Targeting cancer stem cells (CSC), rather than cancer cells in general, is a novel and highly promising strategy for cancer treatment. The defining characteristics of CSC are self-renewal, propagation into heterogeneous lineages of cancer cells, and increased resistance to chemotherapy, thus, attacking the CSC should abolish a tumor's ability to recur or metastasize. Recently, a natural potassium ionophore salinomycin was identified as one of a few available CSC-selective substances, but the precise mechanism of its selectivity remains elusive. Based on hypothesis that the potassium transport is an important process of CSC biology, the objective of the proposed research is to understand fundamental processes of CSC-resistance to therapy and to discover novel compounds with ion homeostasis-modulating properties to selectively target CSC. Project methodology orchestrates research in three principle areas and their close interactions, covering synthesis and characterization of molecules (chemistry), assessment of their activity in biological models (biology, medicine) and computer-aided drug design (computational sciences). We’ll focus on salinomycin and a series of proprietary crown ether compounds, which will be optimized and developed. Their efficacy/selectivity and influence on drug transporters P-gp- and/or ABCG2-mediated efflux of chemoterapeutics will be assessed, as well as the toxicity and in vivo activity of the most promising compound. Deploying cutting-edge techniques in cell and molecular biology, bioimaging, molecular electrophysiology and transgenic cell lines (e.g. a unique CSC model - HMLEshEcad cells) we will strive to recognize novel biomarkers for identification and enrichment of CSC, related to abnormal potassium transport regulation that promote acquisition of CSC phenotype and/or influence their proliferation, death and migratory potential. The ultimate aim is to translate this knowledge into innovative mechanism-based therapeutic approaches.