The molecular events occurring during T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis determine intrinsic defects of leukemic cells as well as altered interpretation of external cues deriving from the microenvironment. Both cell-intrinsic defects and microenvironmental stimuli converge on the activation of regulatory signaling pathways involved in processes enhancing the capacity of self-renewal, overturn- ing the control of cell proliferation, blocking differentiation, and promot- ing resistance to apoptosis. The identification of specific oncogenic sig- naling network profiles involved in the pathogenesis of T-ALL is chal- lenging to gain an integrated, overall features of malignant cells. We used multi-parametric phospho-flow cytometry to simultaneously determine protein expression and protein post-translational modifica- tions (i.e. phosphorylation) at a single cell level in T-ALL cell lines at dif- ferent differentiation stages. We analyzed signaling pathways that are crucial for the survival and proliferation of T-ALL cells, i.e. Notch1, PI3K/Akt, MAPKs and Jak/STAT. Protein expression and signaling prop- erties were measured in baseline and modulated conditions, using bio- logically relevant modulators for T-ALL, i.e. JAG1, CXCL12, IL-7. In unmodulated condition, signaling/expression profiles varied among the cell lines, ranging from a clear positive fluorescence in at least one of the protein measured to no change with respect to isotype. Modulation with physiologic stimuli evoked signaling profiles different from those measured in basal condition. Moreover, profiles are heterogeneous across treatments and cell lines. Pearson’s correlation between nodes (a node is defined as a 3-dimensional vector where each dimension repre- sents a single response to treatment) showed an overall high correlation between signaling/expression statuses following different treatments for each cell lines. Graph diagrams obtained using highly correlated nodes showed topologically different network across cell lines. This study showed that multi-parametric phospho-flow cytometry enables to distinguish signaling network maps that are associated with the T- ALL ontogeny stages. Combinations of different pathway information may identify biologically relevant signaling hubs, thus forming the basis or future studies testing the clinical validity of multi-parametric phos- pho-flow cytometry assay in primary T-ALL cells.
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