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Method Article

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

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DOI:

10.3791/58642

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January 26th, 2019

 ,  ,  ,  , 
1School of Life Sciences, The Chinese University of Hong Kong, 2State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, 3Key Laboratory for Regenerative Medicine, Ministry of Education, The Chinese University of Hong Kong, 4Centre for Novel Biomaterials, The Chinese University of Hong Kong

In This Article

Summary

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Abstract

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Introduction

Cancer has been a leading cause of death, causing heavy burden to countries worldwide1. Breast cancer ranks high both in terms of incidence and mortality in female patients among all types of cancer1. Due to the cancer heterogeneity, a combination of drugs is usually used in chemotherapy to achieve cancer cell death2,3,4. However, since common chemotherapeutic drugs often target DNA5,6, protein synthesis7,8 and/or microtubule dynamics9, rapidly growing cells are affected the most while quiescent cells such as cancer stem cell (CSC)s are usually less affected10. CSCs are, therefore, more likely to survive after the treatment, which later leads to drug resistance and cancer relapse10,11. Hence, elimination of CSCs has become an important topic for cancer treatment and study of the origin of CSCs is necessary.

More studies on the phenomenon of apoptosis reversal have been performed in the recent decade12,13,14,15,16,17,18,19. Before the emergence of this concept, it has been widely accepted that cells will irreversibly undergo apoptosis after caspase activation. Caspases are a family of protein enzymes that play key roles in the initiation and execution stages of apoptosis, including the formation of the apoptotic complex and the cleavage of downstream substrates20. Activation of executioner caspases such as caspase 3 or caspase 7 has been considered as the "point of no return" for apoptosis21. However, researchers recently observed that apoptosis reversal occurs both in vitro and in vivo, during which cells can recover from apoptosis even after caspase activation12,13,14,15,16,17,18,19. Moreover, aggressive features such as higher resistance to the original apoptotic inducer and higher invasiveness are detected in the reversed cancer cells15. Hence, it was proposed that the percentage of CSC-like cells would be higher in the reversed population when compared to the untreated cells, eventually contributing to the more malignant features after apoptosis reversal18.

Previously, many efforts have been made to track the apoptosis reversal in vitro and more importantly, in vivo, which greatly help in confirming the universality of this process16,17,19. However, a systemic study on the consequences of reversed cells is lacking due to the unsatisfactory isolation of cells that have genuinely undergone apoptosis reversal. There is a need to acquire pure apoptotic cells and recover them for further study. Thus, we use the traditionally well-accepted marker of executioner caspase activation as the marker of the "point of no return"21 for apoptosis and utilize fluorescence-activated cell sorting (FACS) to discriminate caspase-activated cells stained with Caspase-3/7 Green Detection dye. The dye is covalently linked to a short amino acid sequence, DEVD, which can be recognized and cleaved by active caspases 3/7. The cleavage helps release the dye, which will translocate from the cytosol to the nucleus where it binds to DNA and emits strong fluorescence. This procedure avoids using a bulk cell population in which some cells may not have undergone apoptosis.

CSCs or tumor-initiating cells have been identified in many solid tumors using a single or a combination of several surface marker(s) and very few numbers of these cells are sufficient to form tumors in immunodeficient mice22,23,24,25,26,27. A combination of CD44 and CD24 has been commonly used in breast CSC studies, and CD44+/CD24- cells have been defined as the breast CSCs26,27,28,29,30. Recently, we have performed a series of experiments to confirm the proposed relationship between apoptosis reversal and CSCs and demonstrate that reversed breast cancer cells gained increased tumor-forming ability in vitro and in vivo with an elevated percentage of cells with CSC markers18. Although we could not exclude the possibility that breast CSCs survive better and thus get enriched after apoptosis reversal, importantly, when we isolate non-stem cancer cells and subject them to apoptosis reversal, CSC will emerge in the originally non-stem cancer cell population, suggesting that non-stem cancer cells can contribute to the increase in the percentage of CSCs during apoptosis reversal.

