Matrix-modulated Air-liquid Interface Microenvironments Reveal Architectural Control of Lung Tumor Phenotypes

Lung adenocarcinoma evolves within a highly structured microenvironment shaped by air exposure, basement-membrane ligands and three-dimensional (3D) organization, yet standard in vitro models capture only fragments of this architecture. Here, we systematically disentangle how air-liquid interface (ALI), extracellular matrix (ECM) cues and dimensionality interact to regulate tumor epithelial states. Using five architectural configurations, we demonstrate that these parameters act synergistically to promote distinct morphological, transcriptional and inflammatory phenotypes not observed under conventional 2D or submerged 3D cultures. ALI broadly enhanced epithelial cohesion, progenitor activation, partial EMT programs and cytokine expression, whereas ECM embedding further induced ALI-mediated mesenchymal activation, matrix remodeling and invasive cytoskeletal organization. Notably, 3D ALI uniquely produced a hybrid alveolar-mucinous phenotype characterized by concurrent SP-C and MUC5B induction, alongside integrated epithelial, basal and mucosecretory identities. These emergent states reflect tumor-relevant plasticity and illuminate architectural controls over lineage infidelity, invasion and inflammatory activation. Together, these findings establish ALI-ECM-dimensionality intersections as critical regulators of lung tumor phenotype and provides a systematic architectural framework for designing more physiologically relevant in vitro lung cancer models.