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Gail M. Seigel, Meerim Choi, Linda Cassidy; Modulation of the Stem Cell Phenotype in ABCG2+ Human Retinoblastoma Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1289.
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Cancer progression and recurrence are major public health issues. Survival of a few chemo-resistant cancer stem-like cells (CSCs) can undermine even the most sophisticated treatment protocol, resulting in cancer recurrence, human suffering and ultimately death. Our group has identified ABCG2+ CSCs in retinoblastoma, a blinding ocular tumor of childhood. In this study, we tested the hypothesis that ABCG2+ retinoblastoma cells can preferentially and reversibly differentiate into mature retinal neurons.
Y79 and WERI-RB27 human retinoblastoma cells were magnetically separated into ABCG2+ and ABCG2- cells and plated as adherent cultures on poly D-lysine coated flasks. ABCG2+ and ABCG2- cells were treated with the reversible differentiation agent Succinylated Concanavalin A (SCA) for 0-96 hours, or treated with SCA for 72 hours and reversed with alpha methyl D-mannoside (AMM) for 24 hours. Appropriate controls included untreated cells, as well as cells treated with AMM alone. We used quantitative immunocytochemistry and qPCR of stem cell markers, retinal progenitor markers, as well as mature retinal markers to determine cell phenotype with and without SCA treatment.
ABCG2+ retinoblastoma cells, when compared with ABCG2- cells, consistently showed greater immunoreactivity to stem cell markers such as Nanog, Musashi-1 and Nestin, less immunoreactivity to retinal progenitor markers such as CHX10, PAX6 and CRX, and less immunoreactivity to mature neural/retinal markers such as Recoverin, PKCalpha, Tuj-1 and PGP9.5. Four day SCA treatment decreased immunoreactivity to Nestin, while increasing immunoreactivity to CRX and PAX6, as well as Recoverin. Reversal of 3-day SCA effects by AMM has been observed after 24 hours with Musashi-1, CRX, beta-tubulin, Rhodopsin and S-100. All of these findings were statistically significant.
Untreated ABCG2+ retinoblastoma cells preferentially express stem cell markers and exhibit lower levels of retinal progenitor markers and mature retinal markers as compared with ABCG2- cells. SCA treatment of ABCG2+ retinoblastoma cells leads to increased levels of retinal progenitor markers and mature retinal markers, with decreased immunoreactivity to stem cell markers. Reversal of immunoreactivity is seen with AMM treatment. This reversible differentiation model provides a unique opportunity to examine the behavior of CSCs, as well as important pathways involved in differentiation of CSCs. Results of these studies may lead to potential therapeutic and differentiation strategies that target stem-like cells not only in retinoblastoma, but in other human malignancies.
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