Claudia Janssen

Ultrasound-guided intramural inoculation of orthotopic bladder cancer xenografts: a novel high-precision approach.

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.

Erythropoietin Accelerates the Regeneration of Ureteral Function in a Murine Model of Obstructive Uropathy

PURPOSE Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. MATERIALS AND METHODS Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and β-common receptor expression in obstructed and unobstructed ureters by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. RESULTS Erythropoietin, erythropoietin receptor and β-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. CONCLUSIONS Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.

Minimally invasive establishment of murine orthotopic bladder xenografts.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse. With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period. In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

A Role for the Hedgehog Effector Gli1 in Mediating Stent-Induced Ureteral Smooth Muscle Dysfunction and Aperistalsis.

OBJECTIVE To better understand the effects of double J stenting on ureteral physiology and function. MATERIALS AND METHODS In total, 24 pigs were stented cystoscopically unilaterally for 48 hours, 1, 2, 4, and 7 weeks. Controls consisted of un-stented animals (n = 4) or the contralateral un-stented ureter in pigs. Ureters were harvested and tested in tissue baths to evaluate their contractility. Ureteral inflammation and expression of Sonic Hedgehog (Shh) and the transcriptional activator Gli1 (the downstream target of active Hedgehog signaling) were assessed histologically and by immunohistochemistry, respectively. RESULTS Indwelling ureteral stents were found to abolish normal ureteral function in all animals. Specifically, ureteral smooth muscle (SM) activity was significantly diminished within 48 hours after stenting and persisted at the 1-week time point. Furthermore, ureteral SM dysfunction was associated with increasing ureteral dilation due to the indwelling stent. Simultaneously, we observed a loss of Gli1 expression in SM cells, with a concomitant increase in ureteral inflammation. Expression of Shh was restricted to the urothelium and was not different between controls, stented, and contralateral ureters. CONCLUSION Stent-induced aperistalsis was associated with diminished SM contractility, increased tissue inflammation, and reduced Gli1 expression in ureteral SM cells, independent of Shh expression. The present study is the first to show that indwelling stents negatively affect ureteral SM activity and identify a role for specific molecular mechanisms involved.

63 Erythropoietin accelerates the regeneration of ureteral function in a murine model of obstructive uropathy

Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. Materials and Methods: Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and b-common receptor expression in obstructed and unobstructed ureters by reverse transcriptasepolymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. Results: Erythropoietin, erythropoietin receptor and b-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. Conclusions: Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.