An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

Summary

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Abstract

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.

Protocol

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

1. The human bladder cancer cell line KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) is grown in RPMI-1640 media supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin.
2. Cultures are maintained at 37 °C, 5% CO₂ with media changes every two to three days.
3. Cells were harvested by trypsin digestion and counted using a hemocytometer. A single cell suspension of 2 x 10⁶ cells in 50 μl was p....

Discussion

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 p21^CIP1/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 mor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KU-7-luc-GFP cells	Caliper Life Sciences	128091
thymic nude mice ( nu/nu)	Simonsen Laboratories	6-7 weeks old	Sim:(NCr) nu/nu fisol
Ultrasound unit	VisualSonics, inc.	Vevo 770	RMV-704 probe
Bioluminescence unit	Caliper Life Sciences	IVIS Spectrum
Exel safelet catheter	Fisher Scientific	14-841-21	24G X ¾"
Silver nitrate	Sigma-Aldrich	S8157
Hamilton syringe	Hamilton Co	80301
LNP-saRNA	Alnylam Pharmaceuticals
Table 1. Specific reagents and equipment.