Stephen Bardot

Laparoscopic Surgical Technique for Internal Drainage of Pelvic Lymphocele

A previously unreported technique of internal drainage of a pelvic lymphocele is described using a laparoscopic approach to create the peritoneal window for drainage in 2 patients. The technique avoids a larger incision and permits a shorter recovery period.

A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis. To develop a patient-derived orthotopic xenograft model to mimic clinical urothelial cell carcinoma progression to metastatic disease, the urothelial cell carcinoma cell line UM-UC-3 and two urothelial cell carcinoma patient specimens were doubly tagged with Luciferase/RFP and were intra-vesically (IB) instilled into NOD/SCID mice with or without lymph node stromal cells (HK cells). Mice were monitored weekly with bioluminescence imaging to assess tumor growth and metastasis. Primary tumors and organs were harvested for bioluminescence imaging, weight, and formalin-fixed for hematoxylin and eosin and immunohistochemistry staining. In this patient-derived orthotopic xenograft model, xenograft tumors showed better implantation rates than currently reported using other models. Xenograft tumors histologically resembled pre-implanted primary specimens from patients, presenting muscle-invasive growth patterns. In the presence of HK cells, tumor formation, tumor angiogenesis, and distant organ metastasis were significantly enhanced in both UM-UC-3 cells and patient-derived specimens. Thus, we established a unique, reproducible patient-derived orthotopic xenograft model using human high-grade urothelial cell carcinoma cells and lymph node stromal cells. It allows for investigating the mechanism involved in tumor formation and metastasis, and therefore it is useful for future testing the optimal sequence of conventional drugs or the efficacy of novel therapeutic drugs.

Abstract A11: Using a patient-derived orthotopic xenograft (PDOX) model to screen drugs targeting renal cell carcinoma (RCC) metastasis: A personalized therapeutic strategy

Renal cell carcinoma (RCC) is the most common solid tumor of the adult kidney, with a mortality of 30-40%(1). RCC is highly metastatic and potentially lethal. 25-30% of RCC patients have metastatic disease at diagnosis, and 40-50% of patients develop metastases later. The 5-year survival for metastatic RCC patients is less than 10%(2), due to its highly-resistant to current therapies. The challenge of managing RCC patients in the clinic often involves trying different drugs one after another until one works, so individualized therapy is urgently needed. Our objective was to establish a patient-derived orthotopic xenograft (PDOX) intra-renal sub-capsular injection NOD/SCID mouse model that recapitulates metastatic RCC for personalized therapeutic strategies. Two luciferase-tagged RCC cell lines (A498-Luc, 769P-Luc) were used to determine the IC50 of sunitinib, pazopanib, and everolimus using bioluminescent imaging (BLI) of in vitro cell viability assay at 48 hrs. A consented human RCC specimen (KiCa-Pt58) was obtained via radical nephrectomy and tumor cells tagged with luciferase. KiCa-Pt58-Luc cells (0.01 million) were then injected sub-capsularly into the left kidneys of NOD/SCID mice. Mice bearing kidney tumors were split into 1 control and 4 sequential treatment groups receiving 40mg/kg sunitinib, 40mg/kg pazopanib, or 5mg/kg everolimus three times per week via gavage. Tumor growth was monitored weekly by BLI. Drug responses were evaluated based on BLI data and decision made for whether to switch to the next sequence drug. At necropsy, primary tumors and mouse lungs were collected for BLI, weights, and histology. The IC50 was 6μM for sunitinib and 3.6μg/ml for pazopanib. In our PDOX model, patient tumors successfully produced primary tumor and spontaneous lung metastasis as seen in RCC patients (45/45). Selected drug dosages delivered by gavage were safe, effective and without adverse effects. Drug sequence of pazopanib switch to everolimus is the best among 4 combinations tested for primary tumor progress and lung metastasis for patient tumor KiCa-Pt58. Our unique PDOX model provides a platform for testing RCC drugs simultaneously for responses in primary tumor progression and metastasis. It could lead to the development of realistic, durable, and individualized treatments for RCC patients. Citation Format: Jessie Gills, Ravan Moret, Xin Zhang, Ashley Richman, John Nelson, Grace Maresh, Christudas Morais, Glenda Gobe, Sunil Talwar, Marc Matrana, Stephen Bardot, Li Li. Using a patient-derived orthotopic xenograft (PDOX) model to screen drugs targeting renal cell carcinoma (RCC) metastasis: A personalized therapeutic strategy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A11.

MP68-15 LYMPH NODE STROMAL CELLS PROMOTE HIGH GRADE UROTHELIAL CELL CARCINOMA CANCER IMPLANTATION, GROWTH, ANGIOGENESIS, AND METASTASIS IN AN ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODEL

INTRODUCTION AND OBJECTIVES: Extracellular vesicles (EV) are small membrane vesicles secreted by most cell types. They are first found in reticulocytes and play important roles in cell-to-cell communication. Recent studies suggest that EV secreted by tumor cells containing tumor specific proteins and miRNAs. Previous study shows that EV purified from muscle invasive bladder cancer promote cancer progression. This study aims to characterize the genetic contents, such as proteins and nucleic acids and establish the bladder cancer specific EV biomarkers METHODS: EV were purified from bladder cancers, immortalized bladder cells, and urine of healthy volunteers and muscle invasive bladder cancer patients. The protein contents were analyzed by mass spectrometry. To prioritize the proteins of interest, we performed gene expression profile analysis of proteins via public microarray repositories from NCBI Gene Expression Omnibus profile #GDS1479 (carcinoma in situ lesions of the urinary bladder) that consists of 5 categories: normal urothelium, Non-muscle invasive BC with cancer in situ (CIS), superficial TCC without CIS, Muscle invasive bladder cancer, and CIS. In addition to protein, we also performed miRNA expression profiling microarray on EV from high grade bladder cancer cells, and immortalized bladder cells (LC Sciences). RESULTS: More than 400 proteins were found in bladder EV. Among them, we found 9 cancer-associated EV proteins that are particularly interesting because their gene expression levels are significantly elevated in cancer stages as compared to normal urothelium. Those proteins are subjected to further functional characterization via gene knockdown/overexpression strategies. The differences in miRNA expression were compared and differences were determined by a student T test (P<0.05). Results showed significant difference miRNAs between cancer cells vs normal cells. Some of cancer-specific miRNAs which have shown to play critical roles in tumorigenesis and cancer progression will be study priority CONCLUSIONS: Our study enables us to establish a panel of EV protein and miRNA signatures that can be further developed into a urine-based method for rapid detection of stage-specific biomarkers which will be valuable for detecting and managing bladder cancer.