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P. E. Kolovou, N. Y. Frank, A. Giani, T. G. Murray, M. H. Frank, B. R. Ksander; ABCB5: A Novel Cancer Stem Cell Marker in Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4764.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma (Rb) is the most common intraocular tumor in children. While chemotherapy has improved the outcome for some patients, treating late stage bilateral Rb remains a challenge. The cancer stem cell theory states that some tumors contain a subset of self-renewing tumor cells with enhanced tumorigenicity and stem cell like properties. A problem in testing this hypothesis is the difficulty in finding markers that allow isolation of these cells. ATP-binding cassette (ABC) transporters act as efflux pumps, and ABCB5, a member of the ABC-B subfamily, identifies cancer stem cells in skin melanoma. Unexpectedly, the normal retina displays a high level of ABCB5. Here we identify for the first time, an ABCB5+ subpopulation of Rb cells with potential drug resistance and/or enhanced tumorigenicity.
Rb143, 116, 125 and 107 cell lines were developed in our laboratory and were validated by Rb gene sequencing. Flow cytometry was used to identify ABCB5+ cells using a monoclonal anti-ABCB5 antibody and isotype control. ABCB5 pump function was measured by flow cytometry using Calcein AM, a pump substrate that becomes fluorescent within viable cells. OCT was used to quantitate intraocular tumors, following orthotopic injection into the sub-retinal space of immunodeficient NOD-scid IL2rg -/- mice.
All Rb cell lines possessed a subpopulation of ABCB5+ cells with a frequency ranging from 3-12%. Moreover the ABCB5 pump was functional. ABCB5+ Rb cells excluded calcein (pump substrate), while ABCB5- Rb cells incorporated calcein. Adding a specific ABCB5 blocking antibody abolished ABCB5+ calcein exclusion. Rb143 cells (originally 3% ABCB5+) were separated by cell sorting into (i) 90% ABCB5+ cells, and (ii) 100% ABCB5- cells. To test the growth potential and drug resistance of these two subpopulations, cells were orthotopically injected in the sub-retinal space of NOD-scid IL2rg -/- mice and tumor growth was measured by OCT.
Retinoblastoma possesses a sub-population of ABCB5+ tumor cells that displays an active pump with the potential of drug resistance and enhanced tumor growth.
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