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Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples
Published on: May 10, 2017
Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological
Maximilian Boesch1, Elisabeth Hoflehner2, Dominik Wolf3
1Institute of Immunobiology, Kantonsspital St. Gallen; Internal Medicine V, Medical University of Innsbruck; Tyrolean Cancer Research Institute (TKFI); Maximilian.Boesch@kssg.ch.
Abstract:
Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.
Insights
This study introduces a novel flow cytometry method to identify and isolate cancer stem cells (CSCs) by detecting their unique dye extrusion properties. This functional assay complements traditional methods for targeting CSCs in cancer research.
Area of Science:
- Cancer Biology
- Stem Cell Research
- Biotechnology
Background:
- Cancer is driven by cancer stem cells (CSCs), making their eradication a key therapeutic objective.
- Identifying and isolating CSCs from heterogeneous tumor samples is crucial for understanding their vulnerabilities and developing targeted therapies.
- Current methods for CSC isolation often rely on phenotypic markers or enzymatic activity, with flow cytometry/FACS being a primary technique.
Purpose of the Study:
- To describe a detailed methodology for identifying and isolating cancer stem cells (CSCs) using a functional assay based on dye extrusion by ATP-binding cassette (ABC) drug transporters.
- To present the side population (SP) assay as a valuable and broadly applicable method for CSC characterization.
- To highlight the compatibility of the SP assay with other flow cytometry techniques for comprehensive CSC sub-characterization.
Main Methods:
- Utilizing flow cytometry with a violet laser to detect the side population (SP) phenotype, characterized by reduced fluorescence due to dye extrusion by ABC drug transporters.
- Employing functional assays with specific inhibitors of ABC drug transporters (e.g., ABCB1, ABCG2) to confirm the CSC nature of the SP population.
- Integrating the SP assay with established flow cytometry evaluations, including surface marker staining, aldehyde dehydrogenase activity detection, and dead cell discrimination (e.g., 7-AAD, propidium iodide).
Main Results:
- The side population (SP) assay effectively identifies a distinct population of cancer cells exhibiting stem cell characteristics.
- SP cancer cells demonstrate functional dye extrusion mediated by ABC drug transporters, which can be inhibited to abrogate the SP phenotype.
- The SP assay is compatible with multi-parameter flow cytometry, enabling simultaneous analysis of surface markers, enzymatic activity, and cell viability.
Conclusions:
- The SP assay provides a robust, functional method for CSC identification and isolation, complementing traditional phenotypic approaches.
- This method offers a valuable tool for cancer research, facilitating the study of CSC biology and the development of novel therapeutic strategies.
- The violet dye-based SP phenotype is readily resolvable on standard flow cytometers, increasing the accessibility and applicability of this technique.
