Zmuda, H. | bioRxiv Science

bioRxiv2023

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)- secretion was the highest with M1 macrophages and Transforming growth factor (TGF)-{beta} 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-{beta} 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. SummaryAuthors 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 StatementFormation 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.

bioengineering↗

bioRxiv2022

Multiscale obstruction sensitivity in collective cell migration

Cellular forces and intercellular cooperation generate collective cell migration. Pathological changes in cell-level genetic and physical properties cause jamming, unjamming, and scattering in epithelial migration. Separately, changes in microenvironment stiffness and confinement can produce varying modes of cell migration. However, it remains unclear whether and how mesoscale disruptions in matrix topology alter collective cell migration. To address this question, we microfabricated matrices with stumps of defined geometry, density, and orientation, which serve as obstructions in the path of collectively migrating healthy mammary epithelial cells. Here, we show that cells lose their speed and directionality when moving through dense obstructions, compared to those sparsely spaced. On flat surfaces, leader cells are significantly stiffer than follower cells, while dense obstructions lead to the overall softening of cells. In moving through dense obstructions, epithelial cells lose the sense of leaders and followers in their physical properties, migration phenotypes, and fluidity. Although Rac inhibition reduces obstruction sensitivity, loss of cell-cell cooperation and induction of leader-like phenotype via -catenin depletion eliminates the effect of matrix obstructions on epithelial migration. Through a lattice-based model, we identify cellular protrusions, polarity, and leader-follower communication as key mechanisms for obstruction-sensitive collective cell migration. Together, microscale cytoskeletal response, mesoscale softening and disorder, and macroscale multicellular communication enable epithelial cell populations to sense topological obstructions encountered in challenging environments. These results reveal that cohesive, healthy populations are more obstruction sensitive than the dysfunctional, aggressive ones. The obstruction-sensitivity could add to the emerging disease mechanotypes such as cell stiffness and traction forces.

biophysics↗