Introduction:

Large animal cancer models offer tremendous research potential to improve and accelerate new therapeutics and treatment modalities, but few exist. Pigs are particularly suitable due to their numerous similarities to humans such as anatomy, physiology, metabolism, immunology, pathology, genetics etc. Larger tumor models also provide a platform for mimicking surgical-treatment-methods under clinically relevant conditions. This is a particular interest in urothelial-cancer for developing endoscopic-therapies in the bladder and upper-urinary-tract to provide effective organ sparing therapies for improving patient care. We sought to develop a urothelial-model using the oncopig (OMC) provided by the University of Illinois Cancer-Center. The OMC carries two mutations (KRASG12DTP53R167H) which can be induced by exposure to the Adeno Virus-Cre (AdCre). To infect urothelial cells, we designed two different procedures to overcome the glycosaminoglycans barrier (GAG-Layer). The aim of this model is to generate urothelial like tumors in the bladder in a targeted manner.

Methods:

Two techniques for virus delivery during cystoscopy were developed and investigated. In total 8 OMCs underwent tumor inoculation, 4 per technique.  In procedure-I a solution of AdCre and Arista or Surgifoam was injected using a 23 g needle at three anatomically distinct locations in the bladder wall in attempting to create a muscle invasive tumor. In procedure-II we attempted to develop superficial tumors by superficially denuding the urothelium in 3 locations and subsequently instilling 100 ml of Virus in buffered solution into the bladder which was emptied after 1 hour. One week after virus exposure pigs underwent a CT scan and a follow-up cystoscopy to evaluate tumor progression. Ultrasound and blood-collection were performed weekly until euthanasia. To analyze tumor-growth over time pigs were euthanized at 3 timepoints (Day: 14, 21, 28). At Necropsy tumors were measured and adjacent organs were examined to evaluate tumor stage. Histopathological examination was performed.

Results:

In total 6/8 (75%) OMC developed endoluminal tumors. Tumor-induction showed identical efficiency for procedure-I and procedure-II, 3/4 (75%). Using Proc-I, one pig received a solution of surgifoam and Ad-Cre and no tumor developed. The other 3 pigs that were injected with AdCre and Arista developed tumors. 2 pigs showed tumors in 2/3 treatment-locations, one pig only in 1/3.  Pigs that underwent Procedure-II, one pig developed tumors in all 3 locations (Fig. A), one pig in 2 and one in 1 location. Growing tumors showed an average of 0.4 cm after 14 days, 1.63 cm after 21 days and 1.66 cm after 28 days. H&E showed a poorly differentiated neoplasm with sarcomatoid and epithelioid features accompanied by inflammatory processes (Fig. B). Tumors showed muscle-invasive-growth. In the Procedure group-I, 2 pigs had T3 tumors after 21 days and 28 days. No perioperative complications were observed during the study period.

Conclusion:

This first description of bladder tumor development in a porcine model using the Oncopig had an good efficiency rate of tumor induction in a short period of time. The model is reproducible, and initial tumors appear to behave in a growth pattern similar to aggressive, high-grade bladder tumors. Our future goals will be refining the virus targeted cell infection process to create more specific tumor growth characteristics and investigate tumor genomics and immune micro-environment to establish a better understanding of the tumor biology.

Funding: Thompson Family Foundation