This article aims to demonstrate the transition from breast non-stem cancer cells to breast CSC-like cells after apoptosis reversal and to detect this transition by flow cytometry. The breast non-stem cancer cells are initially prepared by isolating CD44-/CD24+ breast cancer cells by FACS. Then, apoptosis is induced and confirmed by morphological changes under the microscope. Afterwards, apoptotic cells positively-labelled by Caspase 3/7 Green Detection dye are isolated by FACS and further cultured in the absence of apoptotic inducers for apoptosis reversal. The reversed cells are then stained with CSC markers after 7 days of recovery for flow cytometric analysis. Cells with CD44+/CD24- markers re-appear in the reversed population, suggesting that transition from non-stem cancer cells to CSC-like cells has occurred during apoptosis reversal.

Apoptosis reversal has been observed in multiple cancer cell lines as well as normal primary cells treated with different apoptotic stimuli in vitro12,13. This process has also been traced in Drosophila model in vivo16,17,19. Much information regarding the underlying mechanism of cancer relapse in different cancer disease models and the origin of CSCs can be obtained through the use of the technique as described in this manuscript.

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Protocol

1. Preparation of Breast Non-stem Cancer Cells

  1. Culture MCF-7 and MDA-MB-231 cells in 10 mL of phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a 100 mm dish. Culture T47D in 10 mL of phenol red-free RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, and 1% v/v Penicillin-Streptomycin (PS) in a 100 mm dish. Culture cells at 37 °C in a 5% CO2/95% air cell culture incubator.
    NOTE: Phenol-red affects fluorescence-based detection but also has a potential influence on the growth of breast cancer cells due to its estrogen-like effects31. For example, phenol red could stimulate progesterone receptor and growth of MCF-7 cells31. It could also stimulate the growth of T47D cells31.
  2. Wash cells with 2 mL of phosphate buffered saline (PBS) twice. Add 2 mL of 0.05% trypsin-EDTA to MCF-7 and MDA-MB-231 or 2 mL of 0.25% trypsin-EDTA to T47D cells.
    NOTE: The concentration of trypsin-EDTA needed for separation of cells among different breast cancer cell types vary while an inappropriate concentration may affect the expression pattern of some cell surface markers such as CD44 and CD2432.
  3. Culture the dishes for 5 min at 37 °C under an atmosphere of 5% CO2/95% air. Frequently check the detachment of cells under the microscope to prevent cells from over-digestion by trypsin-EDTA.
  4. When over 90% cells detach, add 5 mL of completed RPMI medium to the dish and pipette it over the cell layer surface several times.
  5. Transfer the cells to a 15 mL conical tube and centrifuge at 300 x g and 4 °C for 5 min.
    NOTE: Usually, around 6 x 106 cells can be obtained from a 100 mm dish when the confluency reaches 70%.
  6. Discard the supernatant and resuspend the pellets at 106 cells/100 μL of FACS buffer (PBS containing 0.5% BSA and 0.1% sodium azide) in a 1.5 mL microcentrifuge tube.
  7. Divide the cells into several tubes. For MCF-7 cells, divide the cells into 4 tubes: two for isotype controls, one for CD24 single staining as CD24 positive control and one for dual staining.
  8. For T47D cells, divide the cells into 3 tubes: two for isotype controls and one for dual staining.
  9. For MDA-MB-231 cells, divide the cells into 4 tubes: two for isotype controls, one for CD44 single staining as CD44 positive control and one for dual staining.
  10. Centrifuge the tubes again at 300 x g and 4 °C for 5 min. Discard the supernatant, and then add Fc block diluted 1:50 in FACS buffer.
  11. Incubate the samples on ice for 20 min in the dark before centrifugation at 300 x g and 4 °C for 5 min. Discard the supernatant.
  12. Add fluorochrome-conjugated monoclonal antibodies against human CD44 (PerCP-Cy5.5) and CD24 (PE) at 1:40 and 1:10 dilutions (in FACS buffer) in the dual staining groups. For positive controls, add CD44 (PerCP-Cy5.5) to MDA-MB-231 and add CD24 (PE) to MCF-7 cells at the same concentration, respectively.
    NOTE: The combination of CD44 and C24 has been commonly used in breast CSC study26,27,28,29,30.
  13. For the isotype control groups, add PerCP-Cy5.5 Mouse IgG2b,κ at a 1:40 dilution as the isotype control for CD44 antibodies and PE Mouse IgG2a,κ at a 1:10 dilution as the isotype control for CD24 antibodies at 106 cells/100 μL.
    NOTE: Isotype controls for the antibodies of interest are recommended as negative controls33.
  14. Incubate the samples (from steps 1.12 and 1.13) at 4 °C in the dark for 30 min. Centrifuge at 300 x g and 4 °C for 5 min. Discard the supernatant.
  15. Wash the pellet twice with 500 μL of PBS and centrifuge at 300 x g and 4 °C for 5 min.
  16. Resuspend the pellet in 0.5 mL of PBS and filter through a 40 µm nylon mesh before running on a fluorescence-activated cell sorter.
  17. For gating and compensation purposes, stain MCF-7 cells with PE-conjugated anti-CD24 antibodies and stain MDA-MB-231 cells with PerCP-Cy5.5-conjugated anti-CD44 antibodies as positive controls. Use cells stained with isotype controls as negative controls (Figure 1).
  18. Collect cells with CD44-/CD24+ markers in round-bottom polystyrene 12 x 75 mm tubes containing 1 mL of collection medium (phenol red-free RPMI 1640 medium supplemented with 20% heat-inactivated FBS and 2% v/v PS). Centrifuge the tubes at 300 x g and RT for 5 min and discard the supernatant.
    NOTE: CSC-like cells in breast cancer cells are defined as CD44+/CD24- cells26,27,28,29,30, and breast non-stem cancer cells are defined as CD44-/CD24+ cells18 (Figure 1).
  19. Plate the sorted breast non-stem cancer cells in the culture dish containing fresh collection medium for further culture at 37 °C in 5% CO2 cell culture incubator. 

