FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression. / Thesis (Master, Biochemistry) -- Queen's University, 2011-09-14 11:49:32.871

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection; however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumour cell killing rapidly extends far beyond the initial sites of infection. This Bystander Effect is due to the virus’ ability to specifically target tumour vasculature through tumour-specific infection of tumour endothelium and the induction of an inflammatory response resulting in tumour-restricted coagulation, acute vascular disruption, apoptosis and necrosis of the tumour core. VSV-infected tumours, reconstructed in three-dimensions from serial histological sections, revealed that the majority of the tumour mass lacks significant blood flow in contrast to uninfected tumours, which exhibit relatively uniform perfusion. VSV infection rapidly induced intravascular coagulation within 6 hours of intravenous administration. The induction of coagulation was dependent on neutrophils and could be prevented with inhibitors of the coagulation pathway. Normal vasculature was not infected by VSV and no increase in coagulation was observed. Vascular collapse was also observed with the oncolytic poxvirus, JX-594, in patients and preclinical models. Biopsies from patients enrolled in a dose escalation trial for JX-594 were immunoreactive for vaccinia antigens and transgene products in high dose cohorts. Tumour-associated vessels from patients treated with JX-594 were infected with JX-594 and expressed virally encoded transgenes. A decrease in blood flow was also observed 5 days post infection. Several viruses, VSV, JX-594, vvDD, Maraba, and Sindbis, were able to rapidly induce widespread bystander cell death in a subset of mouse models. Tumours responded to OV therapy in three ways, and the type of response was determined by two factors - susceptibility to infection and the heterogeneity of the tumour microenvironment. Heterogeneity correlated with E-cadherin expression. Among tumours that supported viral replication, cancers with low E-cadherin expression were susceptible to vascular collapse. E-cadherin positive tumours were susceptible to infection and direct cell killing but resistant to vascular disruption or bystander cell death. If poorly-differentiated tumours were resistant to infection, no acute cell killing was observed. These histological subtypes provide a potential framework for the rational selection of patients, the integration of combination therapies and the creation of designer viruses to improve the success of OV therapy.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells. / Yorkshire Cancer Research

Delta-like ligand 4 (DLL4) is a ligand for the Notch family of receptors. DLL4 is an important regulator of angiogenesis and DLL4 blockade promotes non-productive angiogenesis and delays tumour growth. The aim of this thesis was to investigate the effects of anti-DLL4 therapy in solid tumours in combination with a clinically relevant dose of ionising radiation (5 Gy; IR) and to analyse alterations in the Notch pathway induced by the treatments. Combining both treatments resulted in a greater than additive tumour growth delay in LS174T tumours, compared to either treatment alone. DLL4 blockade dysregulated vasculature and increased necrosis in LS174T and HCT-15 (DLL4-expressing and negative cell lines respectively) tumours within 3 days after treatment, but no changes were observed with IR alone. Additionally, combined IR and anti-DLL4 treatment of FaDu tumours (another DLL4-negative cell line) by our colleagues, also resulted in a supra-additive growth delay. These results show that combining IR with DLL4 blockade is an effective strategy for prolonging tumour growth delay and suggest that the stroma/vasculature provide the main therapeutic target for the anti-DLL4 therapy. Analysis of Notch pathway shows that IR upregulated Jag1 in tumour cells, and may inhibit Notch and downregulate DLL4 in the stroma. These changes may potentially affect tumour vessels and response to anti-DLL4 therapy. In vitro, anti-DLL4 therapy induced proliferation in quiescent contact-inhibited endothelial cells and also appeared to abrogate IR-induced inhibition of migration. These results suggest that DLL4 may be important in maintaining vessel quiescence and that IR may in part decrease migration through Notch signalling. Combining IR and DLL4 blockade to target tumour growth is an effective and well tolerated strategy and warrants further validation and refinement to be translated into clinical practice.

