This work was supported by the government of the Principality of Monaco, as well as by &#39;Le Groupement des Entreprises Mon&#233;gasques dans la Lutte contre le cancer&#39; (GEMLUC) and Flavien Foundation, which provided the means for BD FACS Melody purchase.

The present study was conducted using DAOY wild-type (WT) medulloblastoma cell lines, which were cultured at 37 &#176;C with 5% CO2 in DMEM medium supplemented with 8% FBS. The xCT-deleted cell line was maintained under the same conditions, with experiments carried out in media supplemented with 1 mM N-acetylcysteine (NAC). The cells were regularly screened for mycoplasma using a commercially available Mycoplasma Detection Kit (see Table of Materials) and were cultured up to the 10th passage.
1. Harvesting and seeding the cells
NOTE: All steps are performed using sterile aseptic techniques in a tissue culture laminar flow hood. DAOY medulloblastoma cells are adherent, meaning all unattached cells can be discarded by washing with phosphate-buffered saline (PBS) without Ca2+ and Mg2+.
<ol>
	<li>Take the stock dish (Petri dish, 100 mm diameter) with the cells and remove the floating cells and medium from the plate using an aspirator.</li>
	<li>Add 5-10 mL of PBS to the dish, wash the bottom with circular movements of the dish, and then remove the PBS from the plate using an aspirator.</li>
	<li>Add 1 mL of trypsin (10x dilution) to the dish. Incubate the plate until gentle tapping of the plate dislodges the cells. Take 10 mL of DMEM culture medium supplemented with 8% FBS and mechanically harvest the cells off the dish.</li>
	<li>Transfer the cell suspension into a 15 mL tube.</li>
	<li>Take 500 &#181;L of the cell suspension and put it in a cuvette for counting. Count the number of cells per mL of cell suspension. An automated cell counter was used for this study (see Table of Materials).</li>
	<li>Calculate the volume necessary to take from the cell suspension to have 1,00,000 cells.</li>
	<li>Take a 6-well plate and add the calculated volume of the cell suspension into each well, then add DMEM culture media supplemented with 8% FBS to 2 mL in each well.</li>
</ol>
2. Treatment of the cells
NOTE: The control and treatments are conducted in triplicate. The groups are as follows: Control (DMSO), 1 &#181;M of erastin, 0.3 &#181;M of RSL3, 250 &#181;M of FAC, 2 &#181;M of Ferrostatin 1, 1 &#181;M of erastin + 2 &#181;M of Ferrostatin 1, 0.3 of &#181;M RSL3 + 2 &#181;M of Ferrostatin 1, 250 &#181;M of FAC + 2 &#181;M of Ferrostatin 1. Four 6-well plates are needed for the experiment, as indicated in Table 1). The commercial details of all the necessary reagents are listed in the Table of Materials.
<ol>
	<li>24 h post-seeding, add the corresponding treatment to the well with the cells.</li>
	<li>Leave the dishes at 37 &#176;C and 5% CO2 in the incubator.</li>
</ol>
3. Lipid hydroperoxides staining of the treated cells with BODIPY 581/591 C11 probe
NOTE: The stock solution of the lipid hydroperoxide-specific probe is prepared in DMSO at a concentration of 1 mM. Aliquots of the stock solution are stored at -20 &#176;C in non-transparent tubes. For staining, prepare a 2 &#181;M working solution of the probe in DMEM media supplemented with 8% FBS.
<ol>
	<li>6 h post-treatment, observe the cells under a light microscope (cell rounding should be visible).</li>
	<li>Remove the dishes from the incubator and place them in a tissue culture laminar flow hood. 
	Using an aspirator, remove the media and floating (dead) cells.</li>
	<li>Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.</li>
	<li>Add 2 mL of the working solution of the probe (2 &#181;M final concentration in DMEM supplemented with FBS).</li>
	<li>Leave the dish in the incubator at 37 &#176;C and 5% CO2 for 30 min in the dark (the plates can be wrapped in aluminum foil).</li>
</ol>
4. Flow cytometry analysis of lipid hydroperoxide content in the treated cells
NOTE: All the following steps are performed in the dark (no lights in the laminar flow hood).
<ol>
	<li>Take the dishes out of the incubator and place them in a tissue culture laminar flow hood. 
	Using an aspirator, remove the staining solution.</li>
	<li>Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.</li>
	<li>Repeat the washing step one more time and aspirate any remaining PBS from the well using an aspirator.</li>