2. Apoptotic Induction and Detection

  1. Prepare 1 mM staurosporine12,34 in DMSO. For the treated MCF-7 group, add 25 μL of 1 mM staurosporine to the completed medium of MCF-7 cells to make up to 10 mL of final volume (2.5 μM staurosporine) in a 15 mL conical tube. Pipette to mix the content evenly.
  2. Remove the culture medium from the cells, wash the cells with 2 mL of PBS once. Then add the 10 mL of medium with 2.5 μM staurosporine to MCF-7 cells for 6 h to induce apoptosis when cell density reaches 70% confluency.
  3. Prepare 1 mM paclitaxel35,36 in DMSO. For the treated T47D group, add 12.5 μL of 1 mM paclitaxel to the completed medium of T47D cells to make up to a final volume of 10 mL (5 μM paclitaxel) in a 15 mL conical tube. Pipette to mix the content evenly.
  4. Remove the culture medium from the cells, and wash the cells with 2 mL of PBS once. Then add the 10 mL of medium with 5 μM paclitaxel to the T47D cells for 10 h to induce apoptosis when cell density reaches 70% confluency.
    NOTE: The induction time and the concentration of the inducers that induce apoptosis should be optimized whenever a new cell line is used for the first time.
  5. For the solvent-treated MCF-7 group, add 25 μL of sterile dimethyl sulfoxide (DMSO) to the completed medium of MCF-7 cells to make up to a final volume of 10 mL (i.e., 0.25% v/v DMSO) in a 15 mL conical tube. Pipette to mix the content evenly.
  6. Remove the culture medium from the solvent treated MCF-7 cells, wash with 2 mL of PBS once. Then add the 10 mL of medium with 0.25% v/v DMSO for 6 h as the solvent control for STS.
  7. For the solvent-treated T47D group, add 5 μL of sterile DMSO to the completed medium of T47D cells to make up to 10 mL final volume (i.e., 0.05% v/v DMSO) in a 15 mL conical tube. Pipette to mix the content evenly.
  8. Remove the culture medium from the solvent treated T47D cells, wash with 2 mL of PBS once. Then, add the 10 mL of medium with 0.05% v/v DMSO for 10 h as the solvent control for paclitaxel.
  9. To observe the morphological changes of treated cells, stain cells with 50 nM Mitotracker Red CMXRos and 250 ng/mL Hoechst 33342, and incubate for another 20 min at 37 °C under an atmosphere of 5% CO2/95% air. Observe the typical apoptotic cell morphology under a 60x confocal laser scanning microscope (Figure 2).
    NOTE: Typical apoptosis morphology includes cell shrinkage, membrane blebbing, mitochondria fragmentation and nuclear condensation but with intact cellular membrane12,13,14,15,18,37.

