Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumour in humans. The 5-year survival rate of patients is <10%. GBM has a very high rate of recurrence after treatment, which is thought to involve a subpopulation of cells in the tumour, called cancer stem cells (CSCs). Apart from the self-renewing and multi-lineage differentiation characteristics of CSCs, they have been implicated in resistance to both chemotherapy and radiation. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) induces apoptosis in tumour cells, without causing harm to healthy cells. TRAIL can bind to five different receptors; two death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), and three decoy receptors, DcR1, DcR2 and OPG. To overcome this promiscuous behaviour, TRAIL variants have been designed that have a higher selectivity to either DR4 or DR5. In this study we investigated the sensitivity of various glioblastoma (stem) cell lines for TRAIL using the TRAIL variants alone and in combination with the proteasome inhibitor bortezomib. Recombinant human (rh)TRAIL and the generated TRAIL variants, two DR4-specific (4C7 and 4C9) and one DR5-specific (D269H/E195R) variants, were produced and purified. A panel of GBM cells was used: GSC-23 (with CSC characteristics, provided by H. Colman (Houston, Tx), U87, A172 and normal human astrocytes. First, the basal level of DR4, DR5, DcR1 and DcR2 expression was determined using flow cytometry. All cell lines displayed high DR5 and DcR2 expression, whereas DR4 and DcR1 levels were not detectable. Cell viability assays using resazurin conversion indicated that GSC-23 cells when maintained as neurospheres are resistant to TRAIL and variants, also when combined with bortezomib. TRAIL treated monolayer A172 and U87 cells were stained with AnnexinV and analysed by flow cytometry. A172 cells were sensitive for TRAIL-induced apoptosis, whereas U87 are resistant. Combined treatment with bortezomib effectively sensitized both cell lines for TRAIL-induced apoptosis. Next, a retroviral vector was generated containing the DR4 encoding sequence to examine the effect of enforced DR4 expression in GBM cells. GSC-23-DR4 neurospheres remained resistant to TRAIL and the variants, however, when combined with bortezomib sensitization towards both 4C7, 4C9 and D269H/195ER was seen. In conclusion, our preliminary results show predominantly DR5 expression in GBM suggesting benefit from DR5 selective TRAIL variants. Combined treatment with bortezomib sensitized for TRAIL-induced apoptosis in GSC-23-DR4 neurospheres, whereas GSC-23 cells remained resistant. In ongoing experiments the apoptosis inducing properties of TRAIL and TRAIL variants in GBM stem cells and differentiated derivatives is further examined. Mechanisms of resistance in relation to the effect of bortezomib are further explored to achieve optimal TRAIL-induced apoptosis in GBM (stem) cells.