Measurement of multiple drug resistance transporter activity in putative cancer stem/progenitor cells.
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.
Donnenberg
Vera S
VS
Hillman Cancer Center, Pittsburgh, PA, USA.
Meyer
E Michael
EM
Donnenberg
Albert D
AD
D001562:Benzimidazoles / Q000378:metabolism; D001692:Biological Transport; D001940:Breast / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D018432:Drug Resistance, Multiple; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005374:Filtration; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007834:Lasers; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010641:Phenotype; D020112:Rhodamine 123 / Q000378:metabolism; D013194:Staining and Labeling
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020112:Rhodamine 123; C017807:bisbenzimide ethoxide trihydrochloride