Abstract: Glioblastoma multiforme (GBM) is the most prevalent and lethal primary brain tumor in adults. Despite current therapies, the prognosis of GBM patients remains dismal mainly due to tumor relapse. A subpopulation of cancer cells exhibiting stem cell- and epithelial-mesenchymal-transition (EMT)-like features is putatively responsible for tumor replenishment. Although the Wnt pathway is instrumental for induction of these traits in GBM, the molecular mechanisms are unknown. Single cell transcriptomic and proteomic analysis of specifically located, Wnt-active GBM-cells within the tumor will be performed in collaboration with Z4 (Krijgsveld). The spatio-temporal distribution of Wnt activity will be characterized in collaboration with A. Aulehla (B2). In parallel, we will address the crosstalk of Wnt signaling with other signaling pathways and its impact on stemness and EMT. For example, we could show that the CD95 receptor triggers stem- and EMT-phenotypes and CD95 also influences Wnt and YAP/TAZ activity in GBM stem cells. Reciprocal modulation of Wnt, CD95 and YAP/TAZ activity will therefore be analyzed using corresponding reporter constructs and subcellular localization of signaling components will be analyzed using advanced fluorescence microscopy (Z2). The final induction of stem- and EMT-like phenotypes will be assessed by gain-of-function (GOF) and loss-of function (LOF) experiments in vitro as well as in vivo in preclinical mouse models. We hypothesize that the crosstalk of these three systems to induce malignant transformation and/or to maintain tumorigenesis can be a general mechanism in different types of cancer. We will use single-cell transcriptomics to analyze how CD95-Wnt-YAP/TAZ or other signaling networks contribute to the acquisition of EMT/stem-like features. Thus, we will greatly advance our understanding of the Wnt signaling pathway in tumorigenesis and open new opportunities for the treatment of cancer.