The tumour suppressor p53 is at the centre of the signalling pathway that controls cellular processes crucial in tumourogenesis, cancer progression and tumour clearance. Alterations in the p53 pathway that lead to cancer progression can be good candidates for molecular biomarkers that would assist in the identification of patients with different prognoses, but also serve as good predictors of appropriate targeted therapies. Patient cohorts and cancer cell panels are utilised to seek associations with the attenuation of the p53 pathway and cancer progression. Firstly, the alternatively spliced transcript of the p53 inhibitor HDMX, which is frequently found in tumours with poor prognosis, is studied. The high ratio of the alternatively spliced HDMX-S transcript over the full-length HDMX-FL transcript (HDMX-S/FL) is demonstrated to associate with p53 pathway attenuation in cancer cells and breast carcinomas, and with faster metastatic progression of osteosarcoma and breast cancer patients. Secondly, inherited polymorphism in the HDMX gene is investigated and demonstrated as a unique and highly reproducible eQTL, which identifies patients with different prognoses for metastatic disease in breast cancer and melanoma cohorts. Lastly, a screening approach to identify novel inherited polymorphisms in the p53 pathway genes that associate with metastatic progression of melanoma is developed and implemented, and subsequently in silico and in vitro functional analyses are performed to investigate a mechanism behind the FOXO3 SNP, identified as the strongest candidate, whereby the experimental evidence demonstrate that the causal SNP in the FOXO3 haplotype is controlled by the GATA3 transcription factor. Together, the work presented in this thesis provides strong support for the role of the p53 pathway in the metastatic progression of cancer, and suggests that molecular biomarkers that can detect changes in the activity of p53 pathway genes could offer a robust set of biomarkers for cancer progression applicable to different types of cancer.

Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.

Cancer metastasis is the critical event leading to 90% of cancer related death. Although significant improvement in our understanding on cancer metastasis has been made through years of research, the fundamental mechanism behind this process is still not fully elucidated. For cancer researchers, the “gold standard” for metastasis studies has traditionally been the use of tissue culture and mouse models. Tissue culture offers the simplest system and ease of control but is not able to recapitulate many of the features found in an in vivo tumor microenvironment. On the other hand, mouse model systems offer the most sophisticated and physiologically relevant platforms for studying cancer. However, the lack of control over the in vivo environment in these mouse models and inherent discrepancies from human physiology make results from these models difficult to be translated to clinical trials. The advancement in microfabrication techniques and cancer models developed based on these techniques has shown potential in addressing the gap between in vitro tissue culture and mouse models. Microscopic tumor microenvironments could be built in these in vitro systems to study behavior of human cancer cells. However, the expertise involved in and extra instrumentation needed for implementing these systems have prevented their widespread use by general cancer researchers. In this dissertation, we developed two simple microfabricated systems and demonstrated their application in two aspects of cancer research. The first system is a microfabricated cell patterning stencil, where paracrine signaling can be established and its impact can be measured based on cell migration. Using this tool, we investigated the interaction between melanoma and microenvironmental cells from their common metastasis target organ. Through these simple patterning techniques, we observed significant effects that a given microenvironmental cell line had on the two different melanoma lines, as well as how melanoma affected different microenvironmental cell lines. The second system, a microfluidic device, is able to present individual soluble factors to cancer cells in order to test the response of cancer cells to these physiologically relevant factors. Through this stand-alone system, we found that breast cancer metastasis is influenced by the protein molecules secreted by themselves as well as the local glucose level. Through these findings we believe that our microfabricated systems can benefit the general cancer research community in which a complicated problem can be broken down into manageable pieces and studied on a simple platform in a controlled way. Observation made through these systems can inspire general cancer researchers to form new hypotheses and eventually lead to new findings. / 2017-02-17T00:00:00Z

Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.

Current treatment for spinal metastasis involves a multimodal approach, including bisphosphonates and radiation therapy. Yet, tumour response varies considerably, thus novel treatments or combination therapies are needed to treat these metastases while preserving stability and integrity of the spinal column. Photodynamic therapy (PDT) has been shown to be successful in destroying vertebral osteolytic tumours and enhancing vertebral structure, particularly in combination with bisphosphonates. This thesis aims to evaluate the longer-term effects of PDT alone and in combination with bisphosphonate or radiation therapy on healthy vertebrae, and the short-term effects of PDT combined with radiation therapy on healthy and metastatically-involved vertebrae. The benefits of PDT on vertebral structure, both at short-term and longer-term time-points, were greatest in combination with previous bisphosphonate therapy. Similar effects, to a lesser magnitude, were seen with PDT in combination with radiation therapy. This work supports future translation of PDT for the treatment of spinal metastases.

