Gap junction communication regulates luminal-myoepithelial crosstalk and cell differentiation in a bi-layered human mammary epithelial cell model

Interactions between luminal and myoepithelial cells are essential for proper mammary gland development and function. However, only a few in vitro models allow the study of these interactions and their modulation by extrinsic factors. We developed a layered co-culture system (LCS) that mimics the bilayered architecture of the human breast epithelium, enabling direct contact and bidirectional crosstalk between luminal (MCF-12A) and myoepithelial (MYO1089) cells. We confirmed the formation of adherens and functional gap junctions across the layers. Transcriptomic analysis revealed that co-culture altered gene expression in pathways related to extracellular matrix remodeling, mRNA processing, response to external stimuli, hormonal signaling, receptor activity, and cell cycle regulation. In the LCS, MCF-12A cells exhibited a more luminal-like phenotype, with increased keratin-18 and decreased keratin-14, α-smooth muscle actin (α-SMA), and caldesmon-1 expression compared to 2D monoculture. Conversely, MYO1089 cells showed enhanced expression of myoepithelial markers, including α-SMA, keratin-14, and caldesmon-1. Inhibition of gap junction communication by carbenoxolone disrupted these lineage-specific differentiation patterns. These findings highlight the importance of direct communication in regulating epithelial identity and underscore the LCS as a physiologically relevant model for studying mammary gland biology and external influences that can impact the epithelial differentiation.