The aim of this thesis is to develop a better understanding of the mechanisms and modalities exploited by breast cancer cells during invasion, paying particular attention to those mechanisms employed during cell migration in 3D spheroid culture, a context that more accurately recapitulates the complexities seen in vivo. Using a hydrogel-based model system, we investigated the roles of cellular blebs in cancer migration, developed a novel protocol that enhances optical microscopy images and facilitates high content imaging techniques including mass spectrometry imaging, and then used this new technology to investigate gain of function activity of mutant p53 in breast cancer cell migration in terms of both actomyosin contractility and in lipid abundances and distributions. Chapter 1 begins with an overview of breast cancer, an introduction to cancerous behavior and migration, a discussion of the importance of 3D cell culture and interactions of breast cancer cell and the extracellular matrix, and an overview of the various imaging techniques used in this work. In Chapter 2 we describe a novel mode of breast cancer migration that we term bleb – driven migration. This migratory mode is characterized by invasive cells that are both round and bleb bearing. This migratory mode is highly dependent on actomyosin contractility, a mechanism that is imperative to bleb formation. We show that blebs can actively attach to and rearrange the extracellular matrix via accumulations of β1 integrins at the bleb necks. We show both polarization of blebs as well as collagen alignment in regions of the cell enriched in blebs, both of which are dependent on the expression of β1 integrins by the cell. The discovery of this new migratory mode is important, as many cancers are able to overcome cancer therapies by using escape mechanisms, such as alternate migratory mechanisms. As such, developing a fuller understanding of the migratory modes used by cancer cells is vital in our fight to prevent cancer deaths. Chapter 3 tackles a significant problem associated with working in 3D spheroid culture: obtaining high quality, high resolution images. 3D samples tend to be highly refractive and poorly diffusive, and image acquisition can be severely hindered due to these factors. Further, the depth at which 3D samples can be imaged is limited by the low working distances of high numerical aperture objectives. As such, we developed a protocol that enables the sectioning of invasive spheroids, which we term DISC-3D (dual hydrogel invasive cryosectioning of spheroids in 3D). This protocol enables us to capture images that are higher in resolution and signal to background noise, removes imaging depth related constraints, and enables images to be acquired using dyes and techniques that have not previously been demonstrated in invasive 3D in vitro samples. Using this technology, Chapter 4 then examines the gain of function activity mutant p53 imparts on invasive breast cancer cells. We show that mutant p53 plays a role in increasing actomyosin contractility by promoting targeting of RhoA to the cell membrane. We also show an alteration in lipid expression in mutant p53 bearing cells, a feat that is made possible through use of the DISC-3D technology. Collectively, this work provides insights in to the invasive mechanisms exploited by breast cancer cells. We repeatedly demonstrate the importance of in vitro, 3D cell culture in the study of breast cancer migration. Using such cell culture techniques, we outline previously unknown aspects of breast cancer cellular migration both in regards to the importance of blebs and to the gain of function activity of mutant p53, the latter of which was made possible through use of the DISC-3D protocol. We argue that continued study in this area will provide insights into how breast cancer cells migrate, providing paths and new treatment strategies for preventing such migration.

