Figure 4.

Phosphoprotein signaling networks from multiplex, quantitative single-cell proteomics. All data represented are uniquely measured at the single-cell level. (a) A Monte-Carlo simulation of fluctuations that represent the copy numbers per cell of an activated (such as phosphorylated) form of a protein, as that protein is involved in increasing numbers of regulatory processes. On the right are the experimentally measured fluctuations of HIF-1α from model GBM cancer cells as these cells are exposed to different O₂ partial pressures. The increasingly important role of HIF-1α under hypoxic conditions is evident. Reproduced from [45]. (b) Scatter plot showing protein-protein correlations for two phosphoproteins. The black and red dots represent measurements from 0-cell and 1-cell SCBC microchambers, respectively. Reproduced from [28]. (c) A protein-protein correlation network for model GBM cancer cells following epidermal growth factor (EGF) stimulation (top), and following EGF stimulation + erlotinib (anti-EGF receptor) inhibition (bottom). The weight of the network edges reflects the correlation strength, and a red edge indicates an anti-correlation. Reproduced from [27]. (d) Collective signaling modes, as determined by the eigenvectors of the single-cell protein-protein covariance matrix. Shown are the eigenvectors associated with mTORC1 signaling in model GBM cells, as pO₂ is varied. The composition of the green, red, and blue eigenvectors (top plot) is given in the pie charts below for each value of pO₂ investigated. The amplitude of the mTORC1 associated eigenvectors shows a minimum between 1.5% and 2% pO₂, indicating the loss (and undruggability) of that signaling within this narrow window of pO₂ values. Note that HIF-1α is strongly associated with mTORC1 signaling above 2% pO₂, but not below 2% pO₂, indicating a switch in the structure of the signaling network. The cells studied were model GBM cell lines containing the EDFR variant III (vIII) oncogene (U87 EGFRvIII; panels a, b, d) or the EGRFvIII oncogene plus loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene (EGFRvIII PTEN). Reproduced from [45] with permission.