This thesis considers whether the alteration of telomere function by manipulation of the telomerase enzyme can affect the radiosensitivity of in vivo derived SCCHN cells and hence whether telomere dysfunction inducing strategies are likely to be synergistic with ionising radiation in the management of SCCHN. The role of telomerase in radioprotecting cancer cells is investigated using the ectopic expression of hTERT in cell lines with high and low levels of telomerase. The effect of inhibiting telomerase expression is examined using a dominant negative telomerase gene. This approach had little success in SCCHN cells and so further experiments designed to elaborate the effect of telomerase inhibition on radiosensitivity were carried out using the small molecule reverse transcriptase inhibitor 3’ – azido, 3’ – deoxythymidine (AZT). These showed both telomerase suppression and increased radiosensitivity in cells exposed to AZT in culture. Other suggested factors which may affect the success of radiotherapy for cancer include the missense mutation of the p53 gene. A common polymorphism at codon 72 gives rise to Arginine or proline forms of the protein. This thesis investigates whether this variation affects radiosensitivity in SCCHN cells by assessing a panel of in vivo derived SCCHN cell lines. If the level of telomerase expression does not impact on radiosensitivity, then the use of antitelomerase strategies may be less effective with higher levels of telomerase expression in tumours. Continued selective pressure during tumour progression may mean the emergence of clonal variants with improved telomere function via greater levels of telomerase. This is investigated by anaphase bridge scoring of primary and recurrent archival tumour material. Analysis of cells from primary lesions and then from recurrent disease in the same patient provides information in this regard.

Microarray analysis was performed on 32 head and neck keratinocytes cultures using Affymetrix U133A/B genechips. The panel of cultures included normal cells, mortal and immortal cultures of dysplastic keratinocytes and mortal and immortal cultures from carcinomas, all grown to a standard protocol. The overall GEP revealed that many of the well-established HNSCC molecular markers associated with motility and invasion were up-regulated in the mortal cells, particularly in the mortal carcinomas. Immortal NHSCC cells showed elevated expression of cell-cycle markers and loss of differentiation markers. In addition, a small number of common changes in gene expression in all the carcinomas, regardless of replicative fate, were identified. This included several transcription factors. A series of 49 novel gene expression changes consistently associated with immortality in dysplastic keratinocytes and SCCs were identified. The list included genes involves in cell cycle control, signalling, cellular metabolism and maintenance of cellular structure. Validation of the expression of these genes by western blot demonstrated that, in general, the protein expression of genes agreed with the RNA expression level from the microarray data. However, some heterogeneity was evident. The mortal and immortal gene expression signatures were validated by IHC in the tumours from which the cultures were derived. The tumours that gave rise to immortal cell cultures demonstrated a relatively uniform pattern of staining in relation to the novel markers of immortality. However, those tumours which gave rise to mortal cultures exhibited significant heterogeneity of gene expression pattern, with areas characteristic of both the mortal and immortal phenotype present. These novel markers give us further insight into the mechanisms and importance of keratinocytes immortalization. Surrogate markers of immortality could therefore be valuable for assessment of prognosis and therapy if confirmed in larger in vivo studies.

