Single Living Cell Analysis Decodes Dynamical Signaling Patterns Triggering Different Phenotypes of Cell Migration

A sound understanding of cell migration behaviors and the internal mechanism is crucial for studying cancer metastasis and invasion. Continuous cell tracking and quantifying cellular and molecular dynamics of cell migration at the single-cell level is essential to elucidate rare, dynamic, and heterogeneous cell responses. Yet, a competent comprehensive analytical platform is lacking. Herein, we present an integrated single living cell analysis platform that enables long-term observation of migration behavioral phenotypes in single cells and simultaneous analysis of the signaling proteins and complexes during cell migration. Considering correlation between pathways and phenotypes, this platform is capable of analyzing multiple phenotypes and signaling protein dynamics at a subcellular resolution, reflecting the molecular mechanism of biological behavior. Using the EGFR-PI3K signaling pathway as a proof-of-principle, we explored how the pathway and related regulators, Rho GTPases, promote different migration phenotypes. Signaling pathway protein complexes p85α-p110α and p85α-PTEN were found to reciprocally modulate each other and subsequently regulate the expression level of the small GTPases by EGFR-related signaling pathways, which governs the cell migratory behavior. Thus, this single-cell analysis platform is a promising tool for rapid molecular mechanism analysis and direct observation of migration phenotypes at the single-cell level, providing insights into the molecular mechanism and phenotypes in cell migration.