Abstract 4363: Identification of ABCB5 multidrug transporter in retinoblastoma
Authors: Kolovou Paraskevi E. , Trichonas George , Frank Natasha Y. , Murray Timothy G. , O'Brien Joan M. , Ostrand-Rosenberg Suzanne , Vavvas Demetrios , Frank Markus H. , Ksander Bruce R. 
Published: February 2011

Abstract
Purpose: Retinoblastoma (Rb) is the most common intraocular tumor in children. Chemotherapy has improved the outcome for both unilateral and bilateral early stage disease, yet late stage bilateral Rb remains difficult to treat, and metastasis are often fatal. Treatment with carboplatin, vincristine and etoposide is only effective in 40% of bilateral Rb patients and results in a high incidence of secondary malignancies. ATP-binding cassette (ABC) transporters act as efflux pumps of different substrates, including drugs. ABCB5, a member of the ABC-B subfamily, is expressed on both normal tissues (CNS, mammary gland, testis, retina) and in skin melanoma, where it identifies a subpopulation of cancer stem cells with enhanced tumorigenicity. Unexpectedly, the retina displays the highest level of ABCG5 in normal tissues, suggesting it might be expressed in Rb tumors. Here we identify for the first time in Rb, an ABCB5+ subpopulation of cells that we predict will possess drug resistance and/or enhanced tumorigenicity.
Methods: Rb143, Rb116, Rb125 and Rb107 cell lines were developed in our laboratory from primary explants recovered from patients with large tumors. Sequencing of the Rb gene (27 exons) was used to validate all Rb cell lines. Flow cytometry was used to identify ABCB5+ cells using a monoclonal anti-ABCB5 antibody or isotype control. Calcein AM and/or Aqua fluorescent dye was used to determine viability. ABCB5 pump function was determined by using Calcein AM, a pump substrate that becomes fluorescent within viable cells. Optical coherence tomography (OCT) was used to quantitate the size of intraocular tumors, following orthotopic injection into the sub-retinal space of NOD-scid IL2rg−/− mice.
Results: All Rb cell lines possessed a subpopulation of ABCB5+ cells with a frequency ranging from 3-12%. To determine if the ABCB5 pump was functional, Rb cells were cultured for 30 mins with Calcein AM (ABCB5 pump substrate). ABCB5 positive, but not negative, Rb cells excluded the Calcein AM. The ABCB5 pump mediated Calcein exclusion, which was terminated by the addition of an ABCB5 specific blocking antibody. Rb143 cells (originally 3% ABCB5+ cells) were separated by cell sorting into (i) ABCB5+ cells (90% pure), and (ii) ABCB5- cells (100% pure). To test the growth potential and drug resistance of these two subpopulations in vivo, we developed a murine transient retinal detachment model. Injection of 50μl of PBS into the sub-retinal space induces a retinal detachment, but as the PBS is subsequently absorbed over the next 24 hrs the retina reattaches. Up to 1×106 cells were orthotopically injected beneath the detached retina, resulting in tumor growth in &lt; 1 wk. This model will allow us to determine the drug resistance and growth potential of ABCB5+ and ABCB5- Rb cells.
Conclusion: Retinoblastoma possesses a small sub-population of ABCB5+ tumor cells, which displays an active pump with the potential of drug resistance and preferential tumor growth.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4363.