Background Breast cancer is highly prevalent in older women. Although surgery is the main initial treatment for younger population, research has demonstrated that older women with primary breast cancer have different tumour biology, comorbidities, and patient preferences from younger patients, and hence may benefit from primary endocrine therapy (PETx), such as Tamoxifen and aromatase inhibitors (AIs). However, there is no cost-effectiveness study that compares the use of different treatment strategies in older women. Aim of study This study aimed to evaluate the clinical and cost-effectiveness of using PETx against surgery as the initial treatment strategy in older women with primary breast cancer. Methods This research included four stages. First, a retrospective cohort study was conducted to describe the treatment pathways, and evaluate the effectiveness and costs associated with using different treatment strategies by using a longitudinal database of 1,759 older women, who were diagnosed with primary breast cancer at ages over 70 years old in breast cancer units in Nottingham. Regression analyses, including survival analysis and generalised linear model, were used to identify the covariates, the predict death rates and costs, respectively. Second, a systematic review of economic evaluation studies was conducted using NHS Economic Evaluation Database, Cochrane Library, Ovid Medline, PubMed, and EMBASE to identify full economic evaluations that compared different treatment strategies in postmenopausal women with primary breast cancer. Quality and modelling methodologies of the included studies were assessed and summarised. Finally, a Markov model was conducted from the systematic review to estimate the lifetime costs and quality adjusted life year (QALYs) associated with the use of PETx as against surgery in a hypothetical cohort of older women with primary breast cancer and ER-positive status. The analysis took the UK NHS perspective with a lifetime horizon. Transition probabilities were estimated using parametric survival models derived from the longitudinal database. Resource use and costs were assessed in British pounds of the year 2014 using the longitudinal database. Utilities were derived from literature, including Prescott et al. (2007) (1), Fallowfield et al. (1994), and Peasgood et al. (2010)(2). Both costs and outcomes were discounted by 3.5% annually. A subgroup analysis was conducted for patients with higher oestrogen receptor content (H-score >250 out of 300). PSA was conducted and aggregated results were presented in cost-effectiveness plane and cost-effectiveness acceptability curve. Sensitivity analysis was also conducted to investigate the impact of uncertain parameters and model assumptions. Results Retrospective analysis of the cohort study found that most patients received surgery (n=350; 52.08%) or PETx (n=322; 47.92%) as the initial treatment strategy. The most common adjuvant therapy was endocrine therapy (n=164; 46.86%). Comparing to those received surgery as an initial treatment, patients who received PETx had a significantly higher adjusted risk of breast cancer-related death (HR: 2.15; 95%CI: 1.03, 4.46), and lower costs (mean difference: -£3558.87; 95%CI: -£4018.11, -£3105.39). Overall, 29 economic evaluations were included from the systematic reviews and studies that assessed surgery and none assessed PETx as the initial treatment. Most of the included economic studies used a Markov model with lifetime horizon and one-year cycle length. Nine studies which included subgroup analysis for older women (over 65 years old) used similar economic models and transition states to those that were used for younger women (50 to 65 years old) with primary breast cancer. The key disease-related health states were disease-free, recurrence, and death. Recurrence was mostly separated into locoregional and distant recurrence. These findings were used to design a Markov model in the following decision modelling. In the base-case analysis of decision analytical modelling, the deterministic results showed that patients who received PETx as the initial treatment had lower costs and QALYs compared with patients who received surgery; the mean ICUR was £194 per QALY. However, for patients with H-score >250, PETx was associated with higher costs and lower QALYs; the mean ICUR was -£1849 per QALY. In the probabilistic sensitivity analysis, PETx was associated with lower costs and QALYs. The mean ICUR was £363 per QALY, 95%CI: -6014.99, 5,756.86). However, for patients with H-score >250, PETx was associated with higher costs and lower QALYs. The mean ICUR was -£390.67 per QALY (95%CI: -7,295, 7776.54). The probability of PETx being cost-effective in a situation where the decision-maker is willing to accept (WTA) £21,000 per QALY was 24%. In the subgroup analysis, PETx was associated with slightly lower QALYs and higher costs. The probability of PETx being cost-effective in a situation where the decision-maker is willing to accept £21,000 per QALY was 43%. Conclusion The main findings from this study showed that PETx was not cost-effective compared to surgery. However, this study was mainly based on the effectiveness and cost from a single observational study in a secondary care setting, and other information (evidence) that may bias the effectiveness and cost estimates, including frailty status, adherence, comorbidities, and other unmeasured confounders were not recorded, and thus, may bias the cost-effectiveness results. Future research is recommended to collect further information on patients’ characteristics, including frailty, adherence, and adverse events that may influence the cost-effectiveness results. Moreover, collecting detailed estimation of the resource use in order to provide an accurate estimate for costs.

