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 cocultured human mammary epithelial cells with prepolarized 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 cocultures with M0 (unpolarized) or M2 (anti-inflammatory) macrophages. By contrast, stiff matrices disabled active clustering of epithelial cells due to their enhanced migration and cell-ECM adhesion, regardless of macrophage polarization. We found that the copresence of soft matrices and M1 macrophages reduced focal adhesions, but enhanced fibronectin deposition and nonmuscle myosin-IIA expression, which altogether optimize conditions for epithelial clustering. Upon ROCK inhibition, epithelial clustering was abrogated, indicating a requirement for optimized cellular forces. In these cocultures, TNF-α secretion was the highest with M1 macrophages and TGF-β 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 TGF-β promoted epithelial clustering with M1 coculture 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.