Biomechanical cues within tissue microenvironments are critical for maintaining homeostasis, and their disruption can contribute to malignant transformation and metastasis. Once transformed, metastatic cancer cells can migrate persistently by adapting (plasticity) to changes in the local fibrous extracellular matrix, and current strategies to recapitulate persistent migration rely exclusively on the use of aligned geometries. Here, the controlled interfiber spacing in suspended Crosshatch networks of nanofibers induces cells to exhibit plasticity in migratory behavior (persistent and random) and the associated cytoskeletal arrangement. At dense spacing (3 and 6 µm), unexpectedly, elongated cells migrate persistently (in 1 dimension) at high speeds in 3‐dimensional shapes with thick nuclei, and short focal adhesion cluster (FAC) lengths. With increased spacing (18 and 36 µm), cells attain 2‐dimensional morphologies, have flattened nuclei and longer FACs, and migrate randomly by rapidly detaching their trailing edges that strain the nuclei by ∼35%. At 54‐µm spacing, kite‐shaped cells become near stationary. Poorly developed filamentous actin stress fibers are found only in cells on 3‐µm networks. Gene‐expression profiling shows a decrease in transcriptional potential and a differential up‐regulation of metabolic pathways. The consistency in observed phenotypes across cell lines supports using this platform to dissect hallmarks of plasticity in migration in vitro .—Jana, A., Nookaew, I., Singh, J., Behkam, B., Franco, A. T., Nain, A. S. Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response. FA8EB J. 33, 10618–10632 (2019). www.fasebj.org

Significance: When cells heal a wound or invade a new area, they coordinate their motion. Coordination is often studied by looking at what happens after pairs of cells collide. Postcollision, cells often exhibit contact inhibition of locomotion—they turn around and crawl away from the point where they touched. Past knowledge of repolarization on contact comes from studies on flat surfaces, unlike cells in the body, which crawl along fibers. We discover that cells on single fibers walk past one another, but cells in contact with multiple fibers stick to one another and move as pairs. This outcome changes to walk past after cell division. Our experiments and models reveal how the environment regulates cell–cell coordination after contact.

Investigating the mechanistic influence of the tumor microenvironment on cancer cell migration and membrane blebbing is crucial in the understanding and eventua