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, generated by the peroxidative decomposition of polyunsaturated fatty acids, can react with various macromolecules, leading to a wide variety of pathologies, such as neurodegenerative disorders, diabetes, and chronic heart failure, among others. Nonetheless, these are the consequences of the peroxidation event, and significant effort has been invested in developing good lipid hydroperoxide-specific probes.
One frequently used probe is BODIPY 581/591 C11, which also has its disadvantages that should be taken into consideration. Specifically, BODIPY 591/581 fluorescence color exhibits two emission peaks, one in its reduced state (590 nm) and one in its oxidized state (510 nm). The ratio between fluorescence at these two wavelengths is of particular importance if precise quantification is the aim. However, the intensity of single-emission fluorescence of BODIPY 591/581 nm has been widely used throughout the literature1,25,26,27,28, especially as the reduced intensity of red emission at 510 nm is frequently masked by the spillover of the fluorescence of the oxidized molecule22,29. This is one of the major reasons why the fluorescence of BODIPY 591/581 is usually represented in a qualitative manner as a function of single (green) fluorescence intensity.
Another important limitation of this probe is its fluorescence emission suppression by antioxidants in a concentration-dependent manner30. Therefore, staining should be carried out in media without antioxidant supplementation. In the present protocol, ferrostatin 1, a powerful scavenger of lipid hydroperoxides, was used; nonetheless, before the probe was added, ferrostatin 1 was removed, and the cells were washed with PBS so that the probe was not exposed to its antioxidant activity.
Regarding ferroptosis induction, many commercially available drugs could be used. Here, erastin, an irreversible inhibitor of the xCT transporter31, was employed. Given that it binds covalently to the transporter, even higher concentrations could be used (up to 100 &#181;M) for a shorter period of time (5 min), followed by a washing step31. To evaluate the exact kinetics of ferroptosis upon complete inhibition of xCT, xCT-/- cells were utilized in the absence of NAC. A massive accumulation of lipid hydroperoxides was detectable after 6 h, and virtually no viable cells were detectable 24 h post-seeding. As for RSL3, a highly potent inducer of ferroptosis in the nanomolar range, it exhibited a kinetic similar to the one observed with xCT-/- cells. Its mode of action is still rather controversial, but it seems that it does not affect the activity but instead induces degradation of the GPx4 enzyme32,33.
An important aspect of ferroptosis induction in vitro is cell confluency. It has long been observed that a confluent cell layer is much more resistant to cysteine starvation, xCT or GPx4 inhibition, GSH depletion, etc.34. The main reason given is that physical contact, i.e., confluency, leads to the activation of the Hippo pathway, which, in turn, downregulates metabolism and internal production of reactive oxygen species (ROS). Furthermore, the shuttle of reduced cysteine could also contribute to resistance to incomplete xCT inhibition35. In the present study, a concentration of 1,00,000 cells per well (6-well plate) was used in order to maintain a low confluency with cells that are rather big and flat. However, with smaller and round-shaped cells, such as HD-MB03, this number could be increased to 1,50,000-2,00,000 cells per well (6-well plate).
The final limitation of the present study is the detection of cell death by the propidium iodide (PI) method. This method is user-friendly for detecting any type of cell death, and ferroptosis could be distinguished from other types of cell death by using ferroptosis-specific inhibitors, such as ferrostatin-1. PI staining is based on the fact that dying cells lose membrane integrity, allowing PI to penetrate the cells, intercalate between the DNA bases, and emit a fluorescent signal. However, it is worth noting that lipid hydroperoxidation induces membrane &#34;leakage,&#34; which allows penetration of PI even before cell death occurs. Thus, estimating the kinetics of ferroptosis based on this method alone is challenging.

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><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&amp;nbsp;</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.

