Uncovering the signaling networks of disseminated glioblastoma cells in vivo with INSIGHT

Dysregulation of intracellular signaling networks underpins cancer. However, a systems-level elucidation of how signaling networks within distinct cell subpopulations drive cancer progression in vivo has been unattainable due to technical limitations. We developed INSIGHT (INvestigating SIGnaling network of specific cell subpopulation in Heterogeneous Tissue), a new platform technology combining fluorescence-activated cell sorting with ultra-sensitive mass spectrometry to enable phosphoproteomic characterization of rare and discrete cell subpopulations from fixed tissues. We demonstrated the broad utility of INSIGHT by analyzing the oligodendroglial cell-specific signaling network in the mouse brain. We then applied INSIGHT to investigate the rare, disseminated tumor cell subpopulation in glioblastoma patient-derived xenograft models. INSIGHT uncovered a global rewiring of signaling networks with tumor cell dissemination, marked by a transition from proliferation-associated signaling in the primary tumor cells to signaling associated with postsynapse, neuronal migration, and ion homeostasis in disseminated tumor cells. We reveal interconnections between signaling circuitries within the networks, with numerous proteins, including GluA2, exhibiting altered phosphorylation without protein expression changes, emphasizing the role of post-translational modifications in glioblastoma dissemination. We validated key phosphorylation changes and inferred differentially active kinases with tumor spread to offer new systems-level insights into glioblastoma dissemination mechanisms in vivo. INSIGHT is generally applicable to a wide range of biological systems without genetic engineering and provides quantitative phosphorylation and protein expression data for selected cell subpopulations from heterogeneous tissues.