Ad-Delo3-RGD efficiently kills brain CSC lines under normoxic and hypoxic conditions. In a clonogenic dilution assay, adenovirally infected CSC were diluted in 1:10 steps in stem cell-permissive DMEM-F12 medium and incubated for 4–6 weeks until the medium turned yellow due to metabolic acidification. Control: untreated; TMZ: temozolomide [100 μM]; AdE1-minus: replication-deficient adenovirus dl703; Ad-WT: wild type adenovirus; Ad-Delo3-RGD: YB-1 dependent oncolytic adenovirus. Red color indicates no metabolic activity due to low cell survival whereas yellow color indicates high metabolic activity due to treatment failure, indicating cell survival. A representative experiment is shown.