Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochem-ical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues in-fluence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the per-itumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A mi-gration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stro-mal microenvironment and understanding of the relationships between matrix attributes and cell migra-tion mode can aid ongoing effort s to identify effective cancer therapeutics that address cell plasticity -based therapy resistances.Statement of significanceStromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber -reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of mi-gration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.(c) 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.