By the morbidity inflicted on the mice by the need for laparotomy and mobilization of the bladder. It is also technically challenging to ensure adequate AbMole 4-Demethylepipodophyllotoxin injection into the bladder wall, and this method is associated with a significant learning curve. We have developed a novel approach to address these limitations 
of the intramural inoculation of bladder cancer xenografts, and thereby potentially enhance the accuracy and reproducibility of this model. We have optimized the percutaneous, ultrasound-guided injection of bladder cancer cells into the anterior bladder wall. In addition, we are able to monitor xenograft growth and perfusion in vivo longitudinally during therapy, and we are able to inject therapeutic agents directly into the tumor under ultrasound guidance. Here we demonstrate the feasibility and reproducibility of ultrasoundguided intramural inoculation of orthotopic bladder cancer xenografts as well as subsequent image guided manipulation and monitoring. The mice were anesthetized with isoflurane and mounted on the imaging table with continuous monitoring of vital signs. The abdomen was disinfected with alcohol and sterile ultrasound gel was applied. To visualize the perfusion status of the xenograft tumors, a cine loop was recorded as the reference. A second cine loop was recorded 10 seconds after injection of 120 mL non-targeted microbubbles into the tail vein of anesthetized mice. The point at which microbubbles entered the plane was determined and the background reference was subtracted. The tumor was selected as the contrast region and Reference Subtracted Mean Data were used. Changes of the Contrast Percent Area over time were documented and 2D images were recorded in which any pixel was marked green when a microbubble passed. The existence of reliable animal models is a basic requirement in oncologic research for the in vivo investigation of tumor biology and the testing of novel antineoplastic treatment strategies. Despite the existence of reproducible syngeneic and transgenic orthotopic tumor models of bladder cancer, they are not widely used due to both inherent limitations and complexity of the models, as well as the intensity of associated resource utilization. Orthotopic xenograft models have proven to offer the most flexibility and have the most practical utility, and therefore remain the gold standard for in vivo modeling of bladder cancer. In this work, we have generated a novel in vivo model of orthotopic bladder cancer xenografts via the inoculation of human bladder cancer cells into the murine bladder and have shown it to be highly reproducible. Tumors were established in 98% of inoculated mice using three different human cell lines. Due to excellent optical resolution, the tumor cells can be inoculated by high precision strictly into the anterior bladder wall, thus reducing the rate of obstructive complications and allowing longer growth and treatment periods. Furthermore, the time per inoculation is short in comparison to existing models, and decreases rapidly with additional experience.