Generation of In Vivo‐Inspired 3D Collagen Models for Guided Tumor Invasion In Vitro

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.