Method Article

Analysis of Side Population in Solid Tumor Cell Lines

DOI:

10.3791/60658

⸱

February 23rd, 2021

 ,  ,  ,  , 
1School of Medicine, Nankai University, 2Tianjin Key Laboratory of Tumour Microenvironment and Neurovascular Regulation, 3Collaborative Innovation Center for Biotherapy, Nankai University

In This Article

Summary

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A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Abstract

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Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

Introduction

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Cancer stem cells (CSCs) are subsets of cells with self-renewal ability and multiple differentiation potential, which play a vital role in tumor growth, metastasis, and recurrence1,2. Currently, CSCs have been identified to exist in a variety of malignant tumors, including lung, brain, pancreas, prostate, breast, and liver cancers3,4,5,6,7,8,9. Identification of CSCs in these tumors is mainly based on the presence of surface marker proteins, such as high and/or low expression of CD44, CD24, CD133, and Sca-19,10, but a unique marker that can distinguish CSCs from non-CSCs has not been reported so far. Currently, several techniques are used to identify and purify CSCs in tumor tissue or tumor cell lines. These techniques are designed based on the specific properties of CSCs. Among them, assays and sorting of side population (SP) cells are two of the commonly used methods.

SP cells were originally discovered by Goodell et al.11, when they characterized hematopoietic stem cells in mouse bone marrow cells. When the mouse bone marrow cells were labeled with the fluorescent dye Hoechst 33342, a small group of Hoechst 33342 dimly-stained cells appeared in the two-dimensional dot plot of a flow cytometry assay. Hoechst 33342 is a DNA-binding dye and has at least two binding modes that lead to different spectral characteristics. When viewing fluorescence emission at two wavelengths at the same time, multiple populations can be revealed12. In their assay, the Hoechst 33342 was excited at 350 nm and the fluorescence was measured by using the 450/20 nm band-pass (BP) filter and 675 nm edge filter long-pass (EFLP)11. Compared with whole population of bone marrow cells, this group of cells was enriched with hematopoietic stem cells called SP cells11. SP cells are capable of rapidly expelling Hoechst 33342. The efflux of this dye is related to ATP-binding cassette (ABC) transporters13, which can be inhibited by some agents such as Fumitremorgin C14, Verapamil and Reserpine15,16. After that, different proportions of SP cells were detected in a variety of tissues, organs, tumor tissues, and tumor cell lines17,18,19. These SP cells have many characteristics of stem cells17,19.

This manuscript describes Hoechst 33342 labeling and staining of cultured tumor cells and the analysis of SP cells by flow cytometry. Moreover, optimization of the Hoechst 33342 concentration and the proper blocker selection for a specific tumor cell line using this approach are shown. Finally, the effects of stemness promotion or inhibition signals on the proportion of SP in tumor cells are demonstrated. The experimental examples demonstrate that analysis of SP can be used to explore the effects of various signals, such as gene expression, small inhibitors, activators, cytokines, and chemokines, on tumor stemness. Compared to other methods for isolation and purification of CSCs, such as sorting of CD44+/CD24– population, aldehyde dehydrogenase (ALDH) analysis, and tumor sphere formation assays, this method is easier for manipulation and is cost-effective.

