Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Summary

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

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.

Introduction

The efficacy of primary cancer therapies has improved substantially over the last decade due to compelling advances in the genetic and molecular understanding of the disease and the advent of targeted drugs such as therapeutic antibodies and small molecule inhibitors. In contrast, metastatic and recurrent disease is still typically incurable, and morbidity and mortality remain high in these clinical settings. CSCs represent a distinct subpopulation within tumors and are endowed with canonical stem cell properties such as clonogenicity/tumorigenicity, multi-drug resistance and asymmetric cell division^1,2. Thus, CSCs not only drive metastatic progression and tumor heterogeneity, but also persist during treatment to predispose the patient to relapse. Therapeutic CSC elimination is therefore an important medical need to prevent disease recurrence and allow long-term cure of cancer³.

Identification of vulnerabilities and elucidation of strategies to eradicate CSCs heavily depends on methods that allow their purification from biological samples for subsequent expression profiling and/or functional investigation. In turn, such methods rely on surface, intracellular or functional markers that are specific for these cells. CSC-specific surface markers include, but are not limited to, CD44, CD133, CD24 and CD90, and have been used to identify CSC populations in a variety of tumor entities including breast cancer and colon cancer⁴. Another marker, aldehyde dehydrogenase (ALDH), shows intracellular localization and can be functionally detected providing a respective substrate whose enzymatic conversion produces light. Using this test, CSC populations have been identified in diverse tumor entities as well⁵. A complementary method, commonly referred to as SP analysis and portrayed here in methodological detail, harnesses active dye efflux by ABC drug transporters to identify small populations of fluorescence-dim stem-like cells^6,7,8. To achieve this, a given sample is incubated in the presence of a lipophilic DNA-binding fluorophore which enters all cells through passive diffusion and targets nuclear and mitochondrial DNA for binding. Non-CSCs devoid of ABC drug transporter expression accumulate the dye resulting in bright fluorescence, whereas CSCs actively extrude the dye which reduces fluorescence. Pharmacological inhibition of drug transporter activity abrogates and functionally confirms the SP phenotype and should be used for control purpose. CSC populations exhibiting SP characteristics have been disclosed, amongst others, in ovarian cancer^9,10, prostate cancer^11,12, breast cancer¹³, lung cancer¹⁴, endometrial cancer¹⁵, glioma^16,17 and bone sarcoma¹⁸. Importantly, the SP assay is compatible with both cancer cell lines and primary tumor tissue, even though primary material poses additional challenges such as the requirement for a specific tumor cell discrimination strategy (certain host cell populations can exhibit SP characteristics as well)^19,20.

The two most established SP-conferring drug transporters are ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338^8,9,21; however, other drug transporters can be a molecular determinant of the SP phenotype too (e.g., ABCB5)²². ABCB1 can be efficiently blocked with verapamil whereas the activity of ABCG2 can be specifically abrogated with fumitremorgin C (FTC)^6,19. A particular strength of SP analysis is that it can be combined with other stainings (e.g., surface markers and ALDH) and that it allows live cell recovery thus being compatible with downstream functional investigations. Moreover, SP detection is broadly applicable owing to the high conservation of ABC drug transporters among CSC populations^9,23.

Originally, SP detection has been performed using Hoechst 33342 as a triggering dye²⁴. This dye achieves excellent resolution but requires ultraviolet laser excitation; hence its applicability is naturally limited to high-end flow cytometric instruments. The advent of DyeCycle Violet (DCV)²⁵ has opened new avenues for SP analysis and extended the applicability of this method to standard flow cytometric instruments lacking an ultraviolet laser source (a violet laser source suffices to resolve DCV-SP cells). Importantly, Hoechst 33342 and DCV share common pump specificities, indicating that either dye should identify the same cell populations.

Here, we provide a detailed experimental protocol of DCV-based SP analysis for quick and easy reproduction in independent labs. We thus perceive our article as a resource for CSC researchers that should contribute to the optimization and standardization of this useful cell-biological method.

Protocol

This protocol is in full compliance with standard Institutional Ethical Review Board guidelines. If human or animal tissue is investigated using the protocol depicted here, researchers are obliged to have approval from the relevant review board of their institution or country.

Caution: Standard precautions for the safe handling of biological samples apply to this protocol. This includes wearing gloves and a lab coat, and performing the work under a biosafety cabinet whenever possible.

