Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark 3Dept. of Oncology, Odense University Hospital, Odense, Denmark Cancer stem cells are thought to be responsible for tumorigenic potential and to possess resistance mechanisms against chemotherapy- and radiation-induced cancer cell death, while the bulk of a tumor lacks these capacities. The resistance mechanisms may cause these cells to survive and become the source of later tumor recurrence, highlighting the need for therapeutic strategies that specifically target pathways central to these cancer stem cells. The CD44hi/CD24-/low compartment of human breast cancer is enriched in tumor-initiating cells; however the functional heterogeneity within this subpopulation remains poorly defined. From a triple-negative breast cancer cell line we isolated and cloned CD44hi single-cells that exhibited functional heterogeneity according to cancer stem cell features, such as tumor formation in immunodeficient mice, mammosphere formation, invasion and migration abilities and sensitivity to chemotherapeutics. Quantitative proteomic analysis of these clones revealed several proteins, including kinases, exhibiting altered expression levels while other proteins exhibited altered phosphorylation levels. The quantitative proteomic approach was based on post digestion dimethyl labeling followed by TiO2 enrichment of phosphopeptides. Moreover, analysis of the flowthrough fraction revealed quantitative changes in the total proteome. Transcriptomic analysis using Affymetrix Human Gene 1.0 ST Arrays revealed similar alterations at the gene expression level. We used these single cell clones to further investigate the molecular mechanisms underlying the association between selected proteins, some of them phosphorylated, and cancer stem cell features. Furthermore, phosphosite-expression was investigated with NetworKIN that predicts in vivo kinase-substrate relationships. Our studies reveal pathways that are regulated by protein phosphorylation and include relevant kinase targets for drug discovery. Our results support functional heterogeneity in the breast cancer cell compartment and hold promise for further refinements of prognostic marker profiling of cells with cancer stem cell-like features.