Current treatment for spinal metastasis involves a multimodal approach, including bisphosphonates and radiation therapy. Yet, tumour response varies considerably, thus novel treatments or combination therapies are needed to treat these metastases while preserving stability and integrity of the spinal column. Photodynamic therapy (PDT) has been shown to be successful in destroying vertebral osteolytic tumours and enhancing vertebral structure, particularly in combination with bisphosphonates. This thesis aims to evaluate the longer-term effects of PDT alone and in combination with bisphosphonate or radiation therapy on healthy vertebrae, and the short-term effects of PDT combined with radiation therapy on healthy and metastatically-involved vertebrae. The benefits of PDT on vertebral structure, both at short-term and longer-term time-points, were greatest in combination with previous bisphosphonate therapy. Similar effects, to a lesser magnitude, were seen with PDT in combination with radiation therapy. This work supports future translation of PDT for the treatment of spinal metastases.

<p>Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.</p>

Osteoblasts are highly active cells that are responsible for secreting bone forming components such as collagen type I and matricellular proteins that mediate collagen deposition and mineralisation. SPARC and osteopontin are matricellular proteins that are involved in bone regulation and cell-matrix interactions and are also upregulated in metastatic disease. Secretion of these proteins results in changes to the stromal environment that includes cell migration, angiogenesis, matrix degradation, matrix deposition, bone mineralisation and bone resorption. Signalling pathways not only lead to the expression of target proteins, but also have immediate early effects, for example, on cell adhesion. We asked if the ERK 1 and 2 module of the MAPK pathway was involved in the intracellular trafficking of SPARC and Osteopontin. Membrane trafficking is an essential process that ensures newly synthesised proteins pass from their site of synthesis to the extracellular environment. Using an inhibitor of ERK 1 and 2 activation (U0126), as well as siRNA directed against ERK 1 or 2 individually, a change in intracellular localisation of SPARC and osteopontin was observed in cells treated with U0126 and siRNA against ERK 2 alone, likely in or around the Golgi apparatus. Consistent with the observation above, analysis of protein secretion showed that there was a reduction of total protein secreted (30% reduction) when ERK 1 and 2 activation was prevented together or knock down of ERK 2 alone. A mechanism is proposed where ERK 2 is likely activating a substrate that is allowing SPARC and osteopontin to continue along the secretory pathway. This directly implicates ERK 2 as an important regulator of matricellular protein secretion in osteoblasts. In cancer, Ras mutations can lead to permanent activation of the MAPK pathway leading to cancer cell proliferation and survival, however, we propose another mechanism important in metastasis whereby ERK 2 activation is manipulated to facilitate secretion of matricellular proteins which can then mediate changes to the stromal environment that allow the tumour to metastasise successfully.

The objective of this work is to develop a novel 3-D biological particulate dynamics framework to simulate blood flow in the micro circulation. This entails the amalgamation of concepts from various fields namely blood flow dynamics, solid mechanics, fluid-structure interaction and computational data structures. It is envisioned that this project will serve as a harbinger for implementing a multi-scale simulation model with applications in a vast array of situations from blood flows in heart valves to studying cancer metastasis. The primary motivation for this work stems from the need for establishing a simple, effective and holistic framework for performing blood flow simulations, taking into account the extremely 3-D nature of flow, the particle interactions and fluid structure interaction between blood and its constituent elements. Many current models to simulate blood cells rely on finite element methods which render large scale simulations extremely computationally intensive. The development of a framework for simulating blood flow is tied together with achieving a framework for performing an investigation of cancer metastasis. Cancer initially develops at a primary site and spreads through the body to secondary sites using the circulatory systems of the body - the blood circulatory system and the lymphatic system. It is known that all the cancer cells that enter into the circulation do not survive the harsh environment, though the exact cause of this is still undetermined. Moreover, the mechanical properties of cancer cells are not well documented and appropriate computational models require that experiments be conducted to determine the same. Thus the end goal of this work is to establish a system to analyze and simulate 3-D blood particulate dynamics, including cancer cells, from a holistic standpoint in order to understand more about the phenomenon of blood flow as a whole, and cancer metastasis in particular.

Understanding the dynamics of micro- and nanometer-sized objects like molecules, particles, and living cells in biological systems and biomaterials has become a key component in biomedical research. Consequently, significant progress has been made for the development of imaging platforms, fluorescent probes, and computational tools to visualize and quantify biological processes at different length and time scales. However, despite such advances, achieving a reliable measurement accuracy on the dynamic behavior of these microscopic vehicles in diverse biological contexts is challenging. Subsequently, the motivation behind this dissertation is to develop new robust microfluidic velocimetry techniques to investigate molecular transport and cell migration within an in-vitro microfluidic platform.