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G. M. Seigel, P. E. Kolovou, C. Hackett, S. Narasipura, M. R. King, J. E. Young, S. H. Hayes, B. Ksander; A Tumor-Initiating Subpopulation of Human Retinoblastoma Cells With Stem Cell Properties. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2040.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma is the most common primary eye cancer in children and an important clinical problem facing ocular oncologists. We have previously shown that retinoblastoma is comprised of subpopulation(s) of cells with stem cell-like properties. The cancer stem cell theory states that only a small percentage of these cells within a tumor are able to promote tumor growth and metastasis, whereas the majority of the tumor is made of non-tumorigenic cells. The following study was performed to determine whether ABCG2+ Rb cells exhibit increased tumorigenicity.
Rb143 retinoblastoma cells were stained with 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]. Eyes were enucleated and prepared as 4 micron sections at two and 8 weeks post injection. Sections were stained with H&E or specific antibodies for: HuNu (human nucleus-specific marker), stem cell-associated markers (ABCG2, Musashi-1, P63, and MCM2), and the vascular marker VEGF.
Rb143 cells contained 4% ABCG2+ cells, 96% ABCG2- cells. Cell sorting enriched ABCG2+ cells to >90% purity and ABCG2- cells to >99% purity (adherence of Rb143 cells prevented higher purity). Enriched subpopulations were either cultured in vitro, or injected into the subretinal space of NSG mice. Two weeks after injection, ABCG2 positive Rb cells developed large retinal tumors that grew progressively from the site of injection through the layers of the retina and into the vitreous cavity. By contrast, mice that received ABCG2 negative Rb cells formed small areas of non-invasive Rb cell engraftment that did not show evidence of progressive growth. Eight weeks post-injection, both groups of mice displayed progressively growing tumors, but metastatic tumors were only present in mice that received ABCG2+ Rb cells. All tumors displayed the human nucleus-specific marker HuNu as an indication of human origin, while some cells expressed stem cell markers. In vitro cultures revealed that by eight weeks the cultures enriched for ABCG2- cells contained 4% ABCG2+ cells (likely due to contaminating cells), while cultures enriched for ABCG2+ cells now contained 80% ABCG2- cells (likely due to reversion of ABCG2 positive to negative cells).
These results support our hypothesis that ABCG2+ Rb cells possess increased tumorigenicity and metastatic potential, as compared with ABCG2- Rb cells.
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