

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.

Introduction

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 needed.

Development of novel therapeutics requires preclinical models to evaluate efficacy following initial in vitro assessment. The tumor microenvironment can significantly influence cancer development and responsiveness, which highlights the need for preclinical models in which tumors arise or can be established in the organ of origin. One approach is the development of transgenic models in which tumors arise spontaneously or can be induced in an organ-specific manner. An excellent protocol of a transgenic bladder cancer model has recently been published². The drawback of transgenic models is that tumors tend to develop slowly and with less uniformity than desired. In addition, the cost of maintaining a breeding colony has to be considered. An alternative to transgenic models is orthotopic implantation of tumor cells, which has the benefit of short time frames for tumor establishment in commercially available mice. While some human bladder cancer cell lines can be grown orthotopically (we have successfully used UM-UC-3), it may be desirable to establish tumors in immunocompetent mice. Two murine bladder cancer cell lines, which grow orthotopically are MBT-2 and MB49³. Since MBT-2 cells are contaminated with replicating type C retrovirus⁴, we have chosen MB49 cells for our studies. It is important to note that MB49 cells were isolated from a male mouse and orthotopic implantations are for anatomical reasons performed in female mice. This has the benefit of easy identification of the implanted cells by markers of the Y chromosome, but the gender mismatch can be a drawback for immunological studies.

The bladder epithelium is lined by a glycosaminoglycan (GAG) layer, which functions as a barrier for infection by microorganisms. This barrier can also interfere with implantation of tumor cells and several methods have been developed to overcome this difficulty (Table 1). Electrocautery has been used extensively as a physical means to disrupt the GAG layer ^5-13 and a protocol demonstrating electrocautery has recently been published in JoVE¹⁴. However, should an electrocautery unit not be available, chemical means to destroy the GAG layer such as silver nitrate or poly-L-lysine can also be used^15-24. Tumors are established effectively by a brief exposure of the bladder to a small volume of silver nitrate (5-10 μl, 0.15-1.0 M, ~10 sec) or longer contact with poly-L-lysine (100 μl of 0.1 mg/ml for 20 min) (Table 1). Here we describe a method that uses trypsin to facilitate implantation of MB49 cells.

In an attempt to improve therapeutic approaches for bladder cancer, gene therapy has garnered significant attention. From a clinical point of view, bladder cancer is an ideal target for gene therapy due to easy accessibility of the organ and the ability to locally deliver the payload. Viral vectors that have been explored for bladder cancer gene therapy include an oncolytic herpes simplex virus²⁵, retrovirus²⁶, canarypox virus²⁷, vaccinia virus, AAV, and adenovirus²⁸. In the second part of our protocol, we describe a method for viral delivery that is virtually identical to instillation of the tumor cells. Of interest in our lab is the development of novel approaches to gene delivery, which we assess via bioluminescence using an adenoviral vector that expresses a luciferase transgene. However, the methodology of bladder catheterization can be used for delivery of various agents and therefore has broad applicability.

Protocol

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 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…

Representative Results

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…

Discussion

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…

Materials

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