Quantitative single cell analysis uncovers the life/death decision in CD95 network

CD95/Fas/APO-1 is a member of the death receptor family that triggers apoptotic and anti-apoptotic responses in particular, NF-κB. These responses are characterized by a strong heterogeneity within a population of cells. To determine how the cell decides between life and death we developed a computational model supported by imaging flow cytometry analysis of CD95 signaling. Here we show that CD95 stimulation leads to the induction of caspase and NF-κB pathways simultaneously in one cell. The related life/death decision strictly depends on cell-to-cell variability in the formation of the death-inducing complex (DISC) on one side (extrinsic noise) vs. stochastic gene expression of the NF-κB pathway on the other side (intrinsic noise). Moreover, our analysis has uncovered that the stochasticity in apoptosis and NF-kB pathways leads not only to survival or death of a cell, but also causes a third type of response to CD95 stimulation that we termed ambivalent response. Cells in the ambivalent state can undergo cell death or survive which was subsequently validated by experiments. Taken together, we have uncovered how these two competing pathways control the fate of a cell, which in turn plays an important role for development of anti-cancer therapies.Author summary: Activation of death receptor (DR) family has been reported to activate both apoptotic as well as anti-apoptotic responses. Molecular mechanisms underlying the intricate details of this crosstalk have not been established yet. Here we show that these pathways are triggered simultaneously in one cell. Furthermore, using stochastic computational modeling we uncovered how an individual cell undergoes apoptosis, while other cells survive upon the same DR activation conditions. This was only possible by combination of computational modeling supported by experimental validation based on the state of the art single cell analysis. The latter included cutting edge technology of imaging flow cytometry, which combines microscopy and flow cytometry in one measurement circuit enabling quantitative analysis of endogenous cellular protein levels estimated from a large number of cells simultaneously. This allowed to shed the light on the question how a single cell possibly avoids apoptosis, which is a highly actual topic in the field of cancer research and development of efficient anti-cancer therapies.