	<li>Add 150 &#181;L of a commercially available cell dissociation reagent (see Table of Materials) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Take 250 &#181;L of FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and mechanically harvest the cells off the wells.</li>
	<li>Transfer the cells into the corresponding (previously marked) FACS tube with the filter cap. Place the tube with the cells on ice in the dark until the analysis is performed (analysis should be conducted within an hour after staining). 
	NOTE: FACS machine setup and calibration depend on the machine used (see Table of Materials).</li>
	<li>Create a new experiment and give it a name (Ferroptosis in DAOY - lipid hydroperoxides).</li>
	<li>In the experiment design, choose the laser (488 nm) and the filter (FITC).</li>
	<li>In view data, select the proper cell size (&#62;12 &#181;m), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.</li>
	<li>Add a new plot - histogram, with FITC-A on the y-axis and logarithmic scale.</li>
	<li>Vortex the samples in the FACS tube, place them in the FACS machine and load the sample. Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.</li>
</ol>
5. Propidium iodide (PI) staining of the dead cells upon treatment
NOTE: The experiment design is exactly the same as for lipid hydroperoxide measurement (see step 1 and step 2).
<ol>
	<li>24 h post-seeding, take the dishes out of the incubator and place them in the laminar flow hood.</li>
	<li>Collect the media (with dead cells) in a 15 mL tube.</li>
	<li>Add 1 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then collect the PBS into the tube with the respective media.</li>
	<li>Add 200 &#181;L of trypsin (10x dilution) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Use the respective media + PBS to harvest the cells off the wells.</li>
	<li>Centrifuge the cell suspension at 180 x g for 10 min at room temperature. Remove the supernatant using an aspirator.</li>
	<li>Resuspend the cell pellet in 300 &#181;L of FACS buffer and place it on ice until analysis.</li>
	<li>Just before the analysis, add PI solution (see Table of Materials) to a final concentration of 2 &#181;g/mL.</li>
</ol>
6. Flow cytometry analysis of dead cells 24h post-treatment
NOTE: FACS machine settings and calibration are done as previously indicated (see step 4).
<ol>
	<li>Create a new experiment and give it a name (Ferroptosis in DAOY - cell death).</li>
	<li>In the experiment design, choose the laser (488 nm) and the filter (PE).</li>
	<li>In view data, select the proper cell size (&#62;12 &#181;m), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.</li>
	<li>Add a new plot - histogram, with PE-A on the y-axis and logarithmic scale.</li>
	<li>Vortex the samples in the FACS tube, place them in the FACS machine and load the sample.</li>
	<li>Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.</li>
</ol>
7. Flow cytometry analysis of lipid hydroperoxide content in the DAOY xCT-/- cells
NOTE: xCT-/- cells have been generated as previously described24.
<ol>
	<li>For this experiment, seed the cells as indicated in step 2, but instead of the treatment, supplement the media with 1 mM NAC overnight.</li>
	<li>The next day, maintain NAC in one group and remove it from the other.</li>
	<li>Perform the flow cytometry analysis of lipid hydroperoxide content in the exact same way as described in step 6.</li>
</ol>
8. Analysis of the flow cytometer results
<ol>
	<li>Open the .fcs document in the FlowJo software (see Table of Materials).</li>
	<li>Double click on the individual file to open all events (not only FCS singlets) recorded in the individual sample (SSC-A/FSC-A dot plot). Since the settings are such that the gating done on the machine is not preserved, it is necessary to make the gate once again and name the population (e.g., DAOY WT).</li>
	<li>Double click on the gated population to open another window with the histogram.</li>
	<li>Change the Y-axis to the fluorescent filter used (Comp-FITC/PE-A).</li>
	<li>Change the X-axis to modal (normalize each peak to its mode, i.e., to % of the maximum number of cells found in a particular bin).</li>
	<li>Copy the histogram into the layout editor. Repeat this for all the samples, and every time a new histogram is pasted in the layout editor, drag it over the previous one so that the histograms are overlaid (half-offset).</li>
</ol>

