Cells interact with their immediate fibrous extracellular matrix (ECM); alignment of which has been shown influence metastasis. Specifically, intra-vital imaging studies on cell invasion from tumor-matrix interface and wounds along aligned fibers describe invasion to occur as singular leader (tip) cells, or as collective mass of a few chain or multiple tip cells. Recapitulation of these behaviors in vitro promises to provide new insights in how, when and where cells get the stimulus to break cell-cell junctions and ensue invasion by migrating along aligned tracks. Using Spinneret based Tunable Engineered Parameters (STEP) technique, we fabricated precise layout of suspended fibers of varying diameters (300, 500 and 1000 nm) mimicking ECM dimensions, which were interfaced with cell monolayers to study invasion. We demonstrated that nanofiber diameter and their spacing were key determinants in cells to invade either as singularly, chains of few cells or multiple-chains collectively. Through time-lapse microscopy, we reported that singular cells exhibited a peculiar invasive behavior of recoiling analogous to release of a stretched rubber band; detachment speed of which was influenced with fiber diameter (250, 425 and 400 µm/hr on small, medium and large diameter fibers respectively). We found that cells initiated invasion by putting protrusion on fibers; dynamics of which we captured using a contrasting network of mismatched diameters deposited orthogonally. We found that vimentin, a key intermediate filament upregulated in cancer invasion localized within a protrusion only when the protrusion had widened at the base, signifying maturation. To develop a comprehensive picture of invasion, we also developed strategies to quantify migratory speeds and the forces exerted by cells on fibers. Finally, we extended our findings of cell invasion to report a new wound healing assay to examine gap closure. We found that gaps spanned by crosshatch network of fibers closed faster than those on parallel fibers and importantly, we reported that gaps of 375 µm or larger did not close over a 45-day period. In summary, the methods and novel findings detailed from this study can be extended to ask multiple sophisticated hypotheses in physiologically relevant phenomenon like wound healing, morphogenesis, and cancer metastasis. / Ph. D.

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