Ao longo dos últimos anos, tornou-se evidente que o início e a progressão do câncer dependem de vários componentes do microambiente tumoral, incluindo células imunes e inflamatórias, fibroblastos, células endoteliais, adipócitos e matriz extracelular. Estes componentes e as células neoplásicas interagem entre si e trocam sinais pró e antitumor. O presente estudo teve como objetivo analisar o secretoma de células neoplásicas sob estresse e seu efeito no microambiente tumoral. Para este fim, duas linhagens celulares de carcinoma epidermóide de cabeça e pescoço foram cultivadas em duas condições de estresse: hipóxia e radiação. Os meios condicionados por estas células (secretoma 1) e o seu controle foram utilizados para cultivar células neoplásicas e fibroblastos humanos normais da cavidade oral. Os resultados sugerem que os sinais derivados das células neoplásicas em resposta a estresse dirigem a expressão gênica e proteica, bem como o comportamento celular das células vizinhas. Foram identificadas 38 proteínas celulares e nove proteínas secretadas com expressão aumentada e 61 proteínas celulares e 70 secretadas com expressão reduzida em células neoplásicas sob estresse hipóxico. Também foram identificadas 59 proteínas celulares e 29 proteínas secretadas com expressão aumentada e 59 proteínas celulares e 19 secretadas com expressão reduzida em células neoplásicas e fibroblastos humanos normais tratados com o meio condicionado por células sob estresse hipóxico. O secretome de células sob estresse não foi capaz de induzir proliferação de células neoplásicas e fibroblastos humanos normais, mas promoveu migração e invasão. Os resultados podem contribuir para o melhor entendimento do efeito dos fatores parácrinos liberados pelas células neoplásicas sobre a expressão gênica, bem como sobre o comportamento das células tumorais e estromais / Over the past years, it has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells, fibroblasts, endothelial cells, adipocytes, and extracellular matrix. These components and the neoplastic cells interact with each other providing pro and antitumor signals. The present study aimed to analyze the secretome of cancer cells under stress and their effect on the tumor microenvironment. For this purpose, two cell lines from head and neck carcinomas were cultured in two stress conditions - hypoxia and radiation. The medium conditioned by these cells (secretome 1) and their control were used to grow untreated neoplastic cells and normal human fibroblasts from oral cavity. Our results showed that signals derived from cancer cells in response to stress drive gene and protein expression and cell behavior. Thirty-eight overexpressed cellular and 9 secreted proteins, and 61 underexpressed cellular and 70 secreted proteins were identified in neoplastic cells under hypoxic stress. Fifty-nine overexpressed cellular and 29 secreted proteins, and 59 underexpressed cellular and 19 secreted proteins were identified in neoplastic cells and normal human fibroblasts treated with the medium conditioned by cells under hypoxic stress. The secretome of cells under stress was not able to induce proliferation of cancer cells and normal human fibroblasts, but promoted migration and invasion. The results may contribute to understand the effect of paracrine factors released by neoplastic cell on gene expression as well as on stromal and tumor cells behavior

Classic Hodgkin Lymphoma (cHL) has an annual incidence of 2.4 cases per 100 000 population in the UK, and is one of the most common malignancies diagnosed in young adults aged 15 to 34. The majority of younger patients have a good long-term outcome with between 80 and 90% disease-specific survival but cHL also affects older adults in whom the prognosis is significantly poorer. The role of tumour-associated macrophages (TAM) in cHL has gained much interest, with several studies reporting an association between high numbers of CD68-positive TAM and poor prognosis. There is also a question over the prognostic significance of Epstein-Barr Virus (EBV) infection which is implicated in up to 50% of cHL cases in developed countries. Published data suggests that EBV positivity in elderly patients may be associated with a poorer outcome, whereas in younger adults may be of prognostic benefit. Differences related to age are of interest particularly as an age-related decline in immunity has been linked with the development of certain subtypes of Non-Hodgkin Lymphoma in older patients. In a retrospective study, two separate cohorts of patients with cHL were examined with the aim of identifying: • Differences in the cellular composition of the tumour microenvironment in cHL which has arisen in young and elderly adult patients; • Differences in the cellular composition of the tumour microenvironment in cHL associated with or without EBV infection; • Factors within the tumour microenvironment which may influence prognosis and may be targeted for novel treatments. One group consisted of patients aged between 15 and 34 years at diagnosis and the other, of those aged 60 or over at presentation. Tissue obtained at the time of diagnosis was examined with regard to a number of factors related to the malignant cells and the surrounding microenvironment, including the number and phenotype of macrophages, the number of plasmacytoid dendritic cells and the number of malignant Hodgkin Reed-Sternberg (HRS) cells and non-malignant ‘background’ cells undergoing apoptosis. Comparisons were made between the two age groups, also taking into account the EBV-status of tumours, cHL subtype and gender. Results confirmed the current understanding that EBV-positive cHL is more common in older patients and has a strong, but not exclusive, association with the MCHL subtype. In addition, a strong link between young males and EBV-positive disease was shown. Macrophages were found to vary between the two age groups, in number and phenotype and there were clear differences associated with the presence or absence of EBV infection. While no definite link with outcome and macrophages was identified it was apparent that the implications of macrophages in the tumour microenvironment may differ between the two age groups. The number of apoptotic cells correlated closely with the number of macrophages and in the young the number of HRS cells was associated with prognosis. Investigation of the tumour microenvironment is complex and caution is needed in interpreting studies which do not differentiate between patients according to age, as tumour characteristics may have variable implications in different age groups. In this thesis a number of clinicopathological differences were identified between the two age groups. These point to the need for further larger studies to delineate how such age-related differences may or may not be associated with immune function and how this information could be translated into treatments to improve outcomes.

