Retinoblastoma Tumors Contain Apparent Cancer Cells Not Derived From the Malignant Clone

Abstract

Purpose: : This study was undertaken to test the hypothesis that cells in a solid tumor are solely derived from a malignant clone.

Methods: : Retinoblastoma is an intraocular malignancy unique to human children that decreases in incidence with increasing age and results from mutations in the Rb1 gene. Although some tumors express dysfunctional Rb protein, most tumors are the result of nonsense Rb1 mutations and do not express retinoblastoma protein (Rb). Therefore Rb expression in cells within these tumors indicates a cell that was not derived from the malignant clone. Expression of Rb and other proteins was determined using immunocytochemistry in paraffin embedded tissue blocks from tumors obtained from enucleated eyes of children with retinoblastoma or of cell cultures derived from these tumors. Cultures of tumor cells were grown either in defined serum-free or serum-supplemented media.

Results: : Retinoblastoma tumors contain a small percentage of cells that are Rb positive but are morphologically indistinguishable from the Rb negative cells within the tumor. The Rb positive cells are seen in greater number adjacent to the retina. Similar to the Rb negative cells, some of the Rb positive cells express proteins associated with neural stem cells (CD133, nestin, sox2) while other Rb positive cells express proteins associated with differentiated neural cells (GFAP, NSE, synaptophysin). Tumors cells grown in defined medium form neurospheres that do not express Rb but do express stem cell and differentiation associated proteins in a manner similar to the primary tumor. Tumor cells grown in serum-containing medium are heterogeneous for the expression of Rb.

Conclusions: : Both Rb expressing and non-expressing cells can grow in tissue culture but only Rb negative cells form neurospheres. Retinoblastoma tumors appear to contain both cells of malignant and non-malignant origin that only can be distinguished by their expression of Rb.

Keywords: retinoblastoma • oncology • proliferation