Revealing the Ferroptotic Phenotype of Medulloblastoma

Summary

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.

Abstract

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 "ferroptosis." 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 "bubbling" 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.

Introduction

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 described^7,8. 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 GPx4^11,12,13. 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.

Protocol

The present study was conducted using DAOY wild-type (WT) medulloblastoma cell lines, which were cultured at 37 °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 10^th 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⁺.

1. 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.
2. 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.
3. 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.
4. Transfer the cell suspension into a 15 mL tube.
5. Take 500 µL 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).
6. Calculate the volume necessary to take from the cell suspension to have 1,00,000 cells.
7. 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.

2. Treatment of the cells

NOTE: The control and treatments are conducted in triplicate. The groups are as follows: Control (DMSO), 1 µM of erastin, 0.3 µM of RSL3, 250 µM of FAC, 2 µM of Ferrostatin 1, 1 µM of erastin + 2 µM of Ferrostatin 1, 0.3 of µM RSL3 + 2 µM of Ferrostatin 1, 250 µM of FAC + 2 µM 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.

1. 24 h post-seeding, add the corresponding treatment to the well with the cells.
2. Leave the dishes at 37 °C and 5% CO₂ in the incubator.

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 °C in non-transparent tubes. For staining, prepare a 2 µM working solution of the probe in DMEM media supplemented with 8% FBS.

1. 6 h post-treatment, observe the cells under a light microscope (cell rounding should be visible).
2. 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.
3. 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.
4. Add 2 mL of the working solution of the probe (2 µM final concentration in DMEM supplemented with FBS).
5. Leave the dish in the incubator at 37 °C and 5% CO₂ for 30 min in the dark (the plates can be wrapped in aluminum foil).

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).

1. Take the dishes out of the incubator and place them in a tissue culture laminar flow hood.
   Using an aspirator, remove the staining solution.
2. 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.
3. Repeat the washing step one more time and aspirate any remaining PBS from the well using an aspirator.
4. Add 150 µL 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 µL of FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and mechanically harvest the cells off the wells.
5. 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).
6. Create a new experiment and give it a name (Ferroptosis in DAOY - lipid hydroperoxides).
7. In the experiment design, choose the laser (488 nm) and the filter (FITC).
8. In view data, select the proper cell size (>12 µm), 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.
9. Add a new plot - histogram, with FITC-A on the y-axis and logarithmic scale.
10. 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.

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).

1. 24 h post-seeding, take the dishes out of the incubator and place them in the laminar flow hood.
2. Collect the media (with dead cells) in a 15 mL tube.
3. 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.
4. Add 200 µL 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.
5. Centrifuge the cell suspension at 180 x g for 10 min at room temperature. Remove the supernatant using an aspirator.
6. Resuspend the cell pellet in 300 µL of FACS buffer and place it on ice until analysis.
7. Just before the analysis, add PI solution (see Table of Materials) to a final concentration of 2 µg/mL.

6. Flow cytometry analysis of dead cells 24h post-treatment

NOTE: FACS machine settings and calibration are done as previously indicated (see step 4).

1. Create a new experiment and give it a name (Ferroptosis in DAOY - cell death).
2. In the experiment design, choose the laser (488 nm) and the filter (PE).
3. In view data, select the proper cell size (>12 µm), 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.
4. Add a new plot - histogram, with PE-A on the y-axis and logarithmic scale.
5. Vortex the samples in the FACS tube, place them in the FACS machine and load the sample.
6. Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.

7. Flow cytometry analysis of lipid hydroperoxide content in the DAOY xCT-/- cells

NOTE: xCT^-/- cells have been generated as previously described²⁴.

1. For this experiment, seed the cells as indicated in step 2, but instead of the treatment, supplement the media with 1 mM NAC overnight.
2. The next day, maintain NAC in one group and remove it from the other.
3. Perform the flow cytometry analysis of lipid hydroperoxide content in the exact same way as described in step 6.

8. Analysis of the flow cytometer results

1. Open the .fcs document in the FlowJo software (see Table of Materials).
2. 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).
3. Double click on the gated population to open another window with the histogram.
4. Change the Y-axis to the fluorescent filter used (Comp-FITC/PE-A).
5. 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).
6. 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).

Results

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 µM of erastin, 0.3 µM of RSL3, or 250 µM of FAC. The plates were then placed in the incubator at 37 °C and 5% CO₂. After 6 h, cells ...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
BODIPY 581/591 C11	Thermo Fisher	D3861
Cell counter	Beckman	Coulter Z1
DMEM medium	Gibco	10569010
Erastin	Sigma-Aldrich	E7781-5MG
Ferroamminium citrate	Acros Organics	211842500
Ferrostatin-1	Sigma-Aldrich	SML0583-25MG
Fetal bovin serum (FBS)	Dominique Dutcher	500105N1N
Flow Cytometer	BD Biosciences	FACS Melody
Gibco StemPro Accutase Cell Dissociation Reagent	Thermo Fisher	11599686
N-acetylcysteine	Sigma-Aldrich	A7250
PlasmoTest Mycoplasma Detection Kit	InvivoGen	rep-pt1
propidium iodide	Invitrogen	P3566
RSL3	Sigma-Aldrich	SML2234-25MG
Trypsin - EDTA 10X - 100 mL	Dominique Dutcher	X0930-100