Phenotypic Characterization of Retinoblastoma for the Presence of Putative Cancer Stem-like Cell Markers by Flow Cytometry

Abstract

Retinoblastoma (Rb) is an intraocular tumor that grows rapidly and poses a threat to sight and life. Similar to other tumors, there is increasing speculation that the Rb tumor also contains cancer stem-like cells that could influence the prognosis and response to therapy. This study was undertaken in an attempt to identify putative stem-like cells by characterizing different subpopulations of cells in retinoblastoma. Freshly isolated tumor cells obtained from unfixed eye specimens (n=7) were analyzed for the presence of CD44, ABCG2, CXCR4, CD133, and CD90 using flow cytometry. RT-PCR was performed to analyze the expression of human Syntaxin1A, PROX1, CD133, and NSE in the sorted subpopulation of tumor cells. Two different subpopulations of cells were observed in seven samples. The small cells, assigned FSC(lo)/SSC(lo) (forward scatter low/side scatter low, ranging from 1.7% to 17.7%) were characterized as positive for CD44 and negative for CD133, CXCR4, and CD90. The large cells were designated as FSC(hi)/SSC(lo) (ranging from 2.7% to 35.1%) and characterized as positive for all markers. RT-PCR analysis revealed that sorted cells of FSC(lo)/SSC(lo) subpopulation expressed the retinal progenitor cell markers PROX1 and Syntaxin1A. Retinoblastoma, on flow cytometric analysis, revealed two distinct subpopulations with variable expression of stem cell and retinal progenitor markers. In these populations, the FSC(lo)/SSC(lo) subpopulation appeared to be more primitive, since they expressed stem cell (CD44) and retinal progenitor markers (PROX1 and Syntaxin 1A) combined with a relatively lower percentage of differentiated markers. Moreover, the FSC(hi)/SSC(lo) subpopulation showed a higher percentage of differentiated markers (CD90 and CD133).