1. Preparation of Cells

1. Cancer cell lines
   1. Culture a respective human or murine cancer cell line at 37 °C in 10-30 mL of appropriate medium (e.g., RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin) and harvest the cells at sub-confluency (i.e. 70-90%) using digestion with 0.05% trypsin-EDTA or some other de-attachment procedure. Determine the cell count and check for viability using a counting chamber and trypan blue staining. The fraction of viable cells should exceed 85%.
      NOTE: Examples of human cancer cell lines harboring SP compartments include MCF7/HTB-22 and SKBR3/HTB-30 (breast cancer), A2780 and SKOV-3/HTB-77 (ovarian cancer), and A549 (lung cancer)²⁶.
2. Ex vivo tumor tissue
   1. Take a fresh surgical tumor specimen or alternatively, harvest tumor tissue from a transplantable or genetically-engineered mouse tumor model. Take 200-1,000 mg of tissue and generate a single cell suspension by mechanical disaggregation (e.g., using scissors or a scalpel) and enzymatic digestion (e.g., using a collagenase/dispase/DNase cocktail).
   2. Filter the sample through a 70 µm cut-off strainer. Determine the cell count and check for viability using a counting chamber and trypan blue staining.
      NOTE: Digestion cocktails frequently contain 200 µg/mL collagenase P, 0.8 U/mL dispase and 25 µg/mL DNase I, and incubation times typically range from 20 - 50 min. Ideally, tissue disaggregation is performed according to a protocol optimized for the tissue under investigation ^27,28,29.

2. Test Samples

1. Adjust cell concentration to 1 x 10⁶ cells/mL in fresh culture medium (a nutrient-containing vehicle is important as dye extrusion is an active energy-consuming process). Finally, transfer 1 mL of sample to an ordinary flow cytometry/FACS round-bottom tube (labelled 'test').
   NOTE: For optimal results, it can be necessary that the cell concentration is titrated (for instance 5 x 10⁵-1 x 10⁶-2.5 x 10⁶ cells/mL), but a lower cell concentration generally yields a higher resolution¹⁹. Only for sorting applications where a maximum number of SP cells shall be recovered, it is recommended to increase the cell concentration to up to 1 x 10⁷ cells/mL.
2. Add 10 µM of DCV to the sample and mix well, either by gentle vortexing or resuspending for 3-5x. Proceed to inhibition controls.
   NOTE: Optimal results might depend on titration of the triggering dye (for instance 2.5; 5; 10; 20 µM), but a higher dye concentration generally yields a higher resolution¹⁹.

3. Inhibition Controls

1. Transfer 250 µL of the dye-containing cell suspension to a new flow cytometry/FACS round-bottom tube (labelled 'control'). Add 50 µM of verapamil or 20 µM of FTC and mix well by pipetting.
   NOTE: Verapamil and FTC are the most well-established inhibitors for SP analysis but have different efflux pump specificity: Verapamil inhibits several ABC drug transporters including ABCB1 and ABCB5, whereas FTC is specific for ABCG2^6,19. If the SP status of an individual sample is unknown, it is thus recommended to use both verapamil and FTC in separate tubes.

4. Staining

1. Cap both the 'test' and 'control' tubes and incubate them at 37 °C in the dark for 90 min, with gentle agitation every 15 min. Most of the dye extrusion in SP cells will occur within the first 45 min, but dye accumulation in non-SP cells (contributing to resolution) is only maximal after 75-90 min¹⁹.
2. After staining is complete, wash the cells in 3-4 mL ice-cold PBS and resuspend the pellet in a volume of 100 µL (PBS as well). Keep the cells in the dark and chilled on ice, and proceed to the staining of additional markers. Alternatively, directly perform flow cytometric analysis.

5. Costaining for Other Markers

1. Add a desired panel of fluorochrome-conjugated antibodies to the DCV-stained cells, mix well and incubate the cells at 4 °C in the dark for 20-30 min. Wash the cells in 3 - 4 mL ice-cold PBS and proceed to dead cell discrimination.
   NOTE: Antibody formats that are easily compatible with DCV and virtually do not require compensation include PerCP or PerCP-Cy5.5, PE-Cy7, APC, Alexa Fluor 647, APC-Cy7, APC-H7, BV711, Alexa Fluor 750 and BV786. Formats that are compatible with DCV but may require compensation include FITC, Alexa Fluor 488, PE and BV650. Formats that are virtually not compatible with DCV include BV421, V450 and V500.
2. Add 7-AAD (or PI) to the DCV-stained cells at the concentration recommended by the particular manufacturer and incubate the samples for 5 - 10 min in the dark. Filter the samples through 70 µm cut-off strainers and proceed to flow cytometric analysis.

