A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

Glioma stem Cells are sub-population of cells in the glioma, which are usually resistant to chemo and radiotherapy. Here we described the method for rapid identification of combination therapies, targeting glioma stem cells, instead of differentiated glioma cells. Compared with other methods, which may be a laborious and complicated.

This protocol is a relatively simple and rapid, to identify potential useful drug combinations. Begin by collecting the glioma stem cells or GSCs from the culture medium, and centrifuging them at 70 times G for three minutes at room temperature. After removing the supernatant, digest the cells with accutase for four minutes at 37 degrees Celsius.

Using a 200 microliter tip, pipette the cells repeatedly to dissociate, and resuspend the cell pellet. Add the GSCs at a density of 200, 000 cells in one milliliter of culture medium, into each well of a 12 well culture plate, and incubate them overnight. Oh the next day, add 30 microliters of luciferase-eGFP virus supernatant into each well of the plate.

Centrifuge the cells at 1000 times G for two hours, at 25 degrees Celsius and incubate them overnight. On the following day, replace the medium in the wells by collecting the GSCs, centrifuging them, removing the supernatant, resuspending them in fresh medium, and replating them. Culture the cells for another 48 hours.

Observe the plate under a fluorescent microscope and confirm the appearance of GFP-positive cells. Sort and collect the GSCs with a high fluorescence using a flow cell sorter and culture them further. Coat, 96 well optical bottom plates with an extracellular matrix mixture, such as matrigel, and incubate them for one hour at 37 degrees Celsius.

Remove the excess mixture and gently rinse the plates once with PBS, then seed XG387-Luc cells at a density of 1000 cells in 100 microliters of culture medium, and to each well of the coated plates and culture them overnight. On the following day, observe the cells under a microscope to confirm their attachment to the plate. Next prepare a 200 micromolar temozolomide solution and two micromolar solutions of the targeted agents, in culture medium.

For combination drug screening, remove the blank medium and add the medium containing either 200 micromolar temozolomide or two micromolar targeted agent, or a combination of both, into each well for three technical replicates per treatment. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three days. Take images of the cellular bioluminescence in the plate using the IVIS spectrum imaging system.

Using the built-in software, create multiple circular areas of the region of interest and quantify the cellular bioluminescence. To identify potential candidates that could enhance the antiglioblastoma effect of temozolomide, 20 target selective small molecule inhibitors were analyzed through drug combination screening. The sensitive index values of 13 targeted agents were above zero and five of them were above 0.1.

The sensitive index of the top two candidate drugs UMI-77 and A 83-01, were higher than 0.25, suggesting their potential to synergize with temozolomide. The combined treatment of temozolomide and UMI-77 was evaluated using an anti-proliferative assay in a six by six dose titration matrix. The combination index values were less than one.

And the high single agent values were greater than zero for most of the combinations of temozolomide and UMI-77 at different concentrations. Suggesting an overall synergistic interaction of temozolomide and UMI-77 in both XG387 and XG328 GSCs. This protocol can also be applied in finding drug combination using adherent cancer cells by simply removing the plate coating step.