3. Isolation of Apoptotic Cells and Apoptosis Reversal Procedure

  1. Stain both the apoptotic inducer- and solvent-treated MCF-7 or T47D cells with 3 μM Caspase-3/7 Green Detection dye at the cell concentration of 106/mL in the dark for 30 min at 37 °C under an atmosphere of 5% CO2/95% air.
  2. Filter the cells through a 40 µm nylon mesh before running on the sorter for sorting.
  3. For gating purposes, first gate the major population (R1) and exclude the debris by plotting the graph in dots with forward scatter and side scatter (Figure 3).
  4. Use the untreated cells without adding the Caspase-3/7 Green Detection dye as a negative control to gate the negative region (R2) (Figure 3A).
  5. Use cells treated with staurosporine12,18,34 for 24 h and stained with the dye as a positive control to gate the positive region (R3) (Figure 3B).
  6. Collect positive cells (R3) from the inducer-treated groups in round-bottom polystyrene 12 x 75 mm tubes containing 1 mL of collection medium (same as the collection medium in step 1.18) (Figures 3C-3D). Centrifuge the tubes at 300 x g and RT for 5 min and discard the supernatant.
  7. Collect negative cells (R2) from solvent-treated groups in round-bottom polystyrene 12 × 75 mm tubes with 1 mL of collection medium as in step 3.6 (Figures 3E-3F).
    NOTE: A small portion of cells (such as 10,000 cells) can be collected and re-run on the sorter to check the pattern of active caspases marker. If over 90% of sorted solvent-treated cells are shown in the caspase-negative region or over 90% of sorted inducer-treated cells are shown in the caspase-positive region, these collected cells are believed to be relatively pure (Figures 3C-3F). Otherwise, the sorting region and/or the sorter should be reset.
  8. Resuspend the sorted cells in fresh collection medium and seed them in 12-well tissue culture plates and culture at 37 °C under an atmosphere of 5% CO2/95% air for 7 days for apoptosis reversal.

4. Confirmation of Apoptosis in Caspase-activated Cells

  1. Mix 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 to prepare annexin-binding buffer (pH 7.4). Store the buffer at 4 °C and avoid the buffer to expose to light.
  2. Prepare a 100 µg/mL working solution of propidium iodide (PI) by diluting 5 µL of the 1 mg/mL PI stock solution in 45 µL of annexin-binding buffer. Store the solution at 4 °C and avoid the solution to expose to light.
    NOTE: PI is a potential mutagen and should be handled with care.
  3. Prepare the inducer-treated cells as in steps 2.1 to 2.5.
  4. Collect the inducer-treated MCF-7 or T47D cells by pipetting the medium over the cell layer 3-5x to detach the cells. Transfer the cells to a 15 mL conical tube.
  5. Centrifuge at 300 x g and RT for 5 min. Discard the supernatant.
  6. Resuspend the cells with completed medium at the cell concentration of 106/mL in a 1.5 mL microcentrifuge tube and stain cells with 3 μM Caspase 3/7 Green Detection dye in the dark for 30 min at 37 °C.
  7. Centrifuge at 300 x g and RT for 5 min. Discard the supernatants.
  8. Wash the cells with 1 mL of ice-cold PBS and centrifuge at 300 x g and 4 °C for 5 min. Discard the supernatants.
  9. Resuspend the cells in 100 μL of annexin-binding buffer with the add 5 μL of the annexin V conjugate and 1 µL of 100 µg/mL PI working solution to each 100 μL of cell suspension and incubate the cells for 15 min at room temperature.
    NOTE: Annexin V and PI are commonly used together to label apoptotic cells in flow cytometry38,39,40.
  10. Add 400 µL of annexin-binding buffer, mix gently. Keep the samples on ice before running on a flow cytometer.

