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Published on: May 10, 2017
Measurement of multiple drug resistance transporter activity in putative cancer stem/progenitor cells
Vera S Donnenberg1, E Michael Meyer, Albert D Donnenberg
1Hillman Cancer Center, Pittsburgh, PA, USA.
Abstract:
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.
Insights
Multiple drug resistance (MDR) in cancer, driven by ABC-transporter activity, hinders chemotherapy. This study details methods to detect MDR activity in normal and tumor cells using fluorescent substrates for better therapeutic strategies.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Multiple drug resistance (MDR), mediated by ATP-binding cassette (ABC) transporters, significantly impedes effective antineoplastic therapy.
- Both normal tissue stem cells and cancer cells utilize MDR transporter activity for self-protection, presenting a challenge to achieving a favorable therapeutic ratio.
- MDR transporter activity can be constitutively present or induced by chemotherapeutic agents.
Purpose of the Study:
- To describe a comprehensive protocol for detecting MDR activity in normal and tumor cells.
- To enable the identification of cellular subsets with MDR activity prior to or during therapy.
- To facilitate the optimization of cancer treatment strategies by understanding drug resistance mechanisms.
Main Methods:
- Preparation of single-cell suspensions from tumor and normal tissue specimens.
- Efficient cell surface marker staining for high-throughput analysis.
- Simultaneous flow cytometric measurement of fluorescent substrates (Rhodamine 123 and Hoechst 33342) to assess ABCB1 and ABCG2 transporter activity.
Main Results:
- The described methods allow for the detection of MDR activity in specific cell populations within disaggregated tissues.
- Simultaneous measurement of two distinct fluorescent substrates enables the characterization of multiple ABC transporter functions.
- Provides a foundation for distinguishing between drug-resistant and sensitive cell populations.
Conclusions:
- The presented protocol offers a robust approach for quantifying MDR transporter activity in clinical samples.
- Accurate detection of MDR activity can inform personalized treatment decisions in oncology.
- Understanding and targeting MDR mechanisms are crucial for improving cancer patient outcomes.
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