Supplementary MaterialsDocument S1. axes. The rapid ability of multicellular tissues to
Supplementary MaterialsDocument S1. axes. The rapid ability of multicellular tissues to physically remodel their matrix enables their constituent cells to migrate efficiently along aligned fibers and to quickly change their direction according to other microenvironmental cues, which is important for both normal and disease processes. Introduction Within tissues, cells are surrounded by a fibrous network of extracellular matrix (ECM), an important structural component of the cellular microenvironment that also contains a variety of chemical and mechanical cues that influence cell Bardoxolone methyl pontent inhibitor fate. Cells can sense the mechanical rigidity of their surrounding ECM, and the topography of the polymer network dictates the number and spatial distribution of cell-matrix attachments (1, 2, 3). Individual ECM fibers can also serve as potential paths for cell migration, both in single- and multicellular contexts. For example, alignment of the ECM perpendicular DCN to the boundary of a tumor is thought to precede invasion, with the aligned fibers providing tracks that guide migration of cancer cells, enhancing their migration efficiency and directional persistence (4, 5, 6). Oriented fibers can similarly facilitate metastatic intravasation (7). Moreover, we recently showed that the alignment of collagen fibers directly influences the direction and persistence of collective migration by non-metastatic cells (8). Cell-induced alignment of ECM fibers, therefore, appears to be an important component of the migration process, preceding cellular translocation. Although several studies have focused on the relationship between the structure of the ECM and the behavior of single cells of a particular type (5, Bardoxolone methyl pontent inhibitor 9, 10), little is known about the dynamics of multicellular-tissue-induced ECM alignment in three dimensions (3D), how the alignment process might vary across tissues comprised of different cell types, or what cellular processes drive the alignment of adjacent fibers in a multicellular context. Understanding the dynamic relationship between individual cells, their neighbors, and their surrounding ECM will provide insight into how these interactions might play a role in complex tissue microenvironments. Here, we characterized and quantified the dynamics of tissue-induced alignment of the matrix surrounding 3D multicellular tissues. We used a microfabrication-based approach to vary the initial tissue Bardoxolone methyl pontent inhibitor geometry as well as the constituent cells. We investigated alignment dynamics at the level of both the individual fiber and the network, and we determined the relative roles of contractility and proteolysis in the alignment process. Generally, we found that tissues rapidly aligned their matrix within 24? h primarily by pulling on the adjacent matrix fibers, consistent with previous reports that collagen fibers can be aligned by mechanical strain alone, with the majority of fibers being aligned under 30% strain (5). Comparing different cell types and molecularly altering cell-cell force transmission revealed that strongly cohesive tissues aligned their surrounding matrix faster than weakly cohesive tissues. Altogether, our results provide, to our knowledge, novel insights into the complex relationship between multicellular tissues and their surrounding ECM, as well as how the level of coordination between constituent cells within a tissue contributes to these tissue-matrix interactions. Our findings suggest that matrix alignment occurs primarily due to a physical mechanism driven by tissue-induced strain, which is governed by cell-cell adhesion as well as tissue geometry. This appears to be a universal phenomenon that likely plays a role before collective migration in a variety of biological processes including development and cancer invasion. Materials and Methods Cell culture and reagents Functionally normal EpH4 mouse mammary epithelial cells (11) were cultured in 1:1 Dulbeccos modified Eagles medium (DMEM)/F12 medium supplemented with 2% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 5 plane throughout the depth of the tissue. The projected images of the 3D engineered tissues were then linearized and segmented in ImageJ to quantify collagen alignment relative to the entire surface of the tissues up to a distance of 128?and and em B /em ). These results are consistent with studies using single primary human fibroblasts, which were proven to locally strain collagen gels and reorganize and align the matrix within 5 thereby?h (10). Fibroblast tissue.