Identification of a cancer stem cell enriched side population using Hoechst 33342 based isolation

The surge in interest in identifying and characterizing the tumor initiating cell or cancer stem cell (CSC) has a defining ethos which includes the discovery of novel diagnostic and prognostic markers and facilitation of the understanding of basic biology and cellular signaling pathways. The ultimate aim of this is to generate new therapeutic approaches to the treatment of cancer. However, a major hurdle to this process has been the lack of a truly specific cancer stem cell biomarker. Attention has therefore focused on the identification of CSCs using functional assays based on fundamental stem cell characteristics. One such property is the ability of the stem cells to efflux DNA based dyes such as Hoechst 33342 from the cell via the ABC transporter mechanisms,ABCG1 (MDRP) and ABCG2 (BCRP). This property gives rise to a characteristic side population (SP) which is dentifiable from the cellular milieu using flow cytometry, enabling separation and enrichment of this ub-population of cells from the other terminally differentiated cells. Here we review the development of theHoechst 33342 based method for isolation and characterization of the SP from tumor and non-tumor areas derived from solid tissues, showing that the cells within the SP share many of the characteristics of tumor initiating cells and that this SP characteristic / mechanism is conserved in different tumor tissue. In theabsence of other definitive biomarkers for cancer initiating or “stem” cells the Hoechst 33342 dye efflux assay is a powerful technique for their isolation and characterization