Microenvironmental signals combine to induce non-additive molecular and phenotypic responses in mammary epithelial cells.

Cellular phenotypes are dictated not by single signals but by the integration of multiple extracellular cues, a process that remains poorly understood. Here, we systematically dissect how combinations of Oncostatin M, Transforming Growth Factor β1, and Epidermal Growth Factor shape the behavior of MCF10A mammary epithelial cells. Live-cell imaging revealed that ligand combinations drive emergent phenotypes absent in single-ligand contexts, pointing to the induction of novel molecular programs. Transcriptomic profiling uncovered synergistically regulated genes, while partial least-squares regression linked these transcriptional signatures to quantitative imaging-derived phenotypes and validated them across independent datasets. Functional analysis revealed CXCR2 signaling, upregulated through CREB activation, as a key driver of enhanced cell motility under combinatorial ligand treatment. Together, these findings establish a framework for uncovering how extracellular signals converge to modulate gene expression and phenotype, providing new insight into the principles governing complex epithelial cell behaviors.