Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.
We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.
Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco.
1. Cell Preparation
2. Orthotopic Bladder Tumor Model
3. Ultrasound
4. Bioluminescent Imaging
5. saRNA Preparation
6. Intravesical Administration of Formulated saRNA
7. Representative Results
Bladder tumors stemming from KU-7-luc2-GFP cells can be monitored by luciferase bioluminescent imaging (Figure 1A and C) and bladder ultrasound (Figure 1B and D). Tumors are often detectable within 3-5 days after cell inoculation. We found successful bladder tumor implantation in over 90% of mice. As treatment with intravesical dsRNA ensues, the progression of tumor can be monitored with these modalities. After the animal is sacrificed, tumor growth can also be confirmed using GFP fluorescence. In general, mean bioluminescence correlated with mean ultrasound dimensions for bladder tumors, as seen visually in Figures 1C and 1D. However, after animals were sacrificed and tumor was confirmed by GFP, we found that bioluminescence correlated more closely with the presence of viable tumor than did ultrasound measurements. This was especially apparent among mice treated with saRNA (as opposed to controls) because residual scar tissue in treated mice was often visualized by ultrasound but could not be differentiated from live tumor.
Figure 1. Tumor growth kinetics of bladder tumors. (A) Bioluminescence imaging measures the luciferase activity within bladder tumors to confirm their location and growth. (B) In vivo bladder ultrasound using a 40MHz probe produces tumor images whose dimensions can be accurately measured. (C) Graphs illustrating the tumor’s progressive increase in bioluminescence and ultrasound dimensions. Values represent mean (+/- standard deviation) measurements from 6 animals. Click here to view larger figure.
We present a protocol for the establishment of a mouse orthotopic xenograft bladder tumor model followed by intravesical treatment of the tumors with LNP formulated saRNA that targets the promoter of p21CIP1/WAF1 (p21) for transcriptional activation.2, 3 The resultant tumors can be confirmed and monitored with bioluminescent imaging and bladder ultrasound. Orthotopic models may be superior to intraperitoneal, subcutaneous, or intravenous models by providing a milieu of urine and urothelium that more closely approximates endogenous bladder cancer. A variety of orthotopic mouse bladder tumors have been established using both direct bladder wall injection as well as intravesical instillation.4-8 Intravesical instillation of tumor, as demonstrated here, more closely mimics human bladder tumors which begin superficially in the mucosal epithelium and grow deeper as they progress. Our method of using silver nitrate to accommodate bladders for tumor uptake is inexpensive and technically simple.
Hadaschik et al. described intravesical instillation of bladder tumors in mice and reported reliable bioluminescent tumor assessment.5 We similarly found high rates of successful tumor implantation with catheterization and cell instillation. Our tumors were not only confirmed with bioluminescence but also with in vivo bladder ultrasound, providing an additional method for quantifying tumor progression.
In this protocol, we note several modifications to others’ technique. We employed instillation of silver nitrate solution to disrupt the bladder extracellular glycosaminoglycan layer for tumor cell implantation, instead of electrocautery. We found this to be a cheap, simple, and reliable method that minimized potential for user error. During ultrasound evaluation of the bladder, we found it unnecessary to perform urethral catheterization beforehand. As long as the animal had not been subjected to significant distress to cause urination before undergoing anesthesia, the mouse bladder was reliably distended during imaging to allow for excellent tumor visualization. Without a doubt, urethral catheterization was the least reliable step of the protocol. In our experience, catheterization of athymic nude mice is more difficult than in C57 mice. However, with the technique described in this protocol and video, over 90% of animals were repeatedly catheterized successfully for both tumor instillation and treatment.
The authors have nothing to disclose.
This research is funded by the AACR Henry Shepard Bladder Cancer Research Grant (09-60-30-LI).
Name of the reagent | Company | Catalogue number | Comments (optional) |
KU-7-luc-GFP cells | Caliper | 128091 | |
thymic nude mice ( nu/nu) | Simonsen Laboratories | 6-7 weeks old | Sim:(NCr) nu/nu fisol |
Ultrasound unit | Visualsonics | Vevo 770 | RMV-704 probe |
Bioluminescence unit | Caliper | IVIS Spectrum | |
Exel safelet catheter | Fisher | 14-841-21 | 24G X ¾” |
Silver nitrate | Sigma | S8157 | |
Hamilton syringe | Hamilton company | 80301 | |
LNP-saRNA | Alnylam Pharmaceuticals |
Table 1. Specific reagents and equipment.