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

Abstract

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence 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 peritumoral stromal microenvironment regulate migration mode phenotypes has prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel stiffness 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 developed 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 stiffness distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, multicellular clusters, and collective strands. Taking advantage of the tunability of this material platform, we identify a combination of physical and soluble cues that induces distinct heterogeneous migration modes originating from the same MCF10A spheroid and use this setting to examine a functional consequence of migration mode in apoptotic resistance. We find that cells migrating as part of collective strands are more resistant to staurosporine-induced apoptosis than either disconnected multicellular clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment that help elucidate relationships between matrix attributes and cell migration mode can contribute to ongoing efforts to identify efficacious cancer therapeutics that address migration plasticity-based therapy resistances.

Tomographic Volumetric Bioprinting of Heterocellular Bone-like Tissues in Seconds

“…The observation of increased cell spreading was confirmed by an increased cell surface area in constructs with lower LAP concentration (Figure 3E). In agreement with other studies, we showed increased cell spreading in softer 3D matrices with lower gelatin and photoinitiator concentrations [27,31]. Our results suggest that the physical restrictions might have contributed to limited cell spreading in a 3D covalently crosslinked matrix, and the higher crosslinking ratio decreased the mesh size, which decreased accessibility of cells to nutrients in the medium.…”

A novel jamming phase diagram links tumor invasion to non-equilibrium phase separation

“…It is becoming increasingly clear that physical cues from the ECM can cause spatially heterogeneous migratory modes in initially homogeneous cell populations. Recent work using synthetic hydrogels shows that fiber density and bulk stiffness distinctively contribute to the migratory switch observed spheroid invasion ( Hiraki et al., 2021 ). However, in natural hydrogels, such as collagen, fiber density and stiffness are inexorably related, and our own results show that increasing fiber density stiffens the ECM ( Figures 4 and S8 ), thus potentially increasing the solid stress acting on proliferating spheroids.…”

Magnetic Alignment of Electrospun Fiber Segments Within a Hydrogel Composite Guides Cell Spreading and Migration Phenotype Switching

“…Within partially aligned and aligned fibrous matrices, nuclei also appeared elongated in the direction of fiber alignment ( Figure 7B ). To more directly visualize migration directional bias, we utilized a previously developed custom MATLAB image analysis code to generate heatmap overlays of actin structures ( Figure 7C ) and rose plots of nuclear locations ( Figure 7D ) for 25 spheroids ( Hiraki et al, 2021 ). Non-aligned gels promoted radially uniform cell outgrowths and distribution of nuclei.…”

“…While we did not observe an increase in net invasion depth with fiber alignment, instantaneous migration speeds were not assessed here. As the bulk DVS hydrogel stiffness/crosslinking is separately defined from fiber density and alignment ( Matera et al, 2019 ; Hiraki et al, 2021 ), this hydrogel composite can be used to investigate the individual contributions of fiber alignment and hydrogel stiffness/crosslinking on 3D cell migration speed in future studies ( Riching et al, 2015 ; Wang et al, 2018 ). Further investigation with timelapse imaging could directly assess if fiber alignment increases migration speed during proteolysis-dependent 3D cell migration.…”

“…A previously established custom MATLAB image analysis code was used to extract morphometric data from spheroid migration studies ( Hiraki et al, 2021 ). Briefly, max intensity projections of spheroid nuclei and F-actin channels were separately thresholded and object size filtered to remove background.…”