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 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 used15-24. 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.
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
2. Intravesical Delivery of Adenovirus (8 Days after Implantation of Cells)
CAUTION: This part of the protocol uses adenovirus, which is an infectious agent and should be handled under strict BSL2 guidelines (http://oma.od.nih.gov/manualchapters/intramural/3035/).
All procedures performed with infectious agents were approved by the Institutional Biosafety Committee of the Medical University of South Carolina.
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 variable between animals, which should be taken into account when planning group size for statistical purposes (Figures 2 and 3). If a small animal imaging system is not available, an alternative approach to measure expression of the luciferase transgene is to remove and homogenize the bladder for in vitro analysis. Comparison between in vivo analysis using the IVIS200 small animal system and in vitro analysis using the Steady-Glo kit, indicate excellent correlation between data sets (Figure 3).
Figure 1. Analysis of tumor burden. Weight of nontumor (n=4) and tumor-bearing (n=10) mouse bladders 9 days after instillation of 200,000 MB49 cells. Statistical significance was determined by the Student’s t-test using the Graphpad software. *p= 0.0002. Click here to view larger image.
Figure 2. In vivo analysis of gene expression. Small animal imaging performed 24 and 48 hr after instillation of virus storage buffer (-) or 1 x 109 PFU AdCMV.Luc (+). To visualize luciferase, mice were injected intraperitoneally with 200 μg luciferin/mouse. Expression of the luciferase transgene was measured in vivo using the IVIS200 small animal imaging system. Click here to view larger image.
Figure 3. Comparison of bioluminescence signals obtained by in vivo small animal imaging and an in vitro assay. Twenty-four hours after delivery of virus storage buffer (open circles) or 1 x 109 PFU AdCMV.Luc (closed circles), mice were imaged using the IVIS200 and then sacrificed. Bladders were removed and homogenized for in vitro analysis of the bioluminescent signal using the Steady-Glo kit. Luciferase signal obtained by each method is expressed in arbitrary units (AU). The coefficient of determination (R2) was calculated in with the Data Analysis add-in of Microsoft Excel. Click here to view larger image.
Insult to GAG layer | Cells implanted | Retention time | Tumor detection and Development | reference |
Electrocautery | 0.01-1 x 105 MB49 | Hematuria: 1,000 cells: 0/0; 10,000 cells: 4/6; 100,000 cells: 6/6 Tumors: 1,000 cells: 0/6; 10,000 or more cells: 6/6, 6/6 (100%) | 5 | |
Electrocautery | 5 x 104 MB49-Luc | Tumor incidence: 90% (as in reference11) | 6 | |
Electrocautery | 1 x 105 MB49 | ~3 hr | Tumor incidence: 90% at 50 days | 7 |
Electrocautery | 1 x 105 MB49-PSA | PSA: detected as early as 4 days after implantation | 8 | |
Electrocautery | 1 x 105 MB49 | Tumor incidence: 90% | 9 | |
Electrocautery | 5 x 104 MB49 | 2 hr | Tumor incidence: 83.3% (10/12) on day 21 | 10 |
Electrocautery | 2 x 104 MB49 | 2 hr | Tumor incidence: 100% by day 28 | 11 |
Electrocautery | 1-5 x 104 MB49 | 3 hr | Hematuria: 100% by day 16; Tumor incidence: 100% | 12 |
Electrocautery | 1 x 105 MB49 | 3 hr | Tumor incidence: 97.3% (73/75) | 13 |
PLL (100ul 0.01% 20 min) | 1 x 105 MB49-PSA | 2 hr | Tumor incidence: 100% | 15 |
PLL (0.1 mg/ml) | 1 x 105 MB49-PSA | 2 hr | Tumor incidence: 100% | 16 |
PLL (100 μl 0.1 mg/ml 20 min) | 1 x 105 MB49 | 1 hr | Tumor incidence: 94% (15/16) | 17 |
PLL (0.1 mg/ml) | 1 x 106 MB49 | Hematuria at day 7: 50%, 100% at day 14 | 18 | |
PLL (100 μl 0.1 μg/ml 20 min) or 22% ethanol | 1 x 105 MB49 | 1 hr | Tumor incidence: Unmodified bladder: 0%, PLL: 80-100%; Ethanol 40-80% | 19 |
Silver nitrate (5 μl 0.2 M) | 1 x 106 MB49 | 1 hr | Tumor incidence: 100% | 20 |
Silver nitrate (10 μl 0.15 M 10 sec) | 5 x 105 MB49 | Tumor incidence: 92% (46/50) | 21 | |
Silver nitrate (8 μl 1 M 10 sec) | 5 x 105 MB49 | 2 hr | Hematuria: 100% at day 7; days hematuria 100%; Tumor incidence: 96.7% (29/30 mice) at day 15 | 22 |
Silver nitrate 5 μl 0.2 M | 0.5-2 x 106 MB49-PSA | 1 hr | PSA detected | 23 |
HCl (100 μl 0.1 M 15 sec) | 1 x 106 MB49 | 1 hr | 24 |
Table 1. Tumor detection and development following orthotopic implantation of MB49 cells. Different physical and chemical insults have been used for disruption of the GAG layer to facilitate intravesical growth of MB49 cells. Experimental conditions and results from previous studies using the MB49 orthotopic model are summarized. When available, the information in parentheses in the first column includes volume, concentration, and contact time of agent used for chemical disruption of the GAG layer.
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 require euthanizing up to 25% of the animals by day 14 due to excessive tumor burden. In our experience, mice are not adversely affected by tumor load within 12 days. In addition to standard criteria such as lethargy, poor grooming, and/or loss of appetite excessive tumor burden in this model is evidenced by the inability to urinate.
An important consideration for this protocol is logistics. First, MB49 cells grow rapidly and plating one million cells two days before implantation will yield optimal cultures in log phase on the day of instillation. If the number of animals required for an experiment exceeds the number of animals that can be implanted in one day, MB49 cultures will have to be set up accordingly (on multiple days) to prevent overgrowth of cultures. Second, until the technique has been perfected, users should work with a small group of animals. Experienced users will be able to implant 6 groups of 5 mice each in one day without an assistant (1.5 hr/group including dwell times). However, initially it is helpful to have an assistant prepare and count the cells for instillation. Lastly, location of equipment is important. Ideally the biosafety cabinet and anesthesia equipment are located in the same room.
A potential limitation is that the anesthesia equipment has 5 nosecones, which are in use during dwell time. If large numbers of mice are routinely implanted, several such systems could be used in parallel. In theory, other methods of anesthesia could be used. However, a benefit of isoflurane inhalation anesthesia is that dwell time can be controlled.
Once the technique of catheterization is mastered, this protocol can be applied to a number of experimental conditions. New bladder cancer cell lines or cells derived from primary bladder tumors can be evaluated for their potential to grow orthotopically. Furthermore, it is possible to implant different numbers of cells to determine the threshold amount required for tumor take. Tumor growth rates can also be established. These variations may be particularly useful, if comparisons are made between genetically modified cells to determine the impact of the gene of interest on tumor establishment or growth rate. For monitoring of adhesion, growth rate, or tumor take, cells may be transfected with a reporter gene such as luciferase6. However, an important consideration is the impact of hypoxia and necrosis on the bioluminescence signal29. Catheterization can also be used to deliver a therapeutic agent. We are currently using the orthotopic model to evaluate gene delivery strategies in an in vivo setting. This model will be useful to optimize delivery of therapeutics and to determine their impact on tumor regression and survival.
The authors have nothing to disclose.
This work was supported by NIH R21 CA143505 to Christina Voelkel-Johnson.
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 |