Background and objectives: An increasing number of cancer patients have described experiencing cognitive dysfunctions which are associated with their chemotherapy treatment. This has been called “chemobrain” in patient forums and articles. It has been shown in animal models treated with a range of chemotherapy agents that chemotherapy induces cognitive deficits and a decrease in hippocampal neurogenesis. Antidepressants, anti-inflammatory drugs and physical activity have been proposed to counteract the effect of chemotherapy and provide protection against cognitive impairment associated with cancer treatments. This thesis has used in vivo studies, in rats and Sox1-GFP transgenic mice to firstly evaluate the effect of acute and chronic 5-Fluoruracil (5-FU) treatment on different subpopulations of neural stem cells in the hippocampus. Secondly, the effect of prior treatment with the antidepressant fluoxetine and the anti-inflammatory indomethacin on the impact of chronic treatment with chemotherapy was measured in terms of their effect on cell proliferation, inflammation and DNA damage. The effect of prior physical exercise on acute treatment with 5-Fluoruracil was measured in terms of cell proliferation and differentiation. Methods: Animals were administrated either acute (single injection) or chronic (4 to 6 injections) of 5-FU with or without fluoxetine and indomethacin or physical exercise. Cell proliferation (Ki67), early neural differentiation (DCX), neural stem cells markers (GFAP, Sox1), inflammation (Iba1, Cox2) and DNA damage (γ-H2AX) were quantified by immunostaining either 24hr or 1 week after the last injection of chemotherapy. Morphological studies, to differentiate different stages of neural stem cells development and the state of microglia activation were conducted using confocal imaging. 3 Results: Acute treatment of Sox1-GFP mice with 5-FU caused a decrease in cell proliferation while chronic treatment induced a depletion of the neural stem cell pool and a reduction of the number of microglia. Prior chronic treatment with indomethacin did not prevent the decrease in the number of neural stem cells but did prevent the decline in cell proliferation and microglia. Rats chronically injected with chemotherapy and allowed to recover for 1 week, showed a decreased proliferation and number of microglia, an increased microglia activation, Cox-2 expression and DNA damage. Prior chronic treatment with fluoxetine prevent the decrease in proliferation and number of microglia, the increased microglia activation and DNA damage. Prior treatment with fluoxetine and indomethacin prevented these effects. Physical exercise prior to acute treatment with 5-FU enhanced the overall amount of neurogenesis but, possibly, due to a low dose of chemotherapy it was not possible see a significant effect of the chemotherapy. Conclusions: The chemotherapy agent 5-FU affected the neural stem cell pool and the number of proliferating cells in the hippocampus. Chronic administration with fluoxetine can prevent the decrease in the number of proliferating cells, microglia activation and DNA damage while treatment with Indomethacin protects only against the effect of 5-FU on proliferating cells and microglia activation.

The extracellular matrix (ECM) has emerged as a major regulator of cell behaviours. Changes in extracellular matrix, especially its composition, organization/ dimensionality, and rigidity have been implicated in various aspects of cellular functions including cell growth, migration, and differentiation. In my thesis, I have focused on the effect of two biophysical properties of the extracellular matrix namely dimensionality and rigidity in the inflammatory and fibrotic pathologies of human lung. To study the role of matrix dimensionality, firstly electrospun scaffold based three-dimensional (3D) culture with similar architecture of human lung was developed. By applying this 3D model, inflammatory response was studied in an in vivo like environment by using NF-κB transcription factor activation as a tool for probing inflammatory response in human lung fibroblasts. According to my observations, it was confirmed that the matrix dimensionality together with spatial organisation of cells is crucial in lung inflammatory response, evidenced by the observation of the differences in the level and pattern of inflammatory response between 2D and 3D culture systems. To study the role of matrix rigidity in progression of lung fibrosis, we developed the ECM-based hydrogel platform with tuneable stiffness level relevant to normal and fibrotic lung. By using this disease relevant platform, I have shown that stiff matrix but not soft matrix can induce the myofibroblast differentiation and fibroblast proliferation, the two major features of lung fibrosis. To date, the molecular mechanisms underpinning this cellular mechanosensing process in response to matrix stiffening remains unknown. To achieve this, I further investigated the involvement of two potential mechanosensitive signalling pathways namely, Rho associated coiled coil forming kinase (ROCK) signalling and talin- (focal adhesion adaptor) signalling in this process. Interestingly, my data show that ROCK signalling differentially regulated stiffness induced myofibroblast differentiation between soft normal and stiff fibrotic matrix. Moreover, both ROCK isoforms 1 and 2 are synergistically important in myofibroblast differentiation driven by rigid matrix and the absence of one ROCK isoform can exaggerate myofibroblast differentiation on stiff fibrotic matrix. Regarding talin signalling, my preliminary data confirms that talin1 can control both stiffness induced fibroblast proliferation and myofibroblasts differentiation on stiff matrix. In contrast to talin1, talin2 showed a protective role in controlling myofibroblast differentiation. In conclusion, we have successfully developed two in vitro lung models for studying the effect of matrix dimensionality and rigidity in lung inflammation and fibrosis. Overall my PhD work has elucidated the significant contribution of biophysical cues of external cellular environment in lung inflammation and fibrosis.