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 were observed under the microscope to detect bubbling cells, as indicated by the arrows in Figure 1. This served as an indicator that the drug was effective in inducing ferroptosis before the cells were prepared for lipid hydroperoxide staining and subsequent FACS analysis.
For the detection of lipid hydroperoxide content, the specific dye BODIPY 581/591 C11 was used at a final concentration of 2 &#181;M for 30 min. As this dye is redox-sensitive, it was necessary to remove any treatment from the cells and wash them with pre-warmed PBS. After half an hour of staining, cells were washed twice with PBS, detached using the cell detachment solution, and prepared for FACS analysis. Data obtained at this step showed increased green fluorescence of the dye in the samples treated with all three drugs (Figure 2). However, the most potent effect was observed with 0.3 &#181;M of RSL3 (Figure 2B), while 1 &#181;M of erastin (Figure 2A) and 250 &#181;M of FAC (Figure 2C) showed a somewhat lesser effect after 6 h of treatment.
To establish that the detected lipid hydroperoxide accumulation leads to cell death, specifically ferroptosis, the same treatments outlined in Table 1 were applied for 24 h. For the analysis of dead cells by flow cytometry, both the cells and supernatant were harvested. The live and dead cells were pelleted by centrifugation and then resuspended in FACS buffer for subsequent flow cytometry analysis. The data presented in Figure 3 demonstrated that all three treatments induced massive cell death, which was completely prevented by using the ferroptosis-specific inhibitor, ferrostatin-1. This clearly supports the occurrence of ferroptotic cell death upon xCT inhibition, GPx4 inhibition, or iron overload in medulloblastoma cells.
Given that the three drugs showed somewhat different effects on lipid hydroperoxide accumulation, likely due to differences in potency, a genetic approach was employed to investigate the full potency of the ferroptotic inducers. Previously obtained DAOY xCT-/- cells (Supplementary Figure 1A) were cultured in the same media as their WT counterparts but with NAC supplementation. This positive selection allowed the xCT-/- cells to grow in culture. However, 6 h after NAC was removed from the culturing media, cells began to accumulate lipid hydroperoxides to the same level as WT cells treated with RSL3 (Figure 4). Similarly to pharmacological inhibition, the removal of NAC from the media induced the characteristic bubbling of xCT-/- cells after 6 h (Supplementary Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66645/66645fig01.jpg" /> 
Figure 1: Ferroptotic phenotype of medulloblastoma cells. Representative micrographs of DAOY WT cells treated (or not) for 6 h with 1 &#181;M of erastin (ERA), 0.3 &#181;M of RSL3 or 250 &#181;M of ferroammonium citrate (FAC) in the presence or not of 2 &#956;M ferroptosis inhibitor Ferrostatin-1 (Ferro-1). White arrows indicate round, &#34;bubbling&#34; cells, indicating a ferroptotic phenotype visible under a light microscope. Magnification: 20x, inserts: 40x. Scale bars: 50 &#181;m, insets: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66645/66645fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66645/66645fig02.jpg" /> 
Figure 2: Lipid hydroperoxide as the key marker of ferroptosis in medulloblastoma cells. Representative data of the lipid hydroperoxide staining by flow cytometry in the cells treated for 6 h with (A,B) 1 &#181;M of erastin (ERA), (C,D) 0.3 &#181;M of RSL3, or (E,F) 250 &#181;M of ferroammonium citrate (FAC), in the presence or not of 2 &#181;M ferroptosis inhibitor Ferrostatin-1 (Ferro-1). (A,C,E) Dot plots of the recorded events; (B,D,F) Histogram representation of the lipid hydroperoxide content. <a href="https://www.jove.com/files/ftp_upload/66645/66645fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66645/66645fig03.jpg" /> 
Figure 3: Ferroptotic cell death induction by pharmacological approach. Representative flow cytometry dot plots of propidium iodide-stained cells after 6 h of treatment with 1 &#181;M of erastin (ERA), 0.3 &#181;M of RSL3, or 250 &#181;M of ferroammonium citrate (FAC), in the presence or not of 2 &#181;M ferroptosis inhibitor Ferrostatin-1 (Ferro-1). Gating separates live (Q4) and dead (Q3) populations of the cells. <a href="https://www.jove.com/files/ftp_upload/66645/66645fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66645/66645fig04.jpg" /> 
Figure 4: Ferroptosis induction by genetic approach. Representative data of the lipid hydroperoxide staining by flow cytometry in the DAOY WT and xCT-/- cells after 6 h in the presence or not of 2 &#181;M ferroptosis inhibitor Ferrostatin-1 (Ferro-1). On the left are the histogram representation of the lipid hydroperoxide contents.&#160;On the right are the dot plots of the recorded events. <a href="https://www.jove.com/files/ftp_upload/66645/66645fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plate I&#160;</td>
			<td>CTL I</td>
			<td>CTL II&#160;</td>
			<td>CTL III</td>
		</tr>
		<tr>
			<td></td>
			<td>1 &#181;M erastin I</td>
			<td>1 &#181;M erastin II</td>
			<td>1 &#181;M erastin III</td>
		</tr>
		<tr>
			<td>Plate II&#160;</td>
			<td>0.3 &#181;M RSL3 I</td>
			<td>0.3 &#181;M RSL3 II</td>
			<td>0.3 &#181;M RSL3 III</td>
		</tr>
		<tr>
			<td></td>
			<td>250 &#181;M FAC I</td>
			<td>250 &#181;M FAC II</td>
			<td>250 &#181;M FAC III</td>
		</tr>
		<tr>
			<td>Plate III&#160;</td>
			<td>2 &#181;M Ferro-1 I</td>
			<td>2 &#181;M Ferro-1 II</td>
			<td>2 &#181;M Ferro-1 III</td>
		</tr>
		<tr>
			<td></td>
			<td>1 M erastin + 2 M Ferro-1 I</td>
			<td>1 M erastin + 2 M Ferro-1 II</td>
			<td>1 M erastin + 2 M Ferro-1 III</td>
		</tr>
		<tr>
			<td>Plate IV&#160;</td>
			<td>0.3 M RSL3 + 2 M Ferro-1 I</td>
			<td>0.3 M RSL3 + 2 M Ferro-1 II</td>
			<td>0.3 M RSL3 + 2 M Ferro-1 III</td>
		</tr>
		<tr>
			<td></td>
			<td>250 M FAC + 2 M Ferro-1&#160; I</td>
			<td>250 M FAC + 2 M Ferro-1 II</td>
			<td>250 M FAC + 2 M Ferro-1 III</td>
		</tr>
	</tbody>
</table>
Table 1: Treatment details of the control and experimental cells.
Supplementary Figure 1: Ferroptotic cell death of the DAOY xCT-/- cells. (A) Western blot analysis of the xCT level in DAOY WT and xCT-/- cells in DMEM media supplemented or not with 2 &#181;M of Ferrostatin-1. (B) Representative micrographs of the DAOY xCT-/- cells in the presence (left) and absence (right) of 1mM N-acetylcysteine for 6 h. White arrows indicate round, &#34;bubbling&#34; cells, indicating a ferroptotic phenotype visible under a light microscope. Magnification: 40x. Scale bars: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66645/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>