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Protocol

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1. Cell preparation

  1. Cell digestion and neutralization
    1. Seed tumor cells (such as MDA-MB-231 cells) in a 6 well plate, and culture them in a 37 °C incubator supplied with 5% CO2.
    2. Harvest cells when their density reaches about 90%. Aspirate the culture medium thoroughly and wash the cells 2x with 3 mL of phosphate buffered saline (PBS).
      NOTE: To examine the effects of signaling pathway inhibitors (e.g., FRA1 inhibitor), or activators (e.g., STAT3 activator) on stemness features of tumor cells, tumor cells are seeded in a 6 well plate and pretreated with inhibitors or activators for a certain amount of time before harvest.
    3. Add 500 µL of 0.25% trypsin-EDTA to each well of the 6 well plate, place the plate in an incubator at 37 °C for 1–3 minutes (min), and gently tap the plate to detach the cells.
      NOTE: Prolonged digestion will affect the SP profile due to changes in cell viability.
    4. Add 3 mL of PBS supplemented with 2% fetal bovine serum (FBS) to each well of the 6 well plate to terminate the digestion and gently pipette the cells up and down 3–5x to disperse the cell clumps.
    5. Add 0.5 mL of cell suspension to one well of a new 6 well plate and examine it under a microscope. If cell clumps are observed, pass the cell suspension through a 70 µM cell strainer.
      NOTE: This is an optional step.
    6. Transfer the cells to a 15 mL centrifuge tube. Centrifuge cells at 200 x g for 5 min to pellet cells. Remove the supernatant and resuspend them in 3 mL of PBS supplemented with 2% FBS. Pipette the cells up and down 3–5x to mix thoroughly.
  2. Cell counts
    1. Add 50 μL of the cell suspension into a 1.5 mL microcentrifuge tube and mix it with 50 μL trypan blue solution. Pipette 10 μL of the mixture to count the number of living cells using a standard method, such as a hemocytometer.
    2. Dilute the cells in PBS supplemented with 2% FBS to a final concentration of 1 x 106 cells/mL. Add 1 mL of the cell suspension to a 5 mL polystyrene round bottom test tube and prepare two sample tubes.

2. Cell staining with Hoechst 33342

  1. Add Hoechst 33342 to one tube to reach an appropriate final concentration.
    NOTE: For example, the appropriate concentration of Hoechst 33342 is 3 µg/mL for MDA-MB-231 cells.
  2. Add a blocker (e.g., Fumitremorgin C, Verapamil, or Reserpine) to another tube to an appropriate final concentration and incubate the tube at 37 °C for 30 min before adding the same concentration of Hoechst 33342 as described in step 2.1.
    NOTE: For MDA-MB-231 cells, the appropriate blocker is Reserpine (40 µM). Reserpine is used as a blocking control to verify the absence of cells in the gated SP area.
  3. Prepare several tubes containing cells and add Hoechst 33342 to different concentration gradients. After a flow cytometry assay, choose the proper concentration according to the profile and proportion of SP.
    NOTE: This is an optional step used to define the proper concentration of Hoechst 33342.
  4. Prepare several tubes containing cells, add the blocker to different concentration gradients, incubate the tubes at 37 °C for 30 min, then add Hoechst 33342 to an appropriate concentration. After the flow cytometry assay, choose the proper concentration according to the absence of cells in the gated SP area.
    NOTE: This is an optional step used to define the proper concentration of blocker.
  5. Place the tubes in a 37 °C incubator and incubate them for 60 min, shaking the tubes every 10 min.
    NOTE: Shaking tubes thoroughly is important for staining, because it ensures complete contact between the cells and the dye for better staining results.
  6. After the incubation, centrifuge the cells at 200 x g, 4 °C for 5 min, and aspirate the supernatant carefully, because the cells form very loose and unstable pellets.
  7. Resuspend cells of each tube in 1 mL of ice-cold PBS supplemented with 2% FBS and pipette the cells up and down 3–5x to mix thoroughly. Add 1 μL of 1 mg/mL propidium iodide (PI) to the suspension of each tube to identify dead cells.
    NOTE: All procedures in this step should be performed at 4 °C to inhibit the efflux of Hoechst 33342 from the tumor cells. The tubes should be protected from direct exposure to light.

3. Analysis by flow cytometry

NOTE: Instructions for use of the flow cytometer software (see Table of Materials) are described in this section and Supplementary Figures 1–10.