FACS Analysis of Lipid Hydroperoxides in Ferroptotic Medulloblastoma Cells

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We declare no conflict of interest for the study presented herewith.

The primary hallmark of ferroptotic cell death is the uncontrolled accumulation of lipid hydroperoxides in the plasma membrane. This oxidative damage may occur in an enzymatic or non-enzymatic manner, but in either case, the reaction is iron-dependent/catalyzed, which explains the name of this type of cell death. Lipid hydroperoxidation is often indirectly estimated by measuring the degradation products of lipid hydroperoxidation, such as 4-hydroxy-2,3-trans-nonenal (4-HNE) or malonaldehyde (MDA). These products, generat...

Ferroptosis is a newly contextualized, reactive oxygen species (ROS)-dependent type of cell death1. Besides ROS, iron plays a crucial role(s) in this type of cell death, hence the name2. The final and executive step of ferroptosis is the iron-catalyzed accumulation of oxidative damage of lipids in the plasma membrane that eventually leads to compromised membrane integrity and selective permeability, and, finally, cell death by bubbling. Lipid hydroperoxidation event is a naturally occurring phenomenon; however, its propagation throughout the cellular membrane is prevented by the antioxidant defense of the cell. The major player in this context is Se-protein glutathione peroxidase 4 (GPx4), which can approach the membrane and convert lipid hydroperoxides into their less toxic alcohol derivatives3. The reducing power for GPx4 is mainly, but not exclusively, provided by glutathione (GSH), a tripeptide composed of the non-essential amino acids: glycine, glutamate, and cysteine. The rate-limiting amino acid for the biosynthesis of GSH is cysteine4. Although cysteine is classified as a non-essential amino acid, its requirements can easily exceed its internal production in highly proliferative cells (such as cancer cells). It thus has been re-classified in the group of semi-essential amino acids. The necessary import of cysteine occurs mainly through the Xc- system, which allows the import of the oxidized (dominant) form of cysteine (aka cystine) at the expense of glutamate export5. The Xc- system is composed of a Na+-independent, Cl--dependent transport subunit, known as xCT, and a chaperon subunit, known as CD98. Until recently, the anti-ferroptotic properties of the xCT-GSH-GPx4 axis have been seen as unique and irreplicable6. However, in 2019, an alternative anti-ferroptotic pathway, consisting of ubiquinol (Coenzyme Q10) and its regenerative enzyme - ferroptosis suppressor protein 1 (FSP1), has been described7,8. Soon afterward, yet another lipid hydroperoxide detoxifying system involving GTP cyclohydrolase-1/tetrahydrobiopterin (GCH1/BH4) has been reported9. Nonetheless, the central role of the xCT-GSH-GPx4 axis in the prevention of ferroptosis seems not to be challenged.
Over the past decade, ferroptosis has been extensively studied in a variety of tumor types, showing great potential as an anti-cancer strategy (reviewed by Lei et al.10). Furthermore, it has been reported that cancer cells exhibiting high resistance to conventional chemotherapeutics and/or a propensity to metastasize are surprisingly sensitive to ferroptosis inducers, such as inhibitors of GPx411,12,13. However, in the context of brain tumors, the potential of ferroptotic inducers remains largely understudied. While this type of cell death has been closely associated with cerebral ischemia-reperfusion injury14and neurodegenerative diseases15, its potential in the context of brain tumors has mainly been limited to glioblastoma, the most common malignant craniocerebral tumor (reviewed by Zhuo et al.16). On the other hand, the sensitivity of medulloblastoma, the most common malignant pediatric brain tumor and a leading cause of childhood mortality, to ferroptosis inducers remains largely unexplored. To the best of our knowledge, there is scarce peer-reviewed literature linking ferroptosis and medulloblastoma. Nonetheless, some studies have revealed that iron plays a crucial role in the survival, proliferation, and tumorigenic potential of both medulloblastoma and glioblastoma cancer stem cells (CSCs)17,18, potentially rendering them more vulnerable to ferroptosis induction. This is particularly significant as medulloblastoma is notorious for its subpopulation of CSCs, or tumor-initiating/propagating cells, which appear to be largely responsible for tumor chemoresistance, dissemination, and relapse19.
Sensitivity to ferroptosis induction is typically investigated by measuring lipid hydroperoxide content/accumulation, which may or may not lead to cell death. The most commonly used ferroptosis inducers are (1) erastin, an inhibitor of the xCT transporter20,(2) RSL3, an inhibitor of the GPx4 enzyme2, and/or (3) iron donors, such as ferro-ammonium citrate (FAC)21. Lipid hydroperoxide content is assessed using the selective probe BODIPY 581/591 C1122, which has excitation and emission maxima at 581/591 nm in its reduced state. Upon interaction with and oxidation by lipid hydroperoxides, the probe shifts its excitation and emission maxima to 488/510 nm. Typically, a significant increase in lipid hydroperoxide content precedes ferroptotic cell death. Since ferroptosis is not a programmed cell death, there is no molecular signaling cascade leading to its execution. Therefore, the only way to confirm it is to monitor lipid hydroperoxide content and use specific inhibitors for this type of cell death, such as ferrostatin 123. Ferrostatin 1 is a lipophilic antioxidant that can penetrate the lipid compartment of the cell and detoxify lipid hydroperoxides, thereby preventing ferroptotic events.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BODIPY 581/591 C11</td>
			<td>Thermo Fisher</td>
			<td>D3861</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Beckman</td>
			<td>Coulter Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Erastin</td>
			<td>Sigma-Aldrich</td>
			<td>E7781-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferroamminium citrate</td>
			<td>Acros Organics</td>
			<td>211842500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrostatin-1</td>
			<td>Sigma-Aldrich</td>
			<td>SML0583-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovin serum (FBS)</td>
			<td>Dominique Dutcher</td>
			<td>500105N1N</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACS Melody</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher</td>
			<td>11599686</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>PlasmoTest Mycoplasma Detection Kit</td>
			<td>InvivoGen</td>
			<td>rep-pt1</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodide</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>RSL3</td>
			<td>Sigma-Aldrich</td>
			<td>SML2234-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA 10X - 100 mL</td>
			<td>Dominique Dutcher</td>
			<td>X0930-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

