Measurement of Multiple Drug Resistance Transporter Activity in Putative Cancer Stem/Progenitor Cells
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Multiple drug resistance, mediated by the expression and activity of ABC-transporters, is a major obstacle to antineoplastic therapy. Normal tissue stem cells and their malignant counterparts share MDR transporter activity as a major mechanism of self-protection. Although MDR activity is upregulated in response to substrate chemotherapeutic agents, it is also constitutively expressed on both normal tissue stem cells and a subset of tumor cells prior to the initiation of therapy, representing a built-in obstacle to therapeutic ratio. Constitutive and induced MDR activity can be detected in cellular subsets of disaggregated tissues, using the fluorescent substrates Rhodamine 123 and Hoechst 33342 for ABCB1 (also known as P-gp and MDR1) and ABCG2 (BCRP1). In this chapter, we will describe the complete procedure for the detection of MDR activity, including: (1) Preparing single-cell suspensions from tumor and normal tissue specimens; (2) An efficient method to perform cell surface marker staining on large numbers of cells; (3) Flow cytometer setup and controls; (4) Simultaneous measurement of Hoechst 33342 and Rhodamine123 transport; and (5) Data acquisition and analysis.
Key wordsPre-existing multiple drug resistance ABCB1 activity ABCG2 activity Hoechst 33342 Rhodamine 123 Epithelial tumors Cancer stem cells Flow cytometry
The authors would like to thank Melanie Pfeifer and Amber McCauslin for technical assistance. This work was supported by grants BC032981 and BC044784 from the Department of Defense, the Hillman Foundation, and the Glimmer of Hope Foundation. Vera Donnenberg is a CDMRP Era of Hope Scholar.
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