  1. In the flow cytometer software, click the FOLDER button. Click the Experiment button, then click New Experiment (Supplementary Figure 1A,B).
  2. Click the OK button. “Experiment_001” will show up under the FOLDER (Supplementary Figure 2A,B).
  3. Click the Experiment_001 button to change the folder name to a specific name (e.g., "20191118-SP"). Click Enter. The new name ("20191118-SP") will show up under FOLDER (Supplementary Figure 3A,B).
  4. Click the New Specimen button to add a specimen to the new experiment folder ("20191118-SP"). Click the New Tube button to add a tube to the specimen. Click the Arrowhead button (Supplementary Figure 4A-C).
  5. Click the Parameters button and set up the parameters of the flow cytometer (Supplementary Figure 5).
    1. Use a 610 nm dichroic mirror short-pass (DMSP) to separate the emission wavelengths. Use a 450/20 nm BP filter to collect the blue fluorescence and a 675 nm EFLP to collect the red fluorescence.
      NOTE: Hoechst 33342 is excited with a UV laser at 355 nm and PI is excited at 488 nm.
  6. Run the cell samples on the flow cytometer.
    NOTE: SP cells can be sorted by fluorescence-activated cell sorting (FACS) under sterile conditions. A total of 100,000–500,000 cells should be collected for the follow-up experiments.
    1. Run cells stained with Hoechst 33342 on the flow cytometer.
      1. Place tubes containing cells stained with Hoechst 33342 on the cytometer.
      2. Click the Dot Plot button to display the dot plot, then click the X-axis and set it to “FSC-A”; click the Y-axis and set it to “PI-A”. Display the dot plot of forward scatter pulse area (FSC-A, X-axis set to linear mode) versus the PI fluorescence (Y-axis set to logarithmic scale). Adjust the voltages to show the living cells in the right side and the non-living cells, which are brightly stained with PI, in the upper left corner. Then, establish a polygon gate to exclude dead cells and cell debris (Figure 1A) by clicking the Polygon Gate button to gate the P1 subset, also known as FSC-A, PI-A subset (Supplementary Figure 6A,B).
      3. Click the Dot Plot button to display the dot plot, click the X-axis and set it to “FSC-A”; click the Y-axis and set it to “FSC-W”. Display the dot plot of FSC-A (X-axis) versus forward scatter pulse width (FSC-W, Y-axis). Right-click the dot plot, click the P1 button under the Show Populations button. Click the Rectangular Gate button to create a rectangular gate to gate the P2 subset (also known as FSC-A, FSC-W subset). This will exclude cells with large volumes (Supplementary Figure 7A-C and Figure 1A).
      4. Click the Dot Plot button to display the dot plot, click the X-axis and set it to “SSC-A”; click the Y-axis and set it to “SSC-W”. Display the dot plot of side scatter pulse area (SSC-A, X-axis) versus side scatter pulse width (SSC-W, Y-axis). Right-click the dot plot, and click the P2 button under Show Populations button. Then click the Rectangular Gate button to create a rectangular gate to gate the P3 subset (also known as SSC-A, SSC-W subset). This will obtain a cell population with uniform granularity (Supplementary Figure 8A-C and Figure 1A).
      5. Click the Dot Plot button to display the dot plot, click the X-axis and set it to “Hoechst Red-A”; click the Y-axis and set it to “Hoechst Blue-A”. Display the dot plot of Hoechst Red-A (X-axis) versus Hoechst Blue-A (Y-axis). Right-click the dot plot, click the P3 button under the Show Populations button. Click the Polygon Gate button to create a polygon gate to gate the P4 subset (also known as Hoechst Red-A, Hoechst Blue-A subset). Right-click the dot plot, click the Show Population Hierarchy button to show the population hierarchy.
        NOTE: The dot plot will show three different populations: 1) a G0-G1 phase population near the center of the graph; 2) a S-G2/M phase population near the upper right corner; 3) the SP. The SP is then gated for further analysis (Supplementary Figure 9A-D and Figure 1A). If the dot plot of Hoechst Red-A versus Hoechst Blue-A does not show a SP profile similar to that shown in Figure 1A, the voltages should be adjusted until a similar profile is seen. Meanwhile, adjust all of above gates accordingly.
      6. After determining the gate region of the SP cells, click Acquire Data to collect 20,000–100,000 events from each sample to analyze the percentage of SP cells (Supplementary Figure 10).
    2. Run the Hoechst 33342-stained cells treated with blocker on the flow cytometer.
      1. Place tubes containing blocker on the cytometer and run cells using the same voltages and gates to further check whether the voltages and gates are selected appropriately.
        NOTE: Compared with Hoechst 33342 staining alone, only a very small proportion of cells, if any, should appear in the gated area of the SP (Figure 1B).
      2. Collect 20,000–100,000 events from each sample to analyze the percentage of SP cells.

4. Data analysis

NOTE: Instructions for the use of the flow cytometry analysis software (see Table of Materials) are described in this section and Supplementary Figures 11–16.