Plant natural products have played a pivotal role in the discovery and development of many new drugs for the treatment of various infectious diseases and cancers. The present study was therefore aimed at isolation and identification of novel bioactive compounds from selected plants collected in Malaysia with anti-cancer activity. Plant crude extracts were obtained using solvent extraction methods, while various chromatographic and spectroscopic methods were employed for compound isolation and structure elucidation. Evaluation of pure compounds and crude extracts for anti-cancer activity involved the use of Neutral Red assay (NR), acradine orange – ethidium bromide (AO/EB) staining and cell cycle analysis. Phytochemical investigations of five Malaysian plants, namely, Ficus hispida, F. fistulosa, F. schwarzii, Artocarpus heterophyllus x integer and Macaranga hypoleuca, with the focus on alkaloids and flavonoids, have resulted in the isolation of a total of 24 compounds, of which six are new. The leaf and stem-bark extracts of Ficus hispida yielded two new alkaloids, hispidacine (1) and hispiloscine (2), and a known alkaloid, 13a(S)-(+)-deoxypergularinine (3). Hispidacine represents the first example of an 8,4'-oxyneolignan incorporated an unusual 2-hydroxyethylamine moiety, while hispiloscine represents the first naturally occurring phenanthroindolizidine alkaloid with acetoxy substitution. The leaf and bark extracts of Ficus fistulosa provided two new septicine alkaloids, fistulopsines A and B (4 and 5), together with four known phenanthroindolizidine alkaloids, 13a(R)-(–)-3,6-didemethylisotylocrebrine (6), 13a(S)-(+)-tylocrebrine (7), 13a(S)-(+)-tylophorine (8) and 13a(S)-(+)-septicine (9), and one known non-alkaloid (vomifoliol, 10). The leaves of Ficus schwarzii gave two novel tri-nor-sesquilignan alkaloids, schwarzinicines A and B (11 and 12). The bark of Artocarpus heterophyllus x integer yielded five known compounds, of which four are prenylated flavonoids, namely, cudraflavone C (13), artocarpetin A (14), cycloheterophyllin (15) and artonin J (16), and one natural xanthone, lichexanthone (17). The leaves of Macaranga hypoleuca provided seven known compounds, of which three are flavonoid glycosides, namely, quercetin-3-O--L-arabinopyranoside (18), quercetin-3-O--L-arabinofuranoside (19) and quercetin-3-O-β-D-galactoside (20), three are flavonoid aglycones, namely, quercetin (21), kaempherol (22), and 5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)chroman-4-one (23), and one sterol 3-epi-taraxerol (24). Preliminary screening by NR assay found that the crude extracts (except for Artocarpus heterophyllus x integer) showed growth inhibitory activities against human breast (MDA-MB-231 and MCF-7), lung (A549), and colon (HCT-116) cancer cell lines. Of the 24 pure compounds obtained, hispiloscine (2), fistulopsine A (4), fistulopsine B (5) and cudraflavone C (13) were found to show growth inhibitory activity against colon (HCT-116) and breast (MCF-7) cancer cells. Furthermore, 4 and 5 were found to dominantly arrest cells in G1 phase of the cell cycle without the induction of apoptosis. Cell cycle perturbation of these compounds was found to be reversible for HCT-116 cells at the onset of 72 hours. These results suggest that 4 and 5 have the potential to be further exploited for the development of new anti-cancer agents.

