Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -9, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.