6. Flow Cytometric Analysis

NOTE: It is recommended to switch on the respective flow cytometric instrument during the staining process already so that everything is set as soon as the samples are ready. This includes also standard maintenance procedures such as system performance tracking and tank refilling.

1. Acquire the cells on the flow cytometer and gate them on a bivariate FSC/SSC dot plot (Figure 1B). Exclude doublets and aggregates by comparing the different signals of FSC (i.e. height, width, area) (Figure 1C).
   1. If a dead cell marker has been included, gate on the viable cell fraction (e.g., 7-AAD-negative) (Figure 1D). Visualize the viable singlet cells on a bivariate dot plot for 'blue' and 'red' DCV emission. To this end, display the 'blue' fluorescence channel on the y-axis (e.g., 450/50) and the 'red' fluorescence channel on the x-axis (e.g., 525/50).
   2. Switch both channels to linear mode and adjust the detector voltages correspondingly so that the SP locates to the side of the plot in the lower left quadrant. Conversely, the non-SP locates to the upper right quadrant of the plot reflecting the cell cycle distribution (G1 and G2). Gate the SP and stop the acquisition (Figure 1E).
      NOTE: DCV-defined SP cells can be detected using flow cytometric instruments equipped with a violet laser excitation source (i.e. a 405 nm laser line). It is one specific of SP analysis that the emitted fluorescence is measured in linear mode and in two separate channels displayed on a bivariate dot plot. The 'blue' emission of DCV is measured at approximately 450 nm (e.g., with a 450/40 standard filter) and the 'red' emission of DCV is measured at approximately 510 - 585 nm (e.g., with a 510/50, a 525/50 or a 585/15 filter)¹⁹. Due to the particular chemistry of the assay, the separation between SP and non-SP cells is generally higher in the 'red' fluorescence channel¹⁹.
2. Load the inhibition control(s) and acquire the data. The SP cells have now disappeared or are reduced substantially (Figure 1F). If necessary, refine the SP gate based on these data.
3. To investigate surface marker expression by SP cells, leave the 'red' fluorescence of DCV on the x-axis and display a respective fluorescence channel on the y-axis in logarithmic mode (e.g., APC). Adjust the detector voltages correspondingly and gate the fraction of SP cells staining positive for the investigated marker(s) (Figure 1G). Co-expression of a particular marker by SP cells yields a respective signal in the upper left quadrant of the plot³⁰.
4. Record the data and/or sort the SP cells.

Results

Provided is a representative SP analysis of A2780 cells, a human ovarian cancer cell line previously shown to harbor a SP accounting for less than 1% of cells⁹. Cells were cultured at 37 °C in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin and harvested at 85% confluency using treatment with 0.05% trypsin-EDTA. Cells were washed in PBS and filtered through 70 µm cut-off strainers. The cell count was determined us...

Discussion

Progress in the clinical management of cancer also depends on the development of treatment modalities that target CSCs. Methods allowing the reliable isolation of CSCs from biological samples will accelerate the identification of suitable targets and are therefore of critical importance in this endeavor. SP analysis is an established and valuable technique to identify CSC populations that express ABC drug transporters and the implementation of DCV has extended its applicability to standard flow cytometric instruments. Me...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vybrant DyeCycle Violet	Thermo Fisher Scientific	V35003	Ready to use
Verapamil hydrochloride	Sigma-Aldrich	V4629	Dissolve in ethanol
Fumitremorgin C	Sigma-Aldrich	F9054	Dissolve in DMSO
7-AAD (or propidium iodide - PI)	BioLegend	420403	Ready to use
Standard cell culture reagents (e.g., medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, etc.)	Different suppliers
Sample/cells of interest (e.g., human or murine cancer cell line, human or murine tumor tissue)	Different suppliers
Flow cytometric instrument (FACS; analyzer or cell sorter)	Different suppliers		Violet laser source required
FACS tubes (round-bottom)	Different suppliers		Tubes with cap recommended
70 µm cut-off strainers	Different suppliers		Optional but recommended
Digestion enzyme mix (e.g., collagenase/dispase/Dnase)	Different suppliers		Only relevant for tissue dissociation
Flurochrome-conjugated antibodies against surface markers of interest	Different suppliers		Optional
ALDEFLUOR Kit	STEMCELL Technologies	01700	Optional