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D. H. Hershberger, S. D. Narasipura, M. King, B. Ksander, P. E. Kolovou, C. Hackett, J. Young, S. Hayes, G. M. Siegel; Proteomic Characterization of ABCG2+ Tumor-Initiating Cells in Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3222.
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© ARVO (1962-2015); The Authors (2016-present)
The phenotypic and functional characteristics of cancer stem cells in retinoblastomas relate to the expression of the cell surface protein ABCG2 by this subpopulation of tumor cells. Using the WERI-RB27 and Rb143 retinoblastoma cell lines, we report on the novel proteomic properties that distinguish ABCG2 positive tumor-initiating cells from ABCG2 negative tumor cell populations.
Cell sorting: WERI-RB27 cells were labeled with mouse anti-ABCG2 antibody and secondary goat anti-mouse antibody conjugated with FITC. A BD-FACSvantage machine was used to sort the cells.Proteomics: Following cell sorting, ABCG2 positive and negative populations were incubated at equal numbers (1 million cells) for 3 days. Total protein was then extracted and equal amounts (10-15 ug) were run on a 10% SDS-PAGE gel. Bands of interest were subsequently cut out, destained and sent for Mass Spectometry analysis.Tumor-initiation and Immunocytochemistry: Rb143 retinoblastoma cells were stained with an anti-ABCG2 FITC antibody and separated by a cell sorter into ABCG2 positive and negative subpopulations that were injected into the subretinal space of immunodeficient NOD-scid IL2rg-/- mice (NSG mice) [15,000 cells injected in a total volume of 0.5 ul (15 million cells/ml)]. Eyes were enucleated, embedded and prepared as 4 micron sections at 2 and 8 weeks post injection.
1) Sorted RB cells were analyzed by western blot which showed six bands of interest. Two bands upregulated in ABCG2 immunonegative cells were identified by Mass Spectometry as HSP90-alpha and Beta-tubulin.2) SCID mouse eyes injected with ABCG2 immunopositive cells grew invasive tumors with one animal demonstrating metastatic growth of a similar tumor to the chest wall. Tumors showed immunoreactivity to HSP90 alpha/beta isoforms, as well as survivin, another anti-apoptotic marker that correlates with HSP90 expression in RB.
This study expands the current understanding of the ABCG2+ tumor initiating cells in retinoblastomas. The proteomics unique to ABCG2 positive and negative cell populations may be useful in guiding the development of new chemotherapeutic interventions in retinoblastoma.
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