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

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 death^2,3,4. However, since common chemotherapeutic drugs often target DNA^5,6, protein synthesis^7,8 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....

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 mice^18,24,26,27,

Materials

Name	Company	Catalog Number	Comments
MCF-7	American Type Culture Collection (ATCC)	HTB-22
MDA-MB-231	American Type Culture Collection (ATCC)	HTB-26
T47D	American Type Culture Collection (ATCC)	HTB-133
Reagent
0.05% trypsin-EDTA	Invitrogen	25300054
0.25% trypsin-EDTA	Invitrogen	25200072
Alexa Fluor 680 annexin V conjugate	Invitrogen	A35109
bovine serum albumin	USB	9048-46-8
CaCl2 · 2H2O	Sigma-Aldrich	C-5080
CellEvent caspase-3/7 green fluorescent dye	Invitrogen	C10423
dimethyl sulfoxide	Sigma-Aldrich	D2650
Fc block	Miltenyi Biotec	130-059-901
fetal bovine serum	Invitrogen	16000044	heat-inactivated
HEPES	USB	16926
Hoechst 33342	Invitrogen	H3570
L-glutamine	Invitrogen	25030081
Mitotracker Red CMXRos	Invitrogen	M7512
monoclonal antibodies against human CD24	BD Biosciences	555428	PE Clone:ML5 Lot:5049759 RRID:AB_395822
monoclonal antibodies against human CD44	BD Biosciences	560531	PERCP-CY5.5 Clone:G44-26 Lot:7230770 RRID:AB_1727485
NaCl	Sigma-Aldrich	31434
paclitaxel	Sigma-Aldrich	T7402
PE Mouse IgG2a, κ Isotype Control	BD Biosciences	554648	Clone:G155-178 (RUO) RRID:AB_395491
Penicillin-Streptomycin	Invitrogen	15070-063
PerCP-Cy5.5 Mouse IgG2b, κ Isotype Control	BD Biosciences	558304	Clone:27-35 RRID:AB_647257
phosphate buffered saline	Thermo Fisher Scientific	21600010
propidium iodide	Invitrogen	P1304MP
Roswell Park Memorial Institute 1640 medium	Invitrogen	11835055	phenol red-free
sodium azide	Sigma-Aldrich	S2002
staurosporine	Sigma-Aldrich	S4400
Equipment
100 mm culture dish	Greiner Bio-One	664160
12-well tissue culture plates	Thermo Fisher Scientific	150628
Cell Strainer 40-μm nylon mesh	BD Biosciences	08-771-1
FACSuite software bundle v1.0	BD Biosciences	651360
FACSVerse	BD Biosciences	651155
FluoView FV1000 confocal microscope	Olympus	IX81	60X objective
FV10-ASW Viewer software Ver.4.2b	Olympus	-
round-bottom polystyrene 12 × 75 mm tubes	BD Biosciences	352003
S3e sorter	Bio-Rad	1451006