Proteomic Characterization of ABCG2+ Tumor-Initiating Cells in Retinoblastoma

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinoblastoma • proteomics • flow cytometry