Lianne Beunk

Porous collagen hydrogels are widely used to model dynamic cell‐extracellular matrix (ECM) interactions relevant to inflammation, wound healing, and cancer invasion. To improve the physiological relevance of such assays, it is essential to incorporate architectural ECM characteristics identified in vivo that may affect the mechanical and molecular mechanisms of cell migration, including ECM geometry, alignment, and dimensionality. We present detailed, step‐by‐step protocols for generating three collagen‐hydrogel‐based migration assays that integrate structural guidance cues, either as cleft‐like deformable gaps or tunnel‐like tracks, including track generation by multiphoton (MP) laser ablation. For this application, practical guidance on laser setup and integration in commonly used MP microscopes is provided. We include example applications to compare the migratory behavior of HT1080 fibrosarcoma cells, applied both as single‐cell suspensions and as pre‐formed spheroids, in these spatially controlled models. The data indicate that migration efficiency increases with the presence of guidance cues, highlighting the importance of such cues for modeling invasive cell behavior in 3D. These protocols provide a standardized yet adaptable framework for researchers studying ECM‐guided cell migration and evaluating therapeutic strategies that target cell motility. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Under‐collagen assay Basic Protocol 2 : 3D interface assay Basic Protocol 3 : 3D tissue track assay Support Protocol 1 : Imaging of guidance models by confocal microscopy Support Protocol 2 : Excitation beam adjustment Support Protocol 3 : Generation of stacks of long tracks by three common microscope types

Tumor cell invasion into heterogenous interstitial tissues consisting of network-, channel- or rift-like architectures involves both matrix metalloproteinase (MMP)-mediated tissue remodeling and cell shape adaptation to tissue geometry. Three-dimensional (3D) models composed of either porous or linearly aligned architectures have added to the understanding of how physical spacing principles affect migration efficacy; however, the relative contribution of each architecture to decision making in the presence of varying MMP availability is not known. Here, we developed an interface assay containing a cleft between two high-density collagen lattices, and we used this assay to probe tumor cell invasion efficacy, invasion mode and MMP dependence in concert. In silico modeling predicted facilitated cell migration into confining clefts independently of MMP activity, whereas migration into dense porous matrix was predicted to require matrix degradation. This prediction was verified experimentally, where inhibition of collagen degradation was found to strongly compromise migration into 3D collagen in a density-dependent manner, but interface-guided migration remained effective, occurring by cell jamming. The 3D interface assay reported here may serve as a suitable model to better understand the impact of in vivo-relevant interstitial tissue topologies on tumor invasion patterning and responses to molecular interventions.

The interstitial tumor microenvironment is composed of heterogeneously organized collagen-rich porous networks as well as channel-like structures and interfaces which provide both barriers and guidance for invading cells. Tumor cells invading 3D random porous collagen networks depend upon actomyosin contractility to deform and translocate the nucleus, whereas Rho/Rho-associated kinase-dependent contractility is largely dispensable for migration in stiff capillary-like confining microtracks. To investigate whether this dichotomy of actomyosin contractility dependence also applies to physiological, deformable linear collagen environments, we developed nearly barrier-free collagen-scaffold microtracks of varying cross section using two-photon laser ablation. Both very narrow and wide tracks supported single-cell migration by either outward pushing of collagen up to four times when tracks were narrow, or cell pulling on collagen walls down to 50% of the original diameter by traction forces of up to 40 nN when tracks were wide, resulting in track widths optimized to single-cell diameter. Targeting actomyosin contractility by synthetic inhibitors increased cell elongation and nuclear shape change in narrow tracks and abolished cell-mediated deformation of both wide and narrow tracks. Accordingly, migration speeds in all channel widths reduced, with migration rates of around 45-65% of the original speed persisting. Together, the data suggest that cells engage actomyosin contraction to reciprocally adjust both own morphology and linear track width to optimal size for effective cellular locomotion. Graphic