Phenotypic characterization of retinoblastoma for the presence of putative cancer stem-like cell markers by flow cytometry

Abstract

Purpose: 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. Methods: 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. Results: Two different subpopulations of cells were observed in seven samples. The small cells, assigned FSClo/SSClo (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 FSChi/SSClo (ranging from 2.7% to 35.1%) and characterized as positive for all markers. RT-PCR analysis revealed that sorted cells of FSClo/SSClo subpopulation expressed the retinal progenitor cell markers PROX1 and Syntaxin1A. Conclusions: Retinoblastoma, on flow cytometric analysis, revealed two distinct subpopulations with variable expression of stem cell and retinal progenitor markers. In these populations, the FSClo/SSClo 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 FSChi/SSClo subpopulation showed a higher percentage of differentiated markers (CD90 and CD133). © Association for Research in Vision and Ophthalmology.