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Wesley S. Bond, Lalita Wadhwa, Rebecca L. Penland, Mary Y. Hurwitz, Patricia Chévez-Barrios, Richard L. Hurwitz; Retinoblastoma Tumorsphere Cultures Are Derived from the Malignant Clone and Recapitulate the Original Tumor Genotype and Phenotype In Vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1290.
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This study aims to determine if primary cultures derived from retinoblastoma (Rb) tumors originate from the malignant clone and if secondary tumors formed in mice injected with these cells have a phenotype similar to the original tumor. The etiology of Rb involves the loss of functional retinoblastoma protein (pRb). pRb is encoded by the gene RB1, and mutations in RB1 leading to Rb may be inherited or occur de novo. The genotypes and phenotypes of tumor-derived cultures and xenografts were compared to their original tumors by DNA sequencing and immunostaining for pRb and markers of stem cells and differentiated retinal cell types.
Tumor cells from two Rb patients whose tumors contained de novo mutations in RB1 not present in the germline were cultured in serum-free defined media or media containing serum, giving rise to tumorspheres and attached cells, respectively. Cultured tumorspheres and attached cells were immunostained for pRb, Sox2, Nestin, and CD133. Genomic DNA was isolated from cultured tumorspheres and attached cells and sequenced for mutations in RB1. Tumorspheres were injected into the vitreous of Rag2 knockout mice to form xenograft tumors. Tumors were harvested and immunostained for expression of pRb, Sox2, Nestin, CD133 and markers of epithelial, endothelial, and glial cells.
Tumorspheres lacked expression of pRb, while the attached population demonstrated pRb expression. Sequencing of genomic DNA isolated from the cultures revealed that tumorspheres contained the RB1 mutations found in the original tumor. Tumorspheres and the primary human tumor and secondary murine xenograft tumors exhibited heterogeneous expression of Sox2, Nestin and CD133 and were pRb-negative. The attached cells showed similar expression of Sox2, Nestin and CD133, and most co-express pRb as well as epithelial, endothelial and glial markers.
Tumor cells cultured in serum-free defined media formed tumorspheres that are derived from the malignant clone, while cells cultured in serum-containing media formed an attached population not derived from the apparent cancer cells. Cultured tumorspheres, unlike the attached cell populations, provide an in vitro tool for further investigation of the genomic and phenotypic characteristics of retinoblastoma.
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