Epithelial–mesenchymal cell state heterogeneity predetermines differential phospho-signaling responses to EGF stimulation

Understanding why isogenic cancer cells respond differently to equivalent oncogenic stimuli is vital for optimizing anticancer therapies. Emerging evidence suggests that pre-existing differences in cell state may modulate signaling responses to new stimuli, but the interplay of specific cell states and signals remains unclear. We investigated whether epithelial-mesenchymal (E/M) state, a major axis of cancer cell heterogeneity, influences signaling responses to epidermal growth factor (EGF), a critical oncogenic stimulus in non-small cell lung cancer (NSCLC). We imaged >64,000 A549 NSCLC cells labeled for DNA, F-actin, and alternate signaling markers (p-AKT-S473, p-AKT-T308, p-ERK, or p-S6) after acute stimulation. Quantitative single-cell morphological and spatial profiling defined a stimulus-invariant "E/M state landscape" over which EGF signaling responses were compared. This revealed state-dependent differences in signal-activation magnitudes, dynamics, and subcellular routing. AKT responses exhibited phosphosite- and compartment-specific dynamics across states, with epithelial cells showing strong, transient membrane-localized S473 and higher internalized T308, whereas mesenchymal cells displayed weaker but sustained nuclear and ruffle-localized S473. Regression-based computational multiplexing concurrently inferred all signaling responses per cell, mapping state-dependent divergence in multimolecular signaling trajectories. E/M state thus predetermines distinctive spatiotemporal profiles of EGF-induced signaling, with implications for signaling functions and antisignaling therapy responses across E/M state-diverse tumors.