Revealing the Ferroptotic Phenotype of Medulloblastoma

The ultimate goal of our research is to develop new therapeutics for pediatric cancers such as medulloblastoma. 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, none or insufficiently progenitors within the tumors, as well as developing new therapeutics 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.

To begin seed in 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 microscopy images of the DAOY wild type cells treated with one micromolar of erastin showed round bubbling cells, indicating a ferroptotic phenotype. 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 FITCA 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 erastin.