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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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/o....

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....

Representative 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.......

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,

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.

....

Materials

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

References

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Flow CytometryBreast CancerStem CellsApoptosis ReversalCell IsolationCell CultureCell SortingCD44CD24Trypsin EDTAFACS BufferFc Block

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