Abstract BACKGROUND The plasticity of tumoral cells and the presence of cells displaying stem-like features are two interrelated traits of Glioblastoma (GBM) lesions and both concur in defining its heterogeneity. Particularly, GBM stem-like cells (GSC) can be classified according to trascriptional data in different subgroups, being the Proneural (PN-GSC) and the Mesenchymal (MES-GSC) the most consolidated clusters. GSC are responsible of most of the malignant characteristics of GBM, including therapeutic resistance and tumor recurrence. Therefore, a better understanding of the mechanisms regulating GSC responsiveness to therapy taking into account GSC molecular heterogeneity may help to improve patient’s outcome. Integrin a6 is a commonly used marker for GSC capable to enrich for GSC population and sustain stemness. We investigate the role of integrin a6 in both PN and MES GSC on stemness and radioresistance. MATERIAL AND METHODS The expression of integrin a6 was analyzed in GSC cultures obtained from post-surgical specimens either displaying PN or MES trascriptional traits. Using cell sorting to enrich for integrin a6 expression (integrin a6-high and a6-low) and gene silencing with lentiviral-based shRNA, integrin a6 impact on both GSC cultures was tested. Also, silenced MES-GSC were analysed by means of RNA-seq. The major pathways found altered by integrin a6 silencing were validated at functional level using gliomasphere-based clonogenic assay, extreme limiting dilution assay and gamma-H2AX to monitor DNA damage repair kinetics. RESULTS After sorting GSC cultures by integrin a6 expression, PN-GSC a6-low showed a significant reduction in clonogenic capability and gliomasphere size when compared to a6-high (p<0.0001). On the contrary, sorted MES-GSC did not display any differences. Similar results were obtained following integrin a6 lentiviral silencing. However, RNAseq on silenced MES-GSC revealed a significant impact on cell cycle regulation and DNA damage repair pathways. Indeed, Integrin a6 trascriptional inhibition in MES-GSCs impaired the capacity to clear gamma-H2AX foci after ionizing radiation (p<0.001) and significantly alters MES-GSC capacity to recover from radiation treatment in gliomasphere formation assay (p<0.01). According to the interpretation of the curves with the linear quadratic model, integrin a6 silenced cells displayed higher alpha- and beta- values and lower alpha/beta ratio. The obtained values demonstrate increased radiosensitivity and impaired capacity to repair sublethal DNA damage, in addition to an enhanced sensitivity to fractionated doses. CONCLUSION The data obtained showed that Integrin a6 regulates proliferation and stemness-related features in PN-GSC while supports radioresistance of MES-GSCs. Altogether, we reveal that integrin a6 controls different stem-associated features in GSCs depending on the molecular subtype.