5. Measurement of Breast CSC-like Cells by Flow Cytometry

  1. Harvest the reversed MCF-7 in both inducer- or solvent-treated groups with 0.05% trypsin-EDTA. Harvest T47D cells in both inducer- or solvent-treated groups or with 0.25% trypsin-EDTA as in steps 1.2 to 1.5.
  2. Stain the cells with fluorochrome-conjugated monoclonal antibodies against human CD44 (PerCP-Cy5.5) and CD24 (PE) as in step 1.12. Meanwhile, prepare the isotype controls as in step 1.13.
  3. Run the cells on a flow cytometer and detect the percentage of cells with CD44+/CD24- markers.

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Results

In order to observe the transition from breast non-stem cancer cells to breast CSC-like cells, a first sorting of CD44-/CD24+ breast cancer cells were needed. For the MCF-7 cell line, which has around 0.15% cells with CSC markers in the original population (Figure 1), this step helped exclude the possibility of CSC enrichment during apoptosis reversal. On the contrary, if there were no cells with CSC markers in the original population, s...

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Discussion

This protocol describes a direct and clear way for detecting the transition of breast non-stem cancer cells into breast CSC-like cells as a result of apoptosis reversal. Confirmation of the CSC properties of these reversed cells could be assisted by using in vitro mammosphere formation assay and in vivo xenograft transplantation in immunodeficient mice18,24,26,27,

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Innovative Technology Fund of Innovation Technology Commission: Funding Support from the State Key Laboratory of Agrobiotechnology (CUHK), the Lo Kwee-Seong Biomedical Research Fund and the Lee Hysan Foundation. Y.X. was supported by the postgraduate studentship from the CUHK.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
MCF-7American Type Culture Collection (ATCC)HTB-22
MDA-MB-231American Type Culture Collection (ATCC)HTB-26
T47DAmerican Type Culture Collection (ATCC)HTB-133
Reagent
0.05% trypsin-EDTAInvitrogen25300054
0.25% trypsin-EDTAInvitrogen25200072
Alexa Fluor 680 annexin V conjugateInvitrogenA35109
bovine serum albuminUSB9048-46-8
CaCl2 · 2H2OSigma-AldrichC-5080
CellEvent caspase-3/7 green fluorescent dyeInvitrogenC10423
dimethyl sulfoxideSigma-AldrichD2650
Fc blockMiltenyi Biotec130-059-901
fetal bovine serumInvitrogen16000044heat-inactivated
HEPESUSB16926
Hoechst 33342InvitrogenH3570
L-glutamineInvitrogen25030081
Mitotracker Red CMXRosInvitrogenM7512
monoclonal antibodies against human CD24BD Biosciences555428PE Clone:ML5
Lot:5049759
RRID:AB_395822
monoclonal antibodies against human CD44BD Biosciences560531PERCP-CY5.5 Clone:G44-26
Lot:7230770
RRID:AB_1727485
NaClSigma-Aldrich31434
paclitaxelSigma-AldrichT7402
PE Mouse IgG2a, κ Isotype ControlBD Biosciences554648Clone:G155-178 (RUO)
RRID:AB_395491
Penicillin-StreptomycinInvitrogen15070-063
PerCP-Cy5.5 Mouse IgG2b, κ Isotype ControlBD Biosciences558304Clone:27-35
RRID:AB_647257
phosphate buffered salineThermo Fisher Scientific21600010
propidium iodideInvitrogenP1304MP
Roswell Park Memorial Institute 1640 mediumInvitrogen11835055phenol red-free
sodium azideSigma-AldrichS2002
staurosporineSigma-AldrichS4400
Equipment
100 mm culture dishGreiner Bio-One664160
12-well tissue culture platesThermo Fisher Scientific150628
Cell Strainer 40 μm nylon meshBD Biosciences08-771-1
FACSuite software bundle v1.0BD Biosciences651360
FACSVerseBD Biosciences651155
FluoView FV1000 confocal microscopeOlympusIX8160X objective
FV10-ASW Viewer software Ver.4.2bOlympus-
round-bottom polystyrene 12 × 75 mm tubesBD Biosciences352003
S3e sorterBio-Rad1451006

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Breast Cancer Stem CellsFlow CytometryFluorescence-activated Cell SortingConfocal MicroscopyCaspase-3/7 DetectionCD44 CD24 MarkersNon-stem Cancer CellsTumorigenicity Analysis
Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal
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