Morphological analysis of the appearance and quantity of cells and tissue architecture has been routinely used by pathologists to determine cancer’s histologic grade. In standard practice, however, the analysis is based on only a few morphological features, and is mainly done in a qualitative or semi-quantitative fashion. This makes the analysis subjective and less reproducible. In fact, a single piece of cancer tissue can contain a high level of heterogeneity – two cells of the same type can behave differently depending on their genetic makeup and their microenvironment. This simply means that there is still a vast amount of information hidden in cancer tissues, whose clinical value is not fully realised. With digital slide scanning technology becoming more available, it is now possible to obtain a whole-slide image of a histological section of high-quality and with high-resolution (multi-gigapixel images) in a digital format. Moreover, the volume of data is rapidly growing daily as slides are routinely scanned. This presents an opportunity to advance image analytical techniques and computational algorithms for quantitative analysis of tissue morphology (morphometrics). Morphometrics provides an accurate and reproducible means for the diagnosis and prognostication of cancers. This is the very first step towards effective treatment decision making and personalised medication. In the first part of this thesis, we focus on the development of automated algorithms for quantifying morphological features from histological sections. All the algorithms are specifically designed for histological sections stained by a standard haematoxylin and eosin (H&E) stain. Firstly, we proposed a discriminative dictionary learning based algorithm for learning the appearance of cells. The approach is applied to distinguish mitotic cells from non-mitotic cells in breast cancer histological images. Secondly, we develop a deep learning based framework that utilises a local context information for nuclear detection and classification. We demonstrate the effectiveness of this framework for detecting and classifying various types of cells in colorectal cancer. Lastly, we consider the intestinal gland segmentation problem in histological images of colorectal cancer. We formulate a segmentation algorithm that finds the best polygon matching the shape of each gland in a Bayesian framework. The method is capable of segmenting glands in both benign and malignant cases. In the second part of this thesis, we turn toward a quantitive analysis of tissue morphology of colorectal adenocarcinomas. This is to find potential morphological features that are clinically relevant. We extract morphological features, using the algorithms developed in the first part of this thesis. The results from statistical analyses show that a quantitative measure for aberrance of glandular shape is strongly associated with the histologic grade, and a quantitative measure related to the desmoplasia is associated with the tendency to develop metastasis.

Breast cancer, the most commonly diagnosed type of cancer in women, is a major cause of morbidity and mortality in the western world. Well-established risk factors of breast cancer are mostly related to women’s reproductive history, such as early menarche, late first pregnancy and late menopause. Survival rates have improved due to a combination of factors, including better health education, early detection with large-scale use of screening mammogram, improved surgical techniques, as well as widespread use of adjuvant therapy. At initial presentation, clinicopathological features of breast cancer such as age, nodal status, tumour size, tumour grade, and hormonal receptor status are considered to be the standard prognostic and predictive markers of patient survival, and are used to guide appropriate treatment strategies. Lymphovascular invasion (LBVI), including lymphatic (LVI) and blood (BVI) vessel invasion, has been reported to be prognostic and merit accurate evaluation, particularly in patients with node negative tumours who might benefit from adjuvant chemotherapy. There is a lack of standard assessment and agreement on distinguishing LVI from BVI despite the major challenges in the field. A systematic review of the literatures, examining methods of detection and the prognostic significance of LBVI, LVI and BVI, was carried out. The majority of studies used haematoxylin and eosin (H&E) and classical histochemistry to identify LVI and BVI. Only few recent studies used immunohistochemistry (IHC) staining of the endothelium lining lymphatic and blood vessels, and were able to show clear differences between LVI and BVI. The prognostic significance of LBVI and LVI was well-documented and strongly associated with aggressive features of breast tumours, while the prognostic value and the optimal detection method of BVI were unclear. Assessment and prognostic value of LBVI on H&E sections (LBVIH&E) was examined and compared to that of LVI and BVI detected using IHC with D2-40 for LVI (LVID2–40) and Factor VIII for BVI (BVIFVIII) in patients with breast cancer including node negative and triple negative patients (n=360). LBVIH&E, LVID2–40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node negative patients (206), LBVIH&E, LVID2–40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple negative patients (102), LBVIH&E, LVID2–40 and BVIFVIII were present in 35 (29%), 36 (35%) and 14 (14%) respectively. LBVIH&E, LVID2–40 and BVIFVIII were all significantly associated with tumour recurrence in all cohorts. On multivariate survival analysis, only LVID2–40 and BVIFVIII were independent predictors of cancer specific survival (CSS) in the whole cohort (P=0.022 and P<0.001 respectively), node negative (P=0.008 and P=0.001 respectively) and triple negative patients (P=0.014 and P<0.001 respectively). Assessment of LVI and BVI by IHC, using D2-40 and Factor VIII, improves prediction of outcome in patients with