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

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

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.

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 ...

revealing-the-ferroptotic-phenotype-of-medulloblastoma

Nanyang Technological University

Research

JoVE Journal

Cancer Research

794 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.

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

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Neuroscience

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Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

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Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

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Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma

Pediatric small round blue cell tumors (PSRBCT) are an intriguing and challenging collection of neoplasms. Light microscopy of small round blue cell tumors identifies small round cells. They harbor a generally hyperchromatic nucleus and relatively scanty basophilic cytoplasm. Pediatric small round blue cell tumors include several entities. Usually, they incorporate Wilms tumor, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, retinoblastoma, lymphoma, and small cell osteosarcoma, among others. Even using immunohistochemistry, the differential diagnosis of these neoplasms may be controversial at light microscopy. A faint staining or an ambiguous background can deter pathologists from making the proper diagnostic decision. In addition, molecular biology may provide an overwhelming amount of data challenging to distinguish them, and some translocations may be seen in more than one category. Thus, transmission electron microscopy (TEM) can be extremely valuable. Here we emphasize the modern protocol for TEM data of the neuroblastoma. Tumor cells with tangles of cytoplasmic processes containing neurosecretory granules can diagnose neuroblastoma.

Pediatric small round blue cell tumors are an intriguing and challenging collection of neoplasms. Therefore, transmission electron microscopy (TEM) and professional knowledge of pediatric tumors can be extremely valuable in surgical pathology. Here, we present a protocol to perform TEM for diagnosing neuroblastoma, one of the most common solid tumors in childhood.

Pediatric small round blue cell tumors (PSRBCT) are an intriguing and challenging collection of neoplasms. Light microscopy of small round blue cell ...

Medicine

MOGHE and Epilepsy: Outcomes of Frontal Disconnection Surgery 

Frontal Disconnection for Treating Mild Malformation of Cortical Development with Oligodendroglial Hyperplasia in Epilepsy (MOGHE) in the Frontal Lobe

Malformation of cortical development is an important cause of drug-resistant epilepsy in young children. Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) has been added to the last focal cortical dysplasia (FCD) classification and commonly involves the frontal lobe. The semiology at the onset of epilepsy is dominated by non-lateralizing infantile spasm; the boundaries of the malformation are usually difficult to determine by magnetic resonance imaging (MRI) and positron emission tomography (PET), and electroencephalography (EEG) findings are often widespread. Therefore, the traditional concept and strategy of preoperative evaluation to determine the extent of the epileptogenic zone by comprehensive anatomo-electro-clinical methods are difficult to implement. 
Frontal disconnection is an effective surgical method for the treatment of epilepsy, but there are few related reports. A total of 8 children with histo-pathologically confirmed MOGHE were retrospectively studied. MOGHE was located in the frontal lobe in all patients, and frontal disconnection was performed. The periinsular approach was used in the disconnective procedures, divided into several surgical steps: the partial inferior frontal gyrus resection, the frontobasal and intrafrontal disconnection, and the anterior corpus callosotomy. 
One patient presented with a short-term postoperative speech disorder, while another patient exhibited transient postoperative limb weakness. No long-term postoperative complications were observed. At 2 years after surgery, 75% of patients were seizure-free, with cognitive improvement in half of them. This finding suggested that frontal disconnection is an effective and safe surgical procedure for the treatment of MOGHE instead of extensive resection in the frontal lobe.

Author Spotlight: Studying Clinical Characters and Epilepsy Outcomes After Frontal Disconnection in Patients with MOGHE

Here, we present a protocol to describe the epilepsy outcome and complications of 8 patients with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) in the frontal lobe after frontal disconnection. The procedure is characterized by its simplicity, user-friendliness, and fewer postoperative complications.

Malformation of cortical development is an important cause of drug-resistant epilepsy in young children. Mild malformation of cortical development with ...

Revealing the Ferroptotic Phenotype of Medulloblastoma

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A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Zebrafish Model of Neuroblastoma Metastasis

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma

Frontal Disconnection for Treating Mild Malformation of Cortical Development with Oligodendroglial Hyperplasia in Epilepsy (MOGHE) in the Frontal Lobe