Side Population Is Enriched in Tumorigenic, Stem-Like Cancer Cells, whereas ABCG2+ and ABCG2− Cancer Cells Are Similarly Tumorigenic

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Abstract
Recently, several human cancers including leukemia and breast and brain tumors were found to contain stem-like cancer cells called cancer stem cells (CSC). Most of these CSCs were identified using markers that identify putative normal stem cells. In some cases, stem-like cancer cells were identified using the flow cytometry-based side population technique. In this study, we first show that ∼30% of cultured human cancer cells and xenograft tumors examined (∼30 in total) possess a detectable side population. Purified side population cells from two cell lines (U373 glioma and MCF7 breast cancer) and a xenograft prostate tumor (LAPC-9) are more tumorigenic than the corresponding non–side population cells. These side population cells also possess some intrinsic stem cell properties as they generate non–side population cells in vivo, can be further transplanted, and preferentially express some “stemness” genes, including Notch-1 and β-catenin. Because the side population phenotype is mainly mediated by ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, we subsequently studied ABCG2+ and ABCG2− cancer cells with respect to their tumorigenicity in vivo. Although side population cells show increased ABCG2 mRNA expression relative to the non–side population cells and all cancer cells and xenograft tumors examined express ABCG2 in a small fraction (0.5-3%) of the cells, highly purified ABCG2+ cancer cells, surprisingly, have very similar tumorigenicity to the ABCG2− cancer cells. Mechanistic studies indicate that ABCG2 expression is associated with proliferation and ABCG2+ cancer cells can generate ABCG2− cells. However, ABCG2− cancer cells can also generate ABCG2+ cells. Furthermore, the ABCG2− cancer cells form more and larger clones in the long-term clonal analyses and the ABCG2− population preferentially expresses several “stemness” genes. Taken together, our results suggest that (a) the side population is enriched with tumorigenic stem-like cancer cells, (b) ABCG2 expression identifies mainly fast-cycling tumor progenitors, and (c) the ABCG2− population contains primitive stem-like cancer cells.