node negative and triple negative breast cancer and was superior to the conventional detection method. Breast cancer is recognised as a complex molecular disease and histologically identical tumours may have highly variable outcomes, including different responses to therapy. Therefore, there is a compelling need for new prognostic and predictive markers helpful of selecting patients at risk and patients with aggressive diseases who might benefit from adjuvant and targeted therapy. It is increasingly recognised that the development and progression of human breast cancer is not only determined by genetically abnormal cells, but also dependent on complex interactions between malignant cells and the surrounding microenvironment. This has led to reconsider the features of tumour microenvironment as potential predictive and prognostic markers. Among these markers, tumour stroma percentage (TSP) and tumour budding, as well as local tumour inflammatory infiltrate have received recent attention. In particular, the local environment of cytokines, proteases, angiogenic and growth factors secreted by inflammatory cells and stromal fibroblasts has identified crucial roles in facilitating tumour growth, and metastasis of cancer cells through lymphatic and/or blood vessel invasion. This might help understand the underlying process promoting tumour invasion into these vessels. An increase in the proportion of tumour stroma and an increase in the dissociation of tumour cells have been associated with poorer survival in a number of solid tumours, including breast cancer. However, the interrelationship between these variables and other features of the tumour microenvironment in different subgroups of breast cancer are not clear. Also, whether their prognostic values are independent of other components of the tumour microenvironment have yet to be identified. Therefore, the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease was examined in patients with invasive ductal breast cancer (n=361). The TSP was assessed on the haematoxylin and eosin-stained tissue sections. With a cut-off value of 50% TSP, patients with ≤50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had involved lymph node (P=0.049), Her-2 positive tumours (P=0.029), low-grade peri-tumoural inflammatory infiltrate (P=0.034), low CD68+ macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter CSS (P<0.001). In node negative patients (n=207), high TSP was associated with low CD68+ macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter CSS (P=0.005). In triple negative patients (n=103), high TSP was associated with increased tumour size (P=0.017) high tumour grade (P=0.014), low CD8+ T-lymphocytes infiltrate (P=0.048) and shorter CSS (P=0.041). The 15-year cancer specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. On multivariate survival analysis, a high TSP was associated with reduced CSS in the whole cohort (P=0.007), node negative patients (P=0.005) and those who received systemic adjuvant therapy (P=0.016), independent of other pathological characteristics including local host inflammatory responses. Therefore, a high TSP in invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with invasive ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with breast cancer. Similarly, the relationship between tumour budding, clinicopathological characteristics and outcome was examined in patients with invasive ductal breast cancer (n=474), using routine pathological sections. Tumour budding was associated with several adverse pathological characteristics, including positive lymph node (P=0.009), presence of LVI (P<0.001), and high TSP (P=0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.002). In node negative patients, a high tumour budding was associated with presence of LVI (P<0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.038). On multivariate survival analysis, tumour budding was associated with reduced CSS (P=0.001), independent of nodal status, tumour necrosis, CD8+ and CD138+ inflammatory cells infiltrate, LVI, BVI and TSP. / Furthermore, tumour budding was independently associated with reduced CSS in node negative patients (P=0.004) and in those who have low TSP (P=0.003) and high-grade peri-tumoural inflammatory infiltrative (P=0.012). A high tumour budding was significantly associated with shorter CSS in luminal B and triple negative breast cancer subtypes (all P<0.001). Therefore, tumour budding was a significant predictor of poor survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. These results suggest that tumour budding may promote disease progression through a direct effect on local and distant invasion into lymph nodes and lymphatic vessels. Therefore, detection of tumour buds at the stroma invasive front might therefore represent a morphologic link between tumour progression, lymphatic invasion, spread of tumour cells to regional lymph nodes, and the establishment of metastatic dissemination. Given the potential importance of the tumour microenvironment, the characterisation of intracellular signalling pathways is important in the tumour microenvironment and is of considerable interest. One plausible signalling molecule that links tumour stroma, inflammatory cell infiltrate and tumour budding is the signal transducer and activator of transcription (STAT). The present results suggest that STATs may affect disease outcome through direct impact on tumour cells, and the surrounding microenvironment. The above observations of the present thesis point to the importance of the tumour microenvironment in promoting tumour budding, LVI and BVI. The observations from STATs work may suggest that an important driving mechanism for the above associations is the presence of tumour necrosis, probably secondary to hypoxia. Further work is needed to examine the interaction of other molecular pathways involved in the tumour microenvironment, such as HIF and NFkB in patients with invasive ductal breast cancer.