revealing the ferroptotic phenotype of medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named &#34;ferroptosis.&#34; The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic &#34;bubbling&#34; phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics 

Revealing the Ferroptotic Phenotype of Medulloblastoma

The ultimate goal of our research is to develop new therapeutics for pediatric cancers such as myoblastoma. For this purpose, our team develops new model systems highlighting the genetic background of the disease, as well as exploiting new therapeutic avenues. In the recent years, single cell genomics have revealed how impaired lineage commitment influences clinical outcome of pediatric cancers, as well as response to the treatments, and ultimately the development of treatment resistant cell populations within the tumor.The spectrum of neural progenitor differentiated state appears to underlie the clinical outcome of pediatric cancer. Main challenges are to understand these highly plastic known or insufficiently progenitors within the tumors, as well as developing new therapeutic that are more efficient and less harmful for these diseases. From this standpoint, our research on ferroptosis appears extremely promising.Iron dependence is one of the main vulnerabilities of cancer cells, also known as cancer stem cells, all the essential element. Iron is double-edged sword as its reaction with reactive oxygen species can lead to the specific type of cell death, which is called ferroptosis. Our data clearly showed that ferroptosis is a great potential against this type of cancer cells.

The medulloblastoma cell line DAOY was cultured in a standard DMEM medium supplemented with 8% FBS until it reached approximately 60% confluency. On the day of the experiment, cells were harvested, and 1,00,000 cells per well were plated in 6-well plates, according to Table 1. The following day, cells (in triplicate) were treated with either 1 &#181;M of erastin, 0.3 &#181;M of RSL3, or 250 &#181;M of FAC. The plates were then placed in the incubator at 37 &#176;C and 5% CO2. After 6 h, cells ...

Read this Scientific Article Protocol about Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics at JoVE.com

Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and ...

revealing-the-ferroptotic-phenotype-of-medulloblastoma

Research

JoVE Journal

Cancer Research

Staining of Ferroptosis-Induced Medulloblastoma Cells for Flow Cytometric Detection of Lipid Hydroperoxides

To begin, seed and culture wild type medulloblastoma cell lines in a six well plate. After 24 hours, add the appropriate ferroptosis inducing agent to the cells and incubate the plate at 37 degrees Celsius with 5%carbon dioxide. After six hours of treatment, observe the cells under a light microscope for cell rounding.Using an aspirator, remove the media and floating cells from the plate under a Laminar airflow. To wash the cells, add two milliliters of PBS, swirl gently and aspirate the PBS out. Next, add two milliliters of the probe working solution and incubate the plate in the dark.Light, microscopic images of the DAOY wild type cells treated with one micromolar of Erastin showed round bubbling cells indicating a ferroptotic phenotype.

FACS Analysis of Lipid Hydroperoxides in Ferroptosis-Induced Medulloblastoma Cells

To begin, incubate ferroptosis-induced medulloblastoma cells with body PC-11 and remove the staining solution under a laminar hood. After washing the cells with two milliliters of PBS, incubate the cells with 150 microliters of a cell dissociation reagent. To mechanically harvest the cells, add 250 microliters of flow cytometry buffer, and tap the plate gently.Then, transfer the cells into a labeled flow cytometry tube with a filter cap. Next, turn on the FACS machine. Create a new experiment and choose the FITC filter and 488 nanometer laser.In View Data, select the cell size greater than 12 micrometers and gate the dot plot. Then add a new plot histogram with FITC-A on the axis and logarithmic scale. Then, vortex the sample taken in the tube and load it into the FACS machine.Finally, record 10, 000 forward scatter singlet events. To stain the dead cells, add propidium iodide solution just before the FACS analysis. Flow cytometry analysis of lipid hydroperoxidation, verified ferroptosis-induction in medulloblastoma cells treated with the erastin.

768 Views.  Centre Scientifique de Monaco (CSM).  Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

FACS Analysis of Lipid Hydroperoxides in Ferroptotic Medulloblastoma Cells

Summary