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Background and Aim of the study: Lung cancer is the third most common cancer in the UK. A significant number of these patients are diagnosed with inoperable advanced-stage non-small cell lung cancer. Until recently, the standard of care for these patients was radiotherapy with or without chemotherapy but the overall survival remained poor, with a 5-year survival of only 13%. Recent studies showed significant improvement in overallsurvival in patients who are suitable for, and have received, immunotherapy, in addition to chemotherapy and radiotherapy. Radiotherapy aims to deliver tumoricidal doses to the target volume whilst minimising doses to the surrounding organs at risk (OAR). However, achieving this goal could be challenging especially when treating advanced-stage tumours, as it could increase OAR doses and increase toxicities to an unacceptable level. Furthermore, several factors affect the achieved dose distribution including, patient's geometry, treatment technique, planner's experience, beam geometry, optimisation parameters, and interventions used during treatments. It is therefore important to develop methods to personalise treatment plan optimisation for advanced-stage non-small cell lung cancer (NSCLC) patients to achieve minimum OAR doses without compromising target doses using patient-specific parameters. This study aims to develop knowledge-based planning models (KBP) using patient-specific factors todetermine personalised treatment planning optimisation for advanced-stage NSCLC patients treated with volumetric modulated arc therapy (VMAT) to reduce OAR doses whilst delivering intended doses to the target volume. Methods: Four KBP models were developed using patient-specific dose and volume parameters to predict minimum achievable OAR doses, identify optimal arc parameters, trigger adaptive radiotherapy and estimate doses to adapted gross tumour volume using patients' geometry. The KBP models were verified using independent patient data sets. Change in treatment plan optimisation could increase modulation and affect plandeliverability therefore, several modulation indexes were calculated and plans were measured on the clinical linear accelerator to assess the effect of change in optimisation on treatment plan delivery. Results: The KBP models developed showed that relatively simple models can predict OAR doses and arc parameters and help identify patients for adaptive radiotherapy. The models can accurately estimate personalised and progressive dose escalation. The KBP resulted in a significant reduction in plan variability in all three studied dosimetric parameters, volume of lungs receiving 5Gy (V5), 20Gy (V20) and mean lung dose (MLD)by 4.9% (p=0.007, 10.8% to 5.9%), 1.3% (p=0.038, 4.0% to 2.7%) and 0.9Gy (p=0.012, 2.5Gy to 1.6Gy), respectively. The individualised arc geometry resulted in a significant reduction in lungs (V5 = - 15.1%, MLD = - 1.0Gy) and heart (MHD = - 1.4Gy) doses without compromising target coverage. The models, which were developed to predict changes in PTV coverage (ΔV95^PTV) using a specific biomarker (Programmed death-ligand 1 (PD-L1 expression)) and the difference in 'planning' and 'fraction' planning target volume (PTV) centre of the mass (characterised by mean square difference, MSD), could predict change in PTV coverage within ℗ł 1.0% for 77% of the total fractions. Furthermore, the models developed for predicting personalised and progressive dose escalation predicted doses within 0.4% and 0.7% respectively. Additionally, the plan complexity and deliverability measurements show that plan complexity could increase but may not affect treatment delivery significantly. Conclusion: The studies performed show that altering the 'standard' treatment planning optimisation approach could significantly reduce OAR doses and improve target coverage. This will help reduce toxicities and improve local control and overall survival and outcome for inoperable advanced-stage NSCLC patients.