Smooth muscle (SM) tissue is found in many parts of the body, primarily in sheets or bundles surrounding hollow organs. The main function of the tissue is the regulation of organ tone via its contractile state. Dysfunction of SM in diseases such as asthma and atherosclerosis affect millions worldwide. Current methods for studying SM primarily rely on ex vivo animal tissues or 2D in vitro models. Animal models do not accurately recreate human disease states and 2D models are cultured on stiff surfaces lacking the elastic properties and 3D morphology found in natural extracellular matrix in vivo. Therefore it is desirable to develop both an in vitro model of SM that possesses the ability to contract and a method in which this contraction can be measured. In order to achieve this, primary rat aortic SM cells and primary human airway SM cells were cultured in collagen hydrogels; both free floating in the form of collagen disks and under uniaxial tension in order to generate aligned SM collagen constructs. When stimulated with contractile agonists, these constructs contract in a uniaxial fashion. The design of the constructs allows them to be attached to a force transducer allowing the physical force of contraction to be measured. The force of contraction was dependant on the agonist concentration and could be antagonised by the presence of an L-type calcium channel blocker. In order to improve the alignment and uniformity of the smooth muscle population a range of aligned electrospun scaffolds were produced from polyethylene terephthalate (PET), cross-linked gelatin and cross-linked gelatin methacrylate (GelMa). The average fibre diameter of the scaffolds ranged from approximately 200 nm to several micrometres. Additionally the Young’s moduli of the scaffolds ranged from around 1x105 to 1x108 Pa. In all cases, scaffolds were highly aligned; alignment was achieved by using a rapidly rotating collector mandrel. Culture of primary SM cells upon these scaffolds showed that the cells readily adhered to and proliferated upon the scaffolds over a 10 day culture period. The cells formed a highly aligned population following the topographical cues of the aligned fibrous scaffolds. Additionally, the cells stained positive for SM markers in all cases, indicative of a contractile phenotype. When stimulated with 100 µM UTP, the SM cells were able to contract the gelatin and GelMa scaffolds but not the PET scaffolds. SM seeded GelMa scaffolds were cultured for 10 days prior to attachment to the previously mentioned force transducer apparatus. Upon stimulation the seeded scaffolds contracted generating forces greater than those achieved by the hydrogel model, and was reproducible over several experiments.

Acute myeloid leukaemia (AML) affects about 3000 people in the UK each year. Chemotherapy leads to complete remission in most patients but, up to 70% relapse. The success of allogeneic stem cell transplant (SCT) demonstrates that donor T cells can eliminate residual tumour through a graft-versus-leukaemia effect however, SCT is only viable in a minority of patients. Responses seen with alternative approaches, such as adoptive immunotherapy with tumour specific T cells or inducing T cells by vaccination, have been disappointing, highlighting a clear need for new immunotherapeutic approaches. Immune responses against leukaemia are inhibited by a range of suppressive mechanisms. Identification of novel therapeutic targets capable of unleashing natural and effective immune responses is critical. The complement system is an enzyme cascade with diverse effector functions instituted by both the innate and adaptive immune systems. Recent evidence suggests that complement promotes the progression of malignancy. The role of complement in AML is largely unexplored but the limited data available implies that complement could have a critical role in both the initiation and progression of AML. Sub-lytic complement protects cells against further attack by both lytic doses of complement and other pore-formers including perforin, the cytolytic protein used by both NK and CD8+ T cells. This phenomenon termed complement-induced protection (CIP). Thus cancer cells exposed to sub-lytic complement may be protected from lysis by NK and CD8+ T cells with significant consequences for immune escape. When this hypothesis was tested using an in vitro model of NK cell killing, no evidence of protection against NK cell killing was observed despite clear CIP against further complement attack. Despite a strongly protective phenotype, subsequent microarray analysis revealed no genetic signature for CIP indicating that mechanisms underpinning CIP require no changes in gene expression. A mouse model was then used to test the hypothesis that complement promotes progression of AML. The use of both genetically modified mice lacking various complement components and complement inhibitors in wild-type animals, revealed that complement promotes AML progression. CIP was a potential mechanism by which tumours might escape immune elimination in vivo, however, this was only a small component of the almost complete protection observed in C3 deficient mice. Putative mechanisms include inhibition of CD8+ T cells and promoting AML seeding. Should complement prove to have a key role in human AML, available complement therapeutics could progress rapidly into human studies. These might not only have an independent role in improving spontaneous immune responses, but might also impact on all available or novel immune mediated therapies for AML.