Multi-parametric phospho-flow cytometry in T-call Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells at defined stages of intrathymic T-cell differentiation accounting for 15% of ALL cases in children and 25% in adults. The molecular events occurring during 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, overturning the control of cell proliferation, blocking differentiation, and promoting resistance to apoptosis. To define the features of malignant cells, it is challenging to gain an integrated, overall picture of changes of the entire cells signaling network profiles. We used multi-parametric phospho-flow cytometry to simultaneously determine protein expression and protein post-translational modifications (i.e. phosphorylation) at a single cell level in T-ALL cell lines at different differentiation sages. We analyzed signaling pathways that are crucial for the survival and proliferation of T-ALL cells, i.e. Notch1, phosphatidylinositol-3OH-kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPKs) and Janus kinases/signal transducer activator of transcription Jak/STAT. We measured protein expression and signaling properties in baseline and modulated conditions, using biologically relevant modulators for T-ALL, i.e. JAG1, CXCL12, IL-7. This study showed that signaling network maps obtained by multi-parametric phospho-flow cytometry enables to distinguish different T-ALL maturation stages. Further, combinations of different pathway information may identify biologically relevant signaling hubs. This study forms the basis for future studies testing the clinical validity of multi-parametric phospho-flow cytometry assay for T-ALL disease characterization.