Time-resolved phenotypic profiling maps EGFR and c-Met trafficking signatures.

The epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met/MET) are receptor tyrosine kinases (RTKs) whose signalling depends on endocytic routing, and impaired down-regulation can drive tumour progression and therapy resistance, including MET-driven bypass of EGFR inhibition. However, how routing states couple to signalling and lipid programs, and how this differs between receptors under matched conditions, remains unclear. Using a chemically diverse annotated inhibitor library, we performed time-resolved, pathway-focused high-content imaging in A549 cells to compare co-varying phenotypes across matched EGFR and c-Met assay systems. Late receptor persistence defined operational degradation-inhibitor phenotypes that were heterogeneous and dispersed across a fused phenotypic atlas. Despite this heterogeneity, matched degradation-inhibitor sets showed receptor-selective lipid-trafficking coupling: EGFR persistence was associated with reduced phosphatidylinositol 4,5-bisphosphate-linked readouts and signalling biases, whereas c-Met persistence aligned with increased perinuclear phosphatidylinositol 4-phosphate heterogeneity and altered transferrin-recycling readouts. These results provide a comparative imaging framework for mapping RTK trafficking states and nominate phosphoinositide-linked features as candidate readouts of trafficking rewiring in cancer-relevant contexts.