LAUSR

CD95 (Fas/APO-1) holds a dual role of potential relevance in tumor development. CD95-CD95 ligand (CD95L) signaling regulates apoptotic cell death in CD95-expressing cells, but non-apoptotic, tumor-promoting CD95-CD95L signaling has been likewise described. Therapeutic stimulation of apoptotic CD95 signaling is associated with major clinical side effects. However, inhibition of tumor-promoting CD95 signaling may represent a promising treatment strategy for human cancers where potential tumor-promoting CD95 functions include invasiveness and cancer cell stemness, including glioblastoma. In this study, CD95 and CD95L expression was characterized in human glioma-initiating cells (GIC) in vitro and in vivo. CD95 and CD95L gene knockout (KO) GIC were generated by means of CRISPR-Cas9 and the effects of gene silencing were evaluated by assessing growth, clonogenicity, invasiveness and tumorigenicity in nude mice. CD95 expression and sensitivity to exogenous CD95L-induced apoptosis were confirmed in selected GIC in vitro. CD95L expression was not detected. Upon CD95 KO, all GIC acquired resistance to CD95L-induced apoptosis. Furthermore, despite the confirmed absence of CD95L expression in vitro, CD95 KO S-24 GIC revealed decreased cell growth, inferior sphere forming capacity and decreased invasiveness. These data suggested a CD95L-independent tumor-promoting role of CD95 in S-24 GIC. In vivo, however, CD95 KO did not prolong the survival of glioma-bearing mice. Analyses of further GIC models are ongoing. These data demonstrate that, unlike CD95, CD95L is not expressed in cultured human GIC and that CD95-CD95L interactions are not required for tumor-promoting CD95 signaling. Although CD95 KO is detrimental for S-24 GIC in vitro, CD95 KO alone does not affect survival in S-24 human GIC xenograft-bearing mice.