Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response

Abstract

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). http://www.fasebj.org

Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction

“…To quantitatively describe the formation of 3D-PLPs, we inquired if lateral protrusions could form in the elongated cells, and asked if these exhibited a characteristic morphology that was associated with a specific cell geometry. For this, we used the previously described non-electrospinning spinneret based tunable engineered parameters (STEP 35,36 ) method to fabricate anisotropic/aligned fibers. These could also serve as force sensing nanonets, as we described previously [32][33][34] .…”

The epithelial-mesenchymal transition and the cytoskeleton in bioengineered systems

“…However, “scratching” can leave cell debris or damage soft substrates, and this assay biases towards collective migration [ 9 , 88 , 90 , 91 ] Micropatterned substrates (2.5D) Analogous to cell scattering or wound healing assays, but incorporating topographical features on the substrate Mimics ECM architecture and can affect directional migration via contact guidance, as well as EMT/MET. However, requires some expertise and specialized equipment to fabricate [ 15 , 102 – 104 , 107 – 111 , 113 , 114 ] Transwell/Boyden chamber Plate cells on top of a porous plastic membrane, count the number of cells that migrate across membrane to the other side. Can include a gradient of chemotactic factors across membrane, as well as an ECM coating on the top Standard assay that can be easily quantified.…”

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds

“…We caution that our in vitro fibrous assay attempts to recapitulate the complex native environment in which few cell attachment points (large pore size) force cells to make contact with only a few fibers (12,15,19). Further improvements in density, organization, and size of fibers will provide a comprehensive understanding in energetic pathways used by cells exhibiting diverse modes of cell migration in varying shapes (23) and invasion modes (single versus collective) (43).…”

“…Recently, three-dimensional ECM model systems [gels (14), cell-derived matrixes (12), and microgrooves (20)] provide a more integrative and physiological environment to study cellular behavior. In this study, we used the nonelectrospinning spinneret-based tunable engineered parameters (STEP) fiber manufacturing platform (23,43,45) to determine how bioenergetics in cell-three-dimensional fiber interactions influence cell migration and cell force production.…”