One in two people born after 1960 will develop cancer, with 90% of all cancer deaths arising from metastasis. Conventional 2D in vitro metastasis models do not fully replicate tumour complexity. In vivo models can address tumour complexity, but do not fully represent human tumour biology. Multicellular spheroids are widely used 3D models of cancer. Spheroids contain internal zonal differentiation of oxygen, metabolite, and nutrient gradients, associated with regions of proliferative, quiescent, and necrotic cells. Current in vitro static spheroid methodologies do not recapitulate factors of cellular spread including continuous flow or shear stress. Therefore, better 3D in vitro models to investigate metastasis represent an area of unmet need. This research aims to develop spheroid-on-chip models, providing a novel strategy to investigate cancer spread. The microfluidic devices used in this study feature weirs for spheroid inclusion, and a borosilicate base coverslip for optical clarity. The devices feature an access port allowing direct access to the microwell. Spheroids derived from established cancer cell lines, MCF7 and U-87 MG, were formed off-chip and incorporated into the device by pipetting, before being perfused with complete media at 3 μL min-1 for 72 h. Cell viability was assessed in effluent, using the CytoTox Glo assay, demonstrating spheroid viability is robustly maintained on-chip. In situ analysis of cell viability, through FDA/PI live/dead staining indicated an increased proportion of viable cells and decreased dead cells on-chip compared to off-chip. VEGF (Vascular Endothelial Growth Factor) ELISA showed that VEGF secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. The chip model has been further developed to allow spheroids to be embedded in ECM (extracellular matrix)-like matrices. VEGF ELISA and IL-6 ELISA showed that both VEGF and IL-6 secretion, as evaluated by its presence in conditioned media, was comparable across all testing conditions. ELISA also showed that IL-6 and VEGF was increased in the on-chip models within hydrogel conditions. The device has also allowed the direct imaging of spheroids on chip over 72 h. The analysis of U-87 MG spheroids on chip showed that invasion through Matrigel was comparable to the off-chip static models. Whilst migration on chip, through collagen analysis, was increased over the off-chip counterparts. The work shown offers a novel insight into cancer metastasis; on a more replicative model than the current conventional in vitro techniques.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is the second deadliest form of cancer within the UK. The majority of CRC arises sporadically, as a result of epigenetic alterations transforming normal epithelium to malignant adenocarcinoma. CRC is highly metastatic and as a solid state tumour possesses regions of hypoxia, which promote metastasis. There is evidence for the involvement of the A Disintegrin And Metalloproteinase (ADAM) family in cancer progression and the hypoxic tumour microenvironment, however ADAM 10 is one of the least characterised members in the context of hypoxia-mediated cancer progression. ADAM 10 is strongly implicated in the regulation of normal colon biology, with an ADAM 10 knockout mouse model displaying embryonic lethality. Therefore, ADAM 10 is a key target of CRC research, to determine what effect hypoxia has on ADAM 10 and what implications this may have in the context of CRC progression. In this study, CRC cell lines were examined to characterise the effects of severe hypoxia on ADAM 10. Exposure to severe hypoxia induced an alteration to ADAM 10 expression at both protein and transcript levels, with a previously un-reported doublet band seen for mature ADAM 10 at protein level. The doublet is believed to be a hypoxia-mediated post-translational modification, potentially through alterations in ADAM 10 glycosylation. No effects of either severe hypoxia or ADAM 10 knockdown were seen on CRC cellular migration. However, a downregulation in cellular viability was observed in HCT116 cells after ADAM 10 knockdown, independently of hypoxia, indicating a role of ADAM 10 in the regulation of CRC cell viability. Attempts to elucidate the underlying mechanisms behind this revealed a role for ADAM 10 in the regulation of c-MYC and CCND1 expression in HCT116 cells, however no effect on cell cycle progression was observed after ADAM 10 knockdown. This study has identified an important role for ADAM 10, independently of hypoxia, in the regulation of CRC viability, which may promote the progression and metastasis of CRC.

Breast cancer is the second most represented cancer type worldwide and will affect 1 in 8 women in the UK. Overall patient survival can reach 90% when the disease is diagnosed early but rapidly drops as the tumour progresses and metastasises. The steps required for tumour formation are known but numerous factors are involved and the specific mechanisms controlling this phenomenon are still poorly understood. WSB-1 is a hypoxia-responsive E3 ubiquitin ligase which was found to be upregulated in metastatic tissues, compared to normal or non invasive tissues. In addition, studies demonstrated that WSB-1 was involved in pancreatic cancer, neuroblastoma, and osteosarcoma progression. However, its role in breast cancer has not been particularly studied. The objective of this thesis is to study the role of WSB-1 in breast cancer. Initially, level of WSB1 in patients' tumour cDNA samples was evaluated according to known clinical and biological variables. Impact of high WSB1 levels on patients' distant metastasis-free survival, relapse-free survival and overall survival was also investigated. Then, the effect of WSB-1 knockdown on protein levels of epithelial to mesenchymal transition markers, as well as several matrix metalloproteinases (MMP) transcript and protein levels, and activity was examined. Consequence of these modifications on cell motility (migration, invasion) was studied, using single cells, cellular monolayers and spheroids. Finally, affinity purification followed by mass spectrometry was used to identify novel WSB-1 partners. Overall, in MDA-MB-231 cells, WSB-1 appeared to drive metastasis formation by upregulating MMPs expression and activity, promoting EMT and inducing invasiveness. WSB-1 had a more conflicting effect in MCF7 cells. In fact, WSB-1 effect appeared to be depending on the ER- and ER+ status. Together, these results validated WSB-1 as an important player in breast cancer development, particularly in metastasis formation.