Epithelial multicellular clustering enabled by polarized macrophages on soft matrices.

Formation of epithelial structures of variegated geometries and sizes is essential for organogenesis, tumor growth, and wound repair. Although epithelial cells are predisposed with potential for multicellular clustering, it remains unclear whether immune cells and mechanical cues from their microenvironment influence this process. To explore this possibility, we co-cultured human mammary epithelial cells with pre-polarized macrophages on soft or stiff hydrogels. In the presence of M1 (proinflammatory) macrophages on soft matrices, epithelial cells migrated faster and subsequently formed larger multicellular clusters, compared to co-cultures with M0 (unpolarized) or M2 (anti-inflammatory) macrophages. By contrast, stiff extracellular matrix (ECM) disabled active clustering of epithelial cells due to their enhanced migration and cell-ECM adhesion, regardless of macrophage polarization. We found that the co-presence of soft matrices and M1 macrophages reduced focal adhesions, but enhanced fibronectin deposition and non-muscle myosin-IIA expression, which altogether optimize conditions for epithelial clustering. Upon Rho-associated kinase (ROCK) inhibition, epithelial clustering was abrogated, indicating a requirement for optimized cellular forces. In these co-cultures, Tumor Necrosis Factor (TNF)-α secretion was the highest with M1 macrophages and Transforming growth factor (TGF)-ß secretion was exclusively detectable in case of M2 macrophages on soft gels, which indicated potential role of macrophage secreted factors in the observed epithelial clustering. Indeed, exogenous addition of TGB-ß promoted epithelial clustering with M1 co-culture on soft gels. According to our findings, optimization of both mechanical and immune factors can tune epithelial clustering responses, which could have implications in tumor growth, fibrosis, and would healing. Summary: Authors show proinflammatory macrophages on soft matrices enable epithelial cells to form multicellular clusters. This phenomenon is disabled on stiff matrices due to increased stability of focal adhesions. Inflammatory cytokine secretion is macrophage-dependent, and external addition of cytokines accentuates epithelial clustering on soft matrices. Impact Statement: Formation of multicellular epithelial structures is critical to tissue homeostasis. However, it has not been shown how the immune system and mechanical environment affect these structures. The present work illustrates how macrophage type affects epithelial clustering in soft and stiff matrix environments.