Cancer stem cell characteristics in retinoblastoma.
OBJECTIVE
There is increasing evidence that cancer stem cells contribute to tumor progression and chemoresistance in a variety of malignancies, including brain tumors, leukemias, and breast carcinomas. In this study, we tested the hypothesis that retinoblastomas contain subpopulations of cells that exhibit cancer stem cell properties.


METHODS
The following sources of retinoblastoma cells and tissues were examined for the presence of stem cell markers by immunocytochemistry: retinoblastoma tumors from mice transgenic for the SV40 T-antigen (driven by the beta-luteinizing hormone promoter), cell pellets of human Y79 and WERI-Rb27 retinoblastoma cell lines, and archival human retinoblastoma pathological specimens. Hoechst dye exclusion, mediated by the stem cell surface marker ABCG2 (ATP-binding cassette transporter, G2 subfamily), was assessed by flow cytometry in mouse tumors and WERI-Rb27 cells.


RESULTS
Small numbers of retinoblastoma cells (less than 1%) exhibited immunoreactivity to stem cell markers ABCG2, aldehyde dehydrogenase 1 (ALDH1), MCM2 (minichromosome maintenance marker 2), SCA-1 (Stem cell antigen-1), and p63. Hoechst dye uptake in mouse tumors and WERI-Rb27 cells was enhanced by addition of 50 microM Verapamil, consistent with activity of the calcium-sensitive ABCG2 protein. Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in mouse retinoblastoma tumors (0.3%) and WERI-Rb27 cells (0.1%) in a verapamil-sensitive manner. ABCG2 and ALDH1 positive cells were Hoechst-dim, as seen by dual labeling in vitro.


CONCLUSIONS
Mouse and human retinoblastoma tumor cells contain a small subpopulation of cells that exhibit a cancer stem cell-like phenotype. Especially significant is the expression of ABCG2 in mouse and human tumor cells, a calcium-sensitive cell surface protein that not only acts to exclude Hoechst dye, but also confers resistance to over 20 different chemotherapeutic agents. These findings point to a heterogeneity in retinoblastoma tumors that may have significant impact on future treatment strategies.
Seigel
Gail M
GM
Ross Eye Institute, Department of Ophthalmology, University of Buffalo, Buffalo, NY 14214, USA. gseigel@frontiernet.net
Campbell
Lorrie M
LM
Narayan
Malathi
M
Gonzalez-Fernandez
Federico
F
D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D016147:Genes, Tumor Suppressor; D006801:Humans; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D064111:Minichromosome Maintenance Complex Component 2; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D050697:Retinal Dehydrogenase; D019572:Retinal Neoplasms / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000378:metabolism / Q000473:pathology*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D000950:Antigens, Ly; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D007527:Isoenzymes; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2