Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro-and nano- architectures, which provides signaling cues and influences cell behavior. Mimicking this three dimensional environment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique with controlled fiber diameter (microns to less than 100 nm), orientation and spacing in single and multiple layers are demonstrated as biological scaffolds. Hierarchical nanofiber assemblies were developed to control single cell shape (shape index from 0.15 to 0.57), nuclei shape (shape index 0.75 to 0.99) and focal adhesion cluster length (8-15 micrometer). To further investigate single cell-ECM biophysical interactions, nanofiber nets fused in crisscross patterns were manufactured to measure the "inside out" contractile forces of single mesenchymal stem cells (MSCs). The contractile forces (18-320 nano Newton) were found to scale with fiber structural stiffness (2 -100 nano Newton/micrometer). Cells were observed to shed debris on fibers, which were found to exert forces (15-20 nano Newton). Upon CO? deprivation, cells were observed to monotonically reduce cell spread area and contractile forces. During the apoptotic process, cells exerted both expansive and contractile forces. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology. / Master of Science

Spinneret based Tunable Engineered Parameters (STEP) Platform is a recently reported pseudo-dry spinning and non-electrospinning technique that allows for the deposition of aligned polymeric nano-fibers with control on fiber diameters and orientation in single and multiple layers (diameter: sub 100nm micron, length: mm-cm), deposition (parallelism 2.5 degrees) and spacing (microns)). A wide range of polymers such as PLGA, PLA, PS, and PU have been utilized for their unique material properties in scaffold design. In this thesis two unique bioscaffolds are demonstrated for the measurement of group cell migration for wound closure and single cell contractility force for the study of force modulation. The wound healing assay bridges the gap between confluent reservoirs of NIH3T3 fibroblasts through arrangement of a suspended array of fibers guiding group cell migration along the fiber axis. This platform demonstrates that topographical and geometrical features of suspended fibers play a very important role in wound closure. Spacing, alignment and orientation were optimized to shown an increased rate of closure. In the second complementary assay, we report a fused-fiber network of suspended fibers capable of measuring single cell forces. Results from our experiments demonstrate that force behavior is dependent on mechanical properties such as stiffness and geometry of fiber networks. We also demonstrate changes in spatial and temporal organization of focal adhesion zyxin in response to single cell migration on these networks. / Master of Science

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.

