Abstract B126: Optimization of culture conditions for the establishment of de novo renal cell carcinoma cell lines.
Authors: Lobo Nazleen C. , Gedye Craig , Ailles Laurie E. 
Published: June 2012

Abstract
Renal cell carcinoma, a common malignancy of the genitourinary tract, accounts for approximately 85% of all renal malignancies. Clear cell RCC (ccRCC) is the most common subtype and has a high level of cellular heterogeneity, with a characteristic mutation in the von Hippel-Lindau (Vhl) tumor suppressor gene in a majority of ccRCC cases. While extirpative surgery is most often curative in tumors restricted to the kidney parenchyma, locally advanced and metastatic ccRCC is resistant to conventional chemotherapeutics and radiation therapy.
Several aspects of ccRCC biology, including mechanisms of progression, tumor heterogeneity, and identification of biomarkers and candidate therapeutic targets are being examined. However, these studies typically use cell lines cultured in serum-containing media and passaged for extended periods of time. These lines acquire mutations in vitro, irreversibly corrupting their genotype and phenotype compared to the corresponding patient's tumor. Recent studies in the glioma field have shown that cell lines established in serum-free media maintained their ex vivo phenotype and genotype, and enabled more relevant functional screens like RNA knockdown and chemical cytotoxicity screens. More recently, similar approaches have been used to derive more biologically relevant serum-free cancer cell lines in other cancers, but not in ccRCC. We established a number of lines directly from patient tumors using traditional fetal bovine serum containing culture conditions in ambient (20%) oxygen versus serum-free media containing defined growth factor under 2% oxygen to minimize reactive-oxygen-species-induced mutations. In order to validate both their origin as cancer cells, and maintenance of their genotype and phenotype during cell culture, we used a panel of ten matched cell line pairs (serum and serum-free) and assessed a) cell line and tumor fidelity by STR profiling, b) genomic conservation by copy number variation, and c) Vhl mutation status.
All of the cell lines were found to match the primary patient tissue by STR profiling. Two of the ten lines grown in serum recapitulated the patient tumor Vhl mutation, but none of the lines grown in serum-free media had any mutations characteristic of the tumour, and were thus derived from contaminating normal kidney epithelial cells. These findings suggest that normal cells out-grow ccRCC cells under both culture conditions, but serum-containing media may be more permissive for ccRCC growth. We did not observe any accumulation of additional genetic changes in either culture condition. Our Vhl-mutant cell line fails to survive in the serum-free culture conditions used here, thus providing us with a tool for the optimization of serum-free growth conditions and identification of factors necessary for in vitro growth and survival of ccRCC cells. These conditions will then be used to derive additional lines that may represent more accurate in vitro models of ccRCC. Furthermore, molecular characterization of the matched wild type and mutant lines will allow us to identify markers to more easily distinguish normal cells from cancer cells in vitro. We believe it is important to identify a defined serum-free culture condition that supports ccRCC cell growth, as this will lead to identification of molecules that are essential for ccRCC cell growth and survival, and thus act as potential therapeutic targets.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B126.