Name: flow cytometric detection of newly-formed breast cancer stem cell-like cells after apoptosis reversal
Uploaded: 2019-01-26T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal at JoVE.com

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.

1. Preparation of Breast Non-stem Cancer Cells
<ol>
	<li>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 &#176;C in a 5% CO2/95% air cell culture incu...

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,<s...

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 &#34;point of no return&#34; 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 &#34;point of no return&#34;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.

<table>
	<tbody>
		<tr>
			<td>MCF-7</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>HTB-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 680 annexin V conjugate</td>
			<td>Invitrogen</td>
			<td>A35109</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>USB</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 &#183; 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C-5080</td>
			<td></td>
		</tr>
		<tr>
			<td>CellEvent caspase-3/7 green fluorescent dye</td>
			<td>Invitrogen</td>
			<td>C10423</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block</td>
			<td>Miltenyi Biotec</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>heat-inactivated</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>USB</td>
			<td>16926</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitotracker Red CMXRos</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>monoclonal antibodies against human CD24</td>
			<td>BD Biosciences</td>
			<td>555428</td>
			<td>PE Clone:ML5 
			Lot:5049759 
			RRID:AB_395822</td>
		</tr>
		<tr>
			<td>monoclonal antibodies against human CD44</td>
			<td>BD Biosciences</td>
			<td>560531</td>
			<td>PERCP-CY5.5 Clone:G44-26 
			Lot:7230770 
			RRID:AB_1727485</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>31434</td>
			<td></td>
		</tr>
		<tr>
			<td>paclitaxel</td>
			<td>Sigma-Aldrich</td>
			<td>T7402</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG2a, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>554648</td>
			<td>Clone:G155-178 (RUO) 
			RRID:AB_395491</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 Mouse IgG2b, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>558304</td>
			<td>Clone:27-35 
			RRID:AB_647257</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute 1640 medium</td>
			<td>Invitrogen</td>
			<td>11835055</td>
			<td>phenol red-free</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>staurosporine</td>
			<td>Sigma-Aldrich</td>
			<td>S4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner Bio-One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40-&#956;m nylon mesh</td>
			<td>BD Biosciences</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSuite software bundle v1.0</td>
			<td>BD Biosciences</td>
			<td>651360</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSVerse</td>
			<td>BD Biosciences</td>
			<td>651155</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoView FV1000 confocal microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>60X objective</td>
		</tr>
		<tr>
			<td>FV10-ASW Viewer software Ver.4.2b</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>round-bottom polystyrene 12 &#215; 75 mm tubes</td>
			<td>BD Biosciences</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>S3e sorter</td>
			<td>Bio-Rad</td>
			<td>1451006</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric detection of newly-formed breast cancer stem cell-like cells after apoptosis reversal

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.

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...

Watch this Scientific Journal Video about Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal at JoVE.com 

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal (Video) | JoVE 

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.

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

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

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