Migratory cells in vivo move within a predominantly fibrous microenvironment through the action of forces. These dynamic interactions facilitate mechanosensing, critical to fundamental biological processes in pathophysiology. Naturally, the field of mechanobiology has evolved over the past several decades to decipher the role of forces in mechanotransduction using a variety of force-measurement platforms. A central challenge that has yet to be overcome in the field is connecting forces with the interplay between cell shape and ever-changing environment. Here, through design of specific fibrous architectures, a mechanobiological understanding of force feed-forward loop accounting for shape shifting of the environment and cells is developed. Using the non-electrospinning Spinneret Tunable Engineered Parameters (STEP) technique, two complementary force measurement platforms of varying physical attributes are developed to investigate how the force feed-forward loop impacts cell fate. Nanonet Force Microscopy (NFM) comprised of aligned nanonets is designed to study anisotropic cell shapes, while Crosshatch Force Microscopy (CM) comprised of orthogonal arrangement of fibers is designed to study cell bodies of broad shapes. The combination of shapes achieved on these networks recapitulate mesenchymal shapes observed in vivo, which are used to describe cell behaviors not reported before. The new findings include (i) discovery of a new biological structure, termed 3D-perpendicular lateral protrusions (3D-PLPs) which is proposed to be the missing biophysical link in the remodeling of the ECM and perpetuation of desmoplasia. Using NFM, seven discreet steps in formation of force-exerting PLPs anywhere along the cell body is documented, which allow cells to spread laterally and increase in contractility. Using a variety of fiber networks, it is shown that aligned fibers are necessary for PLP formation and suitable environments for myofibroblast activation, and (ii) a force dipole that links matrix deformability with cell contractility. Aided by machine learning, CFM automates the process of fiber feature recognition to measure forces as cells change shapes during migration and differentiate to osteogenic and adipogenic lineages. The force platforms are applied to investigate (i) the bioenergetic contributors fueling cellular migration and a surprisingly overwhelming impact of glycolytic energetic pathway over the traditionally thought mitochondrial energy production is found. However, neither pathway has substantial impact over the cellular force production, and (ii) quantitate the migratory and contractile response of enucleated cytoplasmic fragments naturally shed by cells. A peculiar contractility driven oscillatory migratory phenotype is found, capable of lasting over tens of hours, and absent in intact cells. Overall, new high spatiotemporal capabilities are developed in mechanobiology to quantitate the force-feed forward loops between cell shape and ECM in pathophysiology. / Doctor of Philosophy / Pathophysiology is the study of abnormal changes in the regular body functions of an organism that are causes or consequences of disease onset. Research in this area is mainly focused on identifying the different factors that cause and propagate the disease states such as cancer. Central to many of these processes are events such as cell migration and remodeling of their surrounding environment. The native microenvironment surrounding cells is highly complex and is composed of many classes of macromolecules, with fibrous components being one of the most important. How cells interact with these environments through application of forces and how this further regulates cellular behavior is vital to advancing our understanding of many of these pathophysiological processes. Currently, there is a lack in our understanding of how this dynamic process referred to as the "force feed-forward loop", is perpetuated. This limitation in our understanding can be attributed to the lack of an in vivo mimicking platform that captures this dynamic interaction and is capable of measuring the forces. To this end, the development of two novel single cell force measurement platforms: Nanonet Force Microscopy (NFM) and Crosshatch Force Microscopy (CFM) is presented. These platforms are fiber based systems, generated with the utilization of previously established non-electrospinning technique of Spinneret based Tunable Engineered Parameters (STEP) technique. Using NFM and CFM, forces were computed in wide range of cell shapes from anisotropic to all other spread morphologies. These platforms were applied to identify a new biological structure called perpendicular lateral protrusions and shown to have potential role in the spreading of tumor microenvironment. Furthermore, the force dynamics in physiological processes such as stem cell differentiation into fat cells or bone cells is also identified. How cellular processes such as migration and force production is fueled is also investigated and found to be not heavily reliant on the commonly understood mitochondrial activity. Finally, sub-cellular components known as cell fragments, which are devoid of nucleus, are also observed to be contractile and migratory in nature, independent of parent cell body. These platforms and findings can be further utilized to advance our current knowledge of the progression of these physiological and pathological processes and serve as diagnostic tools for the early identification of disease onset. Furthermore, based on these findings, strategies can be developed for early intervention to inhibit disease progression or devise bioengineered scaffolds for applications in tissue engineering.

While a large body of work has focused on the transcriptional regulation of cellular identity, the role of the mechanical properties of cells and the importance of their physical interactions with the local environment remains less well understood. In this project, we explored the impact of cytoskeleton-generated forces exerted on the nucleus in the context of early embryonic stem (ES) cell fate decisions. We chose to perturb force generating components in the cytoskeleton – notably the molecular motor non-muscle myosin II - and key structural and chromatin binding proteins in the nuclear envelope, notably, the lamins (LMNA), Lamin B receptor (LBR) and components of the LINC complex (nesprins/KASH). The structural proteins in the nuclear envelope regulate both the mechanical response of the nucleus to force and the stabilization of peripheral heterochromatin (repressed genes). Our hypothesis is that reducing forces transmitted directly to chromatin or increasing tethering of peripheral heterochromatin to the nuclear envelope would restrict access to lineage specific genes sequestered at the nuclear lamina and thereby either impair, or delay, differentiation. We found phenotypes in the capacity of mouse ES cells to specify to the neural lineage following our perturbations: overexpression of LMNA, LBR and KASH proteins resulted in a significant fraction of cells that did not express the neuroectoderm marker Sox1 after four days of differentiation, while inhibiting non-muscle myosin II delayed Sox1 expression in the entire population. Overexpression of LMNA and LBR did not affect the ability of the cells to exit the naive pluripotent state, which raises the possibility that the perturbations are halting the cells in a formative phase prior to lineage specification. Future work will focus on looking at genome-wide transcriptional changes accompanying differentiation combined with an analysis of spatial information of differentially regulated genes.

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.

Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer. / Doctor of Philosophy / Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.