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 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.</li>
	<li>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.</li>
	<li>Culture the dishes for 5 min at 37 &#176;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.</li>
	<li>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.</li>
	<li>Transfer the cells to a 15 mL conical tube and centrifuge at 300 x g and 4 &#176;C for 5 min. 
	NOTE: Usually, around 6 x 106 cells can be obtained from a 100 mm dish when the confluency reaches 70%.</li>
	<li>Discard the supernatant and resuspend the pellets at 106 cells/100 &#956;L of FACS buffer (PBS containing 0.5% BSA and 0.1% sodium azide) in a 1.5 mL microcentrifuge tube.</li>
	<li>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.</li>
	<li>For T47D cells, divide the cells into 3 tubes: two for isotype controls and one for dual staining.</li>
	<li>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.</li>
	<li>Centrifuge the tubes again at 300 x g and 4 &#176;C for 5 min. Discard the supernatant, and then add Fc block diluted 1:50 in FACS buffer.</li>
	<li>Incubate the samples on ice for 20 min in the dark before centrifugation at 300 x g and 4 &#176;C for 5 min. Discard the supernatant.</li>
	<li>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.</li>
	<li>For the isotype control groups, add PerCP-Cy5.5 Mouse IgG2b,&#954; at a 1:40 dilution as the isotype control for CD44 antibodies and PE Mouse IgG2a,&#954; at a 1:10 dilution as the isotype control for CD24 antibodies at 106 cells/100 &#956;L. 
	NOTE: Isotype controls for the antibodies of interest are recommended as negative controls33.</li>
	<li>Incubate the samples (from steps 1.12 and 1.13) at 4 &#176;C in the dark for 30 min. Centrifuge at 300 x g and 4 &#176;C for 5 min. Discard the supernatant.</li>
	<li>Wash the pellet twice with 500 &#956;L of PBS and centrifuge at 300 x g and 4 &#176;C for 5 min.</li>
	<li>Resuspend the pellet in 0.5 mL of PBS and filter through a 40 &#181;m nylon mesh before running on a fluorescence-activated cell sorter.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Plate the sorted&#160;breast non-stem cancer cells in the culture dish containing fresh collection medium for further culture at 37 &#176;C in 5% CO2 cell culture incubator.&#160;</li>
</ol>
2. Apoptotic Induction and Detection
<ol>
	<li>Prepare 1 mM staurosporine12,34 in DMSO. For the treated MCF-7 group, add 25 &#956;L of 1 mM staurosporine to the completed medium of MCF-7 cells to make up to 10 mL of final volume (2.5 &#956;M staurosporine) in a 15 mL conical tube. Pipette to mix the content evenly.</li>
	<li>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 &#956;M staurosporine to MCF-7 cells for 6 h to induce apoptosis when cell density reaches 70% confluency.</li>
	<li>Prepare 1 mM paclitaxel35,36 in DMSO. For the treated T47D group, add 12.5 &#956;L of 1 mM paclitaxel to the completed medium of T47D cells to make up to a final volume of 10 mL (5 &#956;M paclitaxel) in a 15 mL conical tube. Pipette to mix the content evenly.</li>
	<li>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 &#956;M 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.</li>
	<li>For the solvent-treated MCF-7 group, add 25 &#956;L 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.</li>
	<li>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.</li>
	<li>For the solvent-treated T47D group, add 5 &#956;L 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.</li>
	<li>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.</li>
	<li>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 &#176;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.</li>
</ol>
3. Isolation of Apoptotic Cells and Apoptosis Reversal Procedure
<ol>
	<li>Stain both the apoptotic inducer- and solvent-treated MCF-7 or T47D cells with 3 &#956;M Caspase-3/7 Green Detection dye at the cell concentration of 106/mL in the dark for 30 min at 37 &#176;C under an atmosphere of 5% CO2/95% air.</li>
	<li>Filter the cells through a 40 &#181;m nylon mesh before running on the sorter for sorting.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>Collect positive cells (R3) from the inducer-treated groups in round-bottom polystyrene 12 x&#160;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.</li>
	<li>Collect negative cells (R2) from solvent-treated groups in round-bottom polystyrene 12 &#215; 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.</li>
	<li>Resuspend the sorted cells in fresh collection medium and seed them in 12-well tissue culture plates and culture at 37 &#176;C under an atmosphere of 5% CO2/95% air for 7 days for apoptosis reversal.</li>
</ol>
4. Confirmation of Apoptosis in Caspase-activated Cells 
<ol>
	<li>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 &#176;C and avoid the buffer to expose to light.</li>
	<li>Prepare a 100 &#181;g/mL working solution of propidium iodide (PI) by diluting 5 &#181;L of the 1 mg/mL PI stock solution in 45 &#181;L of annexin-binding buffer. Store the solution at 4 &#176;C and avoid the solution to expose to light. 
	NOTE: PI is a potential mutagen and should be handled with care.</li>
	<li>Prepare the inducer-treated cells as in steps 2.1 to 2.5.</li>
	<li>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.</li>
	<li>Centrifuge at 300 x g and RT for 5 min. Discard the supernatant.</li>
	<li>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 &#956;M Caspase 3/7 Green Detection dye in the dark for 30 min at 37 &#176;C.</li>
	<li>Centrifuge at 300 x g and RT for 5 min. Discard the supernatants.</li>
	<li>Wash the cells with 1 mL of ice-cold PBS and centrifuge at 300 x g and 4 &#176;C for 5 min. Discard the supernatants.</li>
	<li>Resuspend the cells in 100 &#956;L of annexin-binding buffer with the add 5 &#956;L of the annexin V conjugate and 1 &#181;L of 100 &#181;g/mL PI working solution to each 100 &#956;L 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.</li>
	<li>Add 400 &#181;L of annexin-binding buffer, mix gently. Keep the samples on ice before running on a flow cytometer.</li>
</ol>
5. Measurement of Breast CSC-like Cells by Flow Cytometry
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Run the cells on a flow cytometer and detect the percentage of cells with CD44+/CD24- markers.</li>
</ol>

The authors have nothing to disclose.

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>&lt;strong&gt;Reagent&lt;/strong&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</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&lt;br/&gt; Lot:5049759&lt;br/&gt; 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&lt;br/&gt; Lot:7230770&lt;br/&gt; 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, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>554648</td><td>Clone:G155-178 (RUO)&lt;br/&gt; 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, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>558304</td><td>Clone:27-35&lt;br/&gt; 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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;mu;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 &amp;times; 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

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.

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

flow-cytometric-detection-newly-formed-breast-cancer-stem-cell-like

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Cancer Research

9.8K Views.  The Chinese University of Hong Kong. De isolaties van echte populaties van apoptotische omkeringscellen waren eerder een probleem, wat ons begrip over de gevolgen van een omkering van apoptose sterk beperkt. Dit protocol stelt ons in staat om een zuiverder populatie van apoptotische omkering cellen te genereren. Het belangrijkste voordeel van deze techniek is dat de gebruikte materialen en apparatuur op grote schaal beschikbaar zijn in verschillende instellingen.Deze techniek is gemakkelijk toepasbaar voor het isoleren va...

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

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Estimating LSC Frequency in Zebrafish T-ALL Using Cell Proliferation Dye Sorting

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Author Spotlight: Identification and Isolation of Quiescent Leukemia Stem Cells from Zebrafish T-ALL

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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

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