  1. Copy the files in fcs format to a computer, open the flow cytometry analysis software, and drag one sample file to the software (Supplementary Figure 11A,B).
  2. Gate cells and obtain the percentage of SP cells.
    1. Double click this sample file, click the X-axis and set it to “FSC-A”; click the Y-axis and set it to “PI-A”. Then, create a polygon gate and click the OK button to obtain the FSC-A, PI-A subset (Supplementary Figure 12A-E).
    2. Double click the FSC-A, PI-A subset file, click the X-axis and set it to “FSC-A”; click the Y-axis and set it to “FSC-W”. Then, create a rectangular gate and click the OK button to obtain the FSC-A, FSC-W subset (Supplementary Figure 13A-E).
    3. Double click the FSC-A, FSC-W subset file, click the X-axis and set it to “SSC-A”; click the Y-axis and set it to “SSC-W”. Then, create a rectangular gate and click the OK button to obtain the SSC-A, SSC-W subset (Supplementary Figure 14A-E).
    4. Double click the SSC-A, SSC-W subset file, click the X-axis and set it to “Hoechst Red-A”; click the Y-axis and set it to “Hoechst Blue-A”. Then, create a polygon gate and click the OK button to obtain the Hoechst Red-A, Hoechst Blue-A subset (Supplementary Figure 15A-E).
    5. Open the Layout Editor by clicking the Open Layout Editor button. Drag the SSC-A, SSC-W subset file to Layout Editor, then click the Click to save layout window to file button to save the image results (Supplementary Figure 16A-C).
  3. Save the workspace to keep the gating information when closing the software.
  4. Perform t-test analyses with statistical analysis software to compare the difference between two groups. A value of P < 0.05 was defined as statistically significant.

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Results

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Four experimental SP analyses were performed according to this method. In the first one, we detected the proportion of SP cells in MDA-MB-231, which is a triple negative human breast cancer cell line, under normal conditions. After cell counting, Hoechst 33342 was added into one tube containing 1 x 106 cells to a final concentration of 3 µg/mL. Reserpine and Hoechst 33342 were added to another tube to final concentrations of 40 µM and 3 µg/mL, respectively. PI was added to both tubes. The dot plot of FSC-A (X-...

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Discussion

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There are several key points to keep in mind for the SP assay. The first is the selection of a proper blocker, such as Verapamil or Reserpine, for each cell line, because the "gate" location of the SP cells is determined according to the position at which a large number of SP cells disappear after the addition of the blocker. For the MDA-MB-231 cell line, Reserpine works well. However, for other cell lines, different blockers might work better.

The second is the concentration of Hoechs...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was funded by the Natural Science Foundation of China 81572599, 81773124, and 81972787; Natural Science Foundation of Tianjin City (China) 19JCYBJC27300; Tianjin People’s Hospital & Nankai University Collaborative Research Grant 2016rmnk005; Fundamental Research Funds for the Central Universities, Nankai University 63191153.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
6 well cell culture plateCORNING35169.5 cm2 (approx.)
ColivelinMCEHY-P1061ASer-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Arg-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro
Fetal bovine serum (FBS)Biological Industries (BIOIND)04-001-1ACS
Flow cytometerBD BiosciencesBD LSRFortessa
Flow cytometer softwareBD BiosciencesFACSDiva
Flow cytometry analysis softwareBD BiosciencesFlowJo
Hoechst 33342Sigma-AldrichB2261bisBenzimide H 33342 trihydrochloride
Polystyrene round bottom test tubeCORNING35205412 x 75 mm, 5 mL
Propidium iodide (PI)Sigma-AldrichP41703,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide
ReserpineSigma-Aldrich83580(3β, 16β, 17α, 18β, 20α)-11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester
SKLB816Provided by Dr. Shengyong Yang, Sichuan University
Trypsin-EDTA (0.25%), phenol redGibco25200072
Verapamil hydrochlorideSigma-AldrichV46295-[N-(3,4-Dimethoxyphenylethyl)methylamino] -2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride

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Tags

Side Population AnalysisCancer Stem CellsHoechst 33342 StainingFlow CytometryABC TransportersCell IsolationStemness PropertiesBlocker ControlPropidium Iodide

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