Cellular and molecular consequences of S100A4-induced motility in rat breast tumour Rama 37 cells

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy

Funding/Support and role of the sponsor: None.

Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy

Funding/Support and role of the sponsor: None.

Total synthesis of furospongolide and related furanolipid analogues as potential anti-tumour agents

This thesis details the design, development and execution of innovative methodology in the total synthesis of the terpene-derived marine natural product, furospongolide. It also outlines the synthetic routes used to prepare a novel range of furanolipids derivatives and subsequent evaluation of their potential as antitumour agents. The first chapter is a review of the literature describing efforts undertaken towards the synthesis of biologically active furanosesterterpenoid marine natural products. A brief discussion on the sources and biological activity exhibited by furan natural products is also provided. In addition, a concise account of the role of hypoxia in cancer, and the increasing interest in HIF-1 inhibition as a target for chemotherapeutics is examined. The second chapter discusses the concise synthesis of the marine HIF-1 inhibitor furospongolide, which was achieved in five linear steps from (E,E)-farnesyl acetate. The synthetic strategy features a selective oxidation reaction, a Schlosser sp3-sp3 cross-coupling, a Wittig cross-coupling and an elaborate one-pot selective reduction, lactonisation and isomerization reaction to install the butenolide ring. The structure-activity relationship of furospongolide was also investigated. This involved the design and synthesis of a library of structurally modified analogues sharing the same C1-C13 subunit. This was achieved by exploiting the brevity and high level of convergence of our synthetic route together with the readily amenable structure of our target molecule. Exploiting the Schlosser cross-coupling allowed for replacement of furan with other heterocycles in the preparation of various furanolipid and thiophenolipid derivatives. The employment of reductive amination and Wittig chemistry further added to our novel library of structural derivatives. The third chapter discusses the results obtained from the NCI from biological evaluation From a collection of 28 novel compounds evaluated against the NCI-60 cancer cell array, six drug candidates were successfully selected for further biological evaluation on the basis of antitumour activity. COMPARE analysis revealed a strong correlation between some of our design analogues and the blockbuster anticancer agent tamoxifen, further supporting the potential of furanolipids in the treatment of breast cancer. The fourth chapter, details the full experimental procedures, including spectroscopic and analytical data for all the compounds prepared during this research.

Statistical considerations in the kinetic analysis of PET-FDG brain tumour studies

Dynamic positron emission tomography (PET) imaging can be used to track the distribution of injected radio-labelled molecules over time in vivo. This is a powerful technique, which provides researchers and clinicians the opportunity to study the status of healthy and pathological tissue by examining how it processes substances of interest. Widely used tracers include 18F-uorodeoxyglucose, an analog of glucose, which is used as the radiotracer in over ninety percent of PET scans. This radiotracer provides a way of quantifying the distribution of glucose utilisation in vivo. The interpretation of PET time-course data is complicated because the measured signal is a combination of vascular delivery and tissue retention effects. If the arterial time-course is known, the tissue time-course can typically be expressed in terms of a linear convolution between the arterial time-course and the tissue residue function. As the residue represents the amount of tracer remaining in the tissue, this can be thought of as a survival function; these functions been examined in great detail by the statistics community. Kinetic analysis of PET data is concerned with estimation of the residue and associated functionals such as ow, ux and volume of distribution. This thesis presents a Markov chain formulation of blood tissue exchange and explores how this relates to established compartmental forms. A nonparametric approach to the estimation of the residue is examined and the improvement in this model relative to compartmental model is evaluated using simulations and cross-validation techniques. The reference distribution of the test statistics, generated in comparing the models, is also studied. We explore these models further with simulated studies and an FDG-PET dataset from subjects with gliomas, which has previously been analysed with compartmental modelling. We also consider the performance of a recently proposed mixture modelling technique in this study.

Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

BACKGROUND: The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. METHODS: Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. RESULTS: Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. CONCLUSIONS: These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

The role of vitamin D signalling in the interaction between mesenchymal mammary tumour cells and macrophages

The transition of epithelial-like tumour cells to those exhibiting mesenchymal characteristics (Epithelial-to-mesenchymal Transition; EMT) is an integral process in breast cancer metastasis. EMT can be promoted by Transforming growth factor-beta (TGF-β) which can be found at high levels in the tumour stroma. Tumour-associated macrophages (TAMs) can also induce EMT in breast cancer cells, which is one way that they promote breast cancer metastasis. Vitamin D signalling has been implicated in EMT suppression and plays a role in modulating macrophage differentiation and stimulating their anti-inflammatory functions. This project had two major aims. First, we aimed to create and verify a unique fluorescent reporter gene construct designed to evaluate the dynamics of EMT in real-time and at the single-cell level. While some components of this reporter system were successfully validated, work to complete the final reporter construct is ongoing. The second and main aspect of this project focused on exploring the ability of 1,25-dihydroxyvitamin D3 (1,25D3) to modulate the interaction between mesenchymal mammary tumour cells and TAMs. Unexpectedly, in short-term treatment (48 hours) studies of 4T1 murine mammary tumour cells, we observed that 1,25D3 and TGF-β signalling work together to increase expression of the mesenchymal markers, Snai1, Fn1, and Col1a1. 1,25D3 and TGF-β also synergistically activate transcription of the gene encoding the 1,25D3-catabolizing enzyme, Cyp24a1. The ability of 1,25D3 and TGF-β to enhance expression of these genes was diminished in a long-term treatment (14 days) of 4T1 cells, and this effect was accompanied by a decrease in cell proliferation. 1,25D3 may also cooperate with cytokines produced by normal macrophages and macrophages considered to be TAM-like. Conditioned media experiments revealed that in the presence of factors from normal macrophages, 1,25D3 enhanced expression of Fn1, and in the presence of factors from TAM-like macrophages, 1,25D3 enhanced expression of Fn1 and Cyp24a1. Rather than mitigating the interaction as hypothesized, 1,25D3 may exacerbate the tumour-promoting effects of the EMT-TAM relationship. Also, signalling pathways involved in the EMT-TAM relationship may synergize with 1,25D3 to upregulate Cyp24a1 expression. These findings are important for understanding the potential of vitamin D compounds to be used in the treatment of breast cancer.

Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells

INTRODUCTION: The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression.

METHODS: Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies.

RESULTS: Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha-induced TRPA1 responses after Biodentine treatment.

CONCLUSIONS: In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses.

Multi-scale colorectal tumour segmentation using a novel coarse to fine strategy

This paper addresses the problem of colorectal tumour segmentation in complex real world imagery. For efficient segmentation, a multi-scale strategy is developed for extracting the potentially cancerous region of interest (ROI) based on colour histograms while searching for the best texture resolution. To achieve better segmentation accuracy, we apply a novel bag-of-visual-words method based on rotation invariant raw statistical features and random projection based l2-norm sparse representation to classify tumour areas in histopathology images. Experimental results on 20 real world digital slides demonstrate that the proposed algorithm results in better recognition accuracy than several state of the art segmentation techniques.

Mesenchymal stromal cells (MSCs) and colorectal cancer - a troublesome twosome for the anti-tumour immune response?

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.