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Neural stem/progenitor cells (NSPCs) play a crucial part in the evolutionary development of the human neocortex. During early postnatal development, NSPCs give rise to immature neurons called neuroblasts within the subventricular zone (SVZ) that utilize unique migratory streams to integrate widely in the cerebral cortex. However, the cellular mechanisms enabling these unique migratory routes through the compacted cellular landscape remain unknown. Special emphasis has been placed on understanding the susceptibility of these brain regions to severe conditions such as congenital heart disease (CHD), resulting in poor neurological outcomes. Owing to its reminiscent complexity to humans, the neonatal piglet (Sus scrofa domesticus), which possesses a highly evolved gyrencephalic neocortex and an expansive outer SVZ, provides a powerful translational model system for the study of how heart dysfunction impacts cortical development from both a modern and evolutionary perspective. The present study provides a detailed characterization of neuroblast migration along their associate substrates in the piglet cortex under normal physiological conditions and how reduced oxygenation (i.e., hypoxia) can impact their vulnerability and/or resistance to injury during a critical period of postnatal development. In this thesis, I investigated the spatiotemporal distribution and developmental origin of SVZ-derived neuroblasts. Following BrdU tracing, multiplex labeling, and confocal microscopy, I show that the porcine brain contains populations of newly generated (BrdU+/DCX+) neurons in the prefrontal cortex that are produced postnatally. Regional analyses using immunohistochemical staining for doublecortin (DCX), a marker expressed by immature neurons, revealed that DCX+ clusters co-express markers of neuronal cell migration (PSA-NCAM), GABAergic interneuron marker (GABA+), and specific transcription factors (SCGN+SP8+) associated with the caudal- and lateral ganglionic eminence progenitor domains in the ventral forebrain. Moreover, I found that DCX+ neuroblasts are encased by astrocytic processes and tightly associated with blood vessels in the SVZ. Additionally, this thesis describes the use of chronic hypoxia as a model to profile neuroblast migration along associated substrates in pathological conditions related to CHD. Together, this work serves as a framework for the functional utilization of the neonatal piglet to understand the impact of substrate-dependent neuronal migration on brain maturation and neurodevelopmental diseases. / Doctor of Philosophy / Congenital heart disease (CHD) remains a significant cause of abnormal fetal brain development, affecting 1-2% of live births per year. Although many surgical strategies have shown promise in increasing quality of life, the current challenges remain the long-term cognitive deficits and diverse neurodevelopmental disabilities due to CHD. Recent studies suggest that dysregulated neurogenesis, which is associated with impaired neocortical development in human fetuses of CHD, may be influenced by altered brain circulation of blood and oxygen deliverance during critical periods of prenatal cortical growth. The brain's subventricular zone (SVZ) niche is essential for producing new neurons following birth to restore, repair, and replace existing neurons in the developing brain. In addition, these newborn neurons undergo long-distance migration from the SVZ to reach their final cortical destinations and ultimately contribute to brain development/plasticity. This study seeks to characterize the migration patterns of newborn neurons and the substrates (e.g., blood vessels or astrocytes), enabling the movement along the unique migratory routes under normal and pathological (i.e., hypoxia) conditions. In short, we found that the vast majority of the SVZ-derived newborn neurons are inhibitory neurons (i.e., interneurons) that originate in the deep region of the brain called the telencephalon and migrate tangentially utilizing blood vessels as scaffolds to the cortex, which is likely to contribute to cortical plasticity. These postnatal piglet findings demonstrate that swine represent a powerful translational model system to study large-brained mammalian cortical development and neuronal migration as it correlates to humans in normal and diseased states.

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

Yes / Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl- channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily due to the expression of two early regulatory proteins termed small and large tumour antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyVmediated cancer formation but thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl- channel modulation can reduce MCPyV STinduced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST upregulates two Cl- channels; CLIC1 and CLIC4, which when silenced, inhibit MCPyV STinduced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are upregulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease, this highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC. / BBSRC DTP studentship (BB/J014443/1) and Royal Society University Research Fellowship to JM (UF100419)

Cancer metastasis is the leading cause of death related to cancer diseases. Once the cancer cells depart the primary tumor site and enter the blood circulation, they spread through the body and will likely initiate a new tumor site. Therefore, understanding the cell migration and stopping the spread in the initial stage is the utmost of importance. In this dissertation, we have proposed a 3-D microenvironment that (partially) mimics the structures, complexity and circulation of human organs for cell migration studies. We have developed the tools to fabricate 3-D complex geometries in PDMS from our previously developed single-mask, single-etch technology in silicon. In this work, 3-D patterns are transferred from silicon structures to glass following anodic bonding and high temperature glass re-flow processes. Silicon is etched back thoroughly via wet etching and the glass is used as master device to create 3-D PDMS structures for use in dielectrophoresis cell sorting applications. Furthermore, this work has been modified to fabricate 3-D master devices in PDMS to create 3-D structures in collagen hydrogels to mimic native tissue structures. We have studied the interaction of endothelial cells with model geometries of blood vessels in collagen hydrogel at different concentrations to mimic the biomechanical properties of tissues varying from normal to tumor under the growth factor stimulation. Finally, we have designed and fabricated a silicon-based transmigration well with a 30um-thick membrane and 8um pores. This platform includes a deep microfluidic channel on the back-side sealed with a glass wafer. The migratory behavior of highly metastatic breast cancer cells, MDA-MB-231, is tested under different drug treatment conditions. This versatile platform will enable the application of more complex fluidic circulation profile, enhanced integration with other technologies, and running multiple assays simultaneously. / Ph. D.