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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/82709 |
| Date | 04 October 2016 |
| Creators | Sharma, Puja |
| Contributors | Biomedical Engineering, Nain, Amrinder, Lee, Jerry S. H,., Robertson, John L., Behkam, Bahareh, Lee, Yong Woo, Davalos, Rafael V. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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