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. / Disease phenomenon like cancer invasion and wound healing have a myriad of things in common including cell migration and the ability of cells to remodel their immediate environment. Often times these singular or group migratory events of cells are initiated and directed by peculiar cells called tip/leader cells that explore cellular environment and make room for migration. While research in the last few decades has yielded a tremendous wealth of information as to how biochemical factors influence their behavior, our understanding of how the biophysical properties of the environment affect their behavior is in its infancy. This lack of understanding can somewhat be attributed to the difficulty in fabricating mechanically welldefined substrate systems that can be tailored to recapture these invasive episodes in a controlled setting outside of a living organism. In this study, we utilize a novel Spinneret based Tunable Engineered Parameters (STEP) technique to fabricate mechanically tunable nanofibers that show close resemble to native cellular environment. Cells were made to interact with these fibers and it was shown for the first time that factors like fiber spacing, diameter and topography can significantly affect the types of leader cells and their trajectory. Furthermore, once these cells come out of the simulated tumors/wounds, their migratory behaviors were still affected by mechanical properties of the fibers. Similarly, we also showed for the first time that the ability of the gaps to close in simulated wound healing settings could be significantly dependent on size, shape, and properties of fibers. These findings offer a novel outlook to our current understanding of single and collective cell behavior and how the biophysical properties of the native cellular environment can affect these behaviors. This can not only expand our understanding of how this invasive episodes occur, but also help us come up with preventive measures to inhibit such episodes for a better prognosis of diseases like cancer and chronic wounds.

Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological process in multiple cell types and diseases.

Collective cell migration is critical for various physiological and pathological processes. In vitro wound healing assay has been widely used to study collective cell migration due to its technical simplicity and ability of revealing the complexity of collective cell migration. This project studies the function and importance of leader cells, the cells pulling cell monolayer migrating into free space, in endothelium and skin epithelial regeneration via plasma lithography enhanced in vitro wound healing assay. Despite leader cells have been identified in in vitro wound healing assays, little is known about their regulation and function on collective cell migration. First, I investigated the role of leader cells in endothelial cell collective migration. I found that the leader cell density is positively related with the cell monolayer migration rates. Second, we used this knowledge to study the effects of arsenic treatment on skin regeneration via in vitro wound healing assay. We found that low concentration of arsenic treatment can accelerate the keratinocyte monolayer migration. We further found that arsenic affected cell migration by modulating leader cell density through Nrf2 signaling pathway. As a conclusion of these studies, we evaluated the function of leader cells in collective cell migration, and elucidated the mechanism of arsenic treatment on skin regeneration.