Mechanical interactions of cells with their immediately surrounding extracellular matrix (ECM) is now known to be critical in pathophysiology. For example, during cancer progression, while uncontrollable cell division leads to tumor formation, the subsequent metastatic migration of cells from the primary tumor site to distant parts of the body causes most cancer-related deaths. The metastatic journey requires cells to be able to adopt different shapes and move persistently through the highly fibrous native ECM, thereby requiring significant spatiotemporal reorganization of the cell cytoskeleton. While numerous studies performed on flat 2-dimensional culture platforms and physiological 3D gels have elucidated cytoskeletal reorganization, our understanding on how cells adapt to natural fibrous microenvironments and regulate their behavior in response to specific ECM biophysical cues including fiber size, spacing, alignment and stiffness remains in infancy. Here, we utilize the non -electrospinning Spinneret tunable engineered parameters (STEP) technique to manufacture ECM mimicking suspended fibrous matrices with precisely controlled fiber diameters, network architecture, inter-fiber spacing and structural stiffness to advance our fundamental understanding of how external cues affect cytoskeleton-based cellular forces in 3-distinct morphological processes of the cell cycle starting from division to spreading and migration. Mechanobiological insights from these studies are implemented to deliver intracellular cargo inside cells using electrical fields. Holistically, we conclude that fibrous environments elicit multiple new cell behaviors never before reported. Specifically, our new findings include (i) design of fiber networks regulates actin networks and cell forces to sculpt nuclei in varying shapes: compressed ovals, tear drop, and invaginations, and drive the nuclear translocation of transcription factors like YAP/TAZ. In all these shapes, nuclei remain rupture-free, thus demonstrating the unique adaptability of cells to fibers, (ii) dense crosshatch networks are fertile environments for persistent 1D migration in 3D shapes of rounded nuclei and low density of actin networks, while sparse fiber networks induce 2D random migration in flattened shapes and well-defined actin stress fibers, (iii) actin retraction fiber-based stability regulates mitotic errors. Cells undergoing mitosis on single fibers exhibit significant 3D movement, and those attached to two fibers can have rotated mitotic machinery, both conditions contributing to erroneous division, and (iv) a bi-phasic force response to electroporation that coincides with actin cytoskeleton remodeling. Cells on suspended fibers can withstand higher electric field abuse, which opens opportunities to deliver cargo of varying sizes inside the cell. Taken altogether, our findings provide new mechanobiological understanding of cell-fiber interactions at high spatiotemporal resolution impacting cell migration, division and nuclear mechanics-key behaviors in the study of pathophysiology. / Doctor of Philosophy / Cancer, one of the major pathophysiological conditions, progresses within the living body through spreading of malignant cells from the primary tumor to distant secondary sites, ultimately leading to life-ending outcomes. Such spreading of cancer also known as cancer metastasis requires mechanical interactions of cells with their immediately surrounding microenvironment or the extracellular matrix (ECM). Cells utilize their cytoskeleton, a dynamic internal network of filamentous proteins, to adopt various morphologies, exert mechanical forces and physically remodel their local environment as they navigate through the highly fibrous native ECM. While previous research has elucidated how biochemical factors and bulk matrix properties regulate such cytoskeletal organization and single cell behavior, our understanding of how cells adapt to fibrous environments and respond to local biophysical cues like fiber diameter, spacing, alignment and stiffness remains in infancy. Here we use the non -electrospinning Spinneret tunable engineered parameters (STEP) to generate suspended nanofiber networks of tunable geometric and mechanical properties to mimic the native cellular environment. We discover that cells elongated within these ECM-mimicking environments utilize a unique cytoskeletal caging structure to regulate the shape and response of their nuclei in a fiber -diameter and organization-dependent manner. Additionally, we demonstrate that these elongated cell morphologies often observed during metastatic cancer cell movements, is achievable not only in aligned fibers but can also be induced by dense networks of fibers in a crossing organization. Specifically, such dense crosshatch networks allow cells to migrate persistently at high speeds while cells on sparsely spaced networks demonstrate slower and random movements. As cells elongated during interphase rounded up to undergo division, we find that the underlying fiber-geometry modulates mitotic dynamics through differential levels of actin retraction fiber-mediated stability, leading to significant alterations in orientation of mitotic machinery and mitotic spindle defects. Finally, we utilize these mechanobiological insights on cytoskeletal organization and cell shape control to optimize intracellular delivery of cargo using high-voltage electric fields. We demonstrate suspended cells are capable of withstanding higher electric fields and identify multistage cell contractility recovery dynamics, which correlate with cytoskeletal disruption and reassembly. Taken altogether, our findings provide a comprehensive understanding of the fibrous ECM-mediated regulation of the cytoskeletal organization and its impact in cell migration, division and nuclear mechanics. Knowledge obtained from this study will improve our understanding of cancer metastasis and provide predictive data for in vivo cellular response, essential for cytoskeleton-targeting cancer therapies.