3D Tumor Spheroid Cytotoxicity Assay

The procedure was adapted from previous work [28 (link)]. Cells (i.e., U87MG, U118MG, U138MG, and GL261) were plated in 96-well CellCarrier Spheroid ULA Microplates™ (Perkin Elmer^®) at 2000 cells/well in culture medium, including 3–4 replicates per experimental condition. Spontaneously formed tumor spheroids appeared after 48 to 74 h of growth. Then, spheroids were treated with TMZ or Fingolimod in culture medium containing fluorescent DNA intercalating agent, which detects dead cells that are stained when their plasma membrane is compromised (Sytox™ green Dead Cell Stain, reference S7020, ThermoFisher Scientific™). Confluence and the positive fluorescence area were monitored by image-based analysis using the Incucyte™ system (Sartorius^®, Goettingen, Germany). One image every 4 h was acquired. Spheroid growth was determined by analyzing the total spheroid surface (in pixel²). All data were normalized to the control condition per experiment. Cytotoxicity was determined as the fluorescent positive area in pixel² among the spheroid area. Cytotoxicity was defined as a percentage (%). Two to three independent experiments were performed per test substance. All experiments were performed with 3 to 4 technical replicates.