IN VIVO CHARACTERIZATION OF SYNGENEIC, ORTHOTOPIC MOUSE MODEL OF COX-2 POSITIVE RENAL CELL CANCER

Author

Purba Singh, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2013

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Wilber, Andrew

Abstract

Renal cell carcinoma (RCC) is the third most common genito-urinary cancer. Beyond surgery, few other treatment options for RCC exist and about one-third of the patients who have had nephrectomy develop metastasis subsequently. The treatment of the metastatic disease remains a clinical challenge. Hence, novel therapeutic options are necessary for the patients with metastatic RCC. Immunotherapy is the most common mode of treatment in RCC presently; however, it contributes to a large number of toxic side effects to the patients. The immunotherapeutic regimens currently used to treat metastatic renal cancer are recombinant human interleukin -2 (IL-2) and recombinant human interferon alpha alone or in combination. However, the uses of these high dose cytokines are limited by their toxicity and poor patient response rates. Preclinical studies in animal tumor models of RCC are required to address the newer and effective therapeutic approaches for late stage metastatic RCC. A suitable animal model for studying RCC is lacking. Hence, development of a novel animal model would largely contribute in testing newer therapeutics and combating the metastatic disease. Cyclooxygenases are group of enzymes that catalyze the conversion of arachidonic acid to prostaglandins (PGE2). It comprises of two isoforms Cox-1 and Cox-2. Previous studies have implicated the potential role of Cox-2 in carcinogenesis and the expression of Cox-2 have been reported in colorectal, lung, breast, gastric and esophageal carcinomas. Cox-2 is also highly expressed in RCC and a potential biomarker in RCC. Based on emerging clinical evidences on the role of Cox-2 in several malignancies, we hypothesize that overexpression of Cox-2 in RCC promotes tumor growth and metastasis. Selective Cox-2 inhibitors act by inhibiting PGE2 synthesis and have been shown to retard tumor formation, metastasis, and angiogenesis. They induce apoptosis and inhibit the PGE2 induced immunosupression. Thus, a specific Cox-2 inhibitor like indomethacin or NS398 would be able to inhibit the tumorigenic properties of Cox-2, thereby attenuating tumor growth and dissemination to other organs. In this context, we injected the Cox-2 engineered Renca cell lines (COX2 -ve and COX2 +ve) in the subcapsular space of kidney of a Balbc/Cr mice. This resulted in the tumor growth, which was monitored by bioluminescence imaging (BLI) for a period of three weeks post-inoculation. Metastases were evident in distant organs such as lung, liver, spleen and lymph nodes. This was expressed as luciferase activity per milligram of protein of the particular tissue normalized with the background luciferase activity per milligram of the tissue from control (non-injected mice). Thus, the animal model was established and validated in our preliminary studies. To address the potential role of PGE2 in the tumor microenvironment, tumors were harvested and then processed for assessment by histology and immunohistochemistry. Initial characterization studies included immunohistochemical assessment of tumor vasculature as elucidated by staining with specific markers for lymphatic vessels (LYVE-1), blood endothelium (CD31) and tumor infiltrating macrophages (Cd11b). Macrophage recruitment close to the LYVE-1+ structures were also determined by the double positive events obtained by staining with both CD11b and LYVE-1. A large number of peripheral (18%±2.79), intratumoral (12%±3) and marginal (7%±2.01) LYVE-1+ve structures were found in PGE2 producing tumor than in the control tumor. Although, the frequency of blood vessels in both the tumor types were unaltered, however, an increased vascular area was obtained in the COX2 +ve tumor than in COX2 -ve tumor. There was a significant increase in the frequency of infiltrating macrophages in the peripheral (25%± 3.86), intratumoral (10%± 3.93) and tumor-kidney margin of COX-2 positive tumors (10%± 2.34) than in the COX-2 negative tumors (12% ± 2.36, 0% and 0%) respectively. Frequency of the blood vessels in both the tumors were unaltered, however, a significant increase in the mean fluorescent intensity (MFI) in the peripheral region (4.3±0.1) of COX2 +ve tumor was observed when compared to COX2 -ve tumors (2.3±0.05). These preliminary studies indicate the potential role of PGE2 in promoting tumor vasculature, increased macrophage recruitment within the tumors and tumor-kidney margins. In our initial studies, a significant enrichment of LYVE-1+ve macrophages were observed in the kidney-tumor sections of the COX2 +ve mice, which might indicate that, PGE2 may promote differentiation of the macrophages into a lymphatic phenotype. Thus, this animal model would further help in thorough characterization of other immune infiltrating cells like CD8+ T cells and NK cells and thereby lead us to identify the cause of immune dysregulation in renal cell carcinoma due to RCC derived soluble factors like PGE2 and TGF-beta. Furthermore, treatment with Cox-2 inhibitors like NS-398 should retard tumor growth, metastases, immune cell dysregulation, and tumor vasculature.