Evaluating GBM Cell Invasiveness

A modified Boyden chamber technique (Greiner Bio-One, Kremsmünster, Austria) with Matrigel-coated membranes (Corning, Amsterdam, Netherlands) was used to examine the invasive behavior of GBM cells after treatment. Before seeding, cells were cultured for 24 h in a serum-free medium. After that, cells were seeded with serum-free medium supplemented with the cytostatic agents in the Matrigel-coated inserts (ThinCerts, 8 µm, Greiner Bio-One). To stimulate the cells to migrate through the membrane, the chamber below was filled with full medium. After 72 h, invading cells were quantified by WST-1 staining (1:10 in serum-free medium, Merck KGaA, Darmstadt, Germany). The analysis was done after 3 h incubation using the Tecan Reader at an excitation/emission of 480/650 nm. To document the invasiveness of tumor spheroids after treatment, 96-ULA well plates (Greiner Bio-One, Kremsmünster, Austria) were placed on ice after 4 days of sphere formation; half of the medium was removed, and cytostatic drugs were added including EGF (1%, ImmunoTools, Friesoythe, Germany) to stimulate the invasion into U-bottom wells containing ice-cold matrigel (Corning, Amsterdam, The Netherlands). The spheroids were monitored for 10 days and images were taken on days 0, 5, and 10 using the Leica microscope DMI 4000B (Leica, Heidelberg, Germany).