<p>Cancer metastasis is the cause of about 90% of cancer patients' deaths. Despite significant improvements in the past three decades in understanding the molecular bases of oncogenic transformation of cancer cells, little is known about the molecular mechanisms underlying tumour cells' alteration of their microenvironment, entrance into the circulation, and colonization of distant organs. In recent years, accumulating evidence has indicated that tumour microenvironment, which consists of a variety of cell types and extracellular matrix components&#65292;plays an important role in regulating the metastatic abilities of carcinoma cells. Co-opted by cancer cells, those stromal cells promote tumour progression via multiple mechanisms, including enhancement of tumour invasiveness, elevation of angiogenesis, and suppression of immune surveillance activity. </p><p>Using a series of human breast cancer cell lines with different metastatic potentials <italic>in vivo</italic>, we performed an unbiased screen examining expression of miRNAs, and found that miR-126 and miR-126*, whose expression are regulated by methylation of the promoter of their host gene Egfl7 inside tumour cells, were significantly negatively correlated with metastatic potential. Using both mouse xenograft models and <italic>in vitro</italic> assays, we showed that this pair of miRNAs suppressed breast cancer metastasis through shaping the tumour microenvironment without changing tumour cell autonomous properties. Specifically, miR-126 and miR-126* act independently to suppress the sequential recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes into the primary tumour stroma, consequently inhibiting lung metastasis by breast tumour cells. Mechanistically, these miRNAs directly inhibit the production of stromal cell-derived factor-1 alpha (Sdf-1&alpha;, also known as Cxcl12), and indirectly suppress the expression of chemokine (C-C motif) ligand 2 (Ccl2) by the cancer cells within the tumour mass in an Sdf-1&alpha;-dependent manner. In addition, in contrast with the majority of reports which have shown incorporation of only the guiding strand of the miRNA duplex into the mRNA-targeting RNA induced silencing complex (RISC), both strands of the miR-126 RNA duplex are maintained at a similar level and suppress Sdf-1&alpha; expression independently. </p><p>Collectively, we have determined a dynamic process by which the composition of the primary tumour microenvironment could be altered via a change in the expression of two tumour-suppressive miRNAs derived from a single miRNA precursor to favor metastasis by breast cancer cells. Importantly, this work provides a prominent mechanism to explain the clinical correlation between reduced expression of miR-126/126* and poor metastasis-free survival of breast cancer patients.</p> / Dissertation

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

Cervical cancer still represents the fourth most common cause of cancer deaths in women worldwide. Human papilloma virus (HPV) infection plays a role in cervical carcinoma initiation, but other genomic changes are needed for pre-malignant abnormalities to fully develop to cancer. This often happens through genomic instability caused by the virus oncoproteins. Several integrative genomic analysis studies have found that one of the most common imbalances in cervical squamous cell carcinoma (SCC) is copy number gain and amplification of chromosome 5p. In this region, copy number gain of the OSMR gene was found to correlate significantly with adverse outcome independent of the tumour stage (p=0.046). Furthermore, this copy number gain correlated with Oncostatin M receptor (OSMR) overexpression and sensitised these cells to Oncostatin M (OSM) leading to increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation, cell migration, invasion and proangiogenic signalling. The aim of this PhD project was to study the role of OSMR overexpression in the SCC tumour microenvironment (TME) and tumour growth in vivo and to study the role of hypoxia inducible factor driven hypoxia signalling in OSMR overexpressing SCC cells and their tumour microenvironment. OSMR overexpression was found to sensitise tumour cells to induce Hypoxia inducible factor 1a and 2a (HIF1a, HIF2a) signalling in normoxic conditions, to promote pro-angiogenic signalling. Furthermore, hypoxic conditions were found to enhance OSM signalling in OSMR overexpressing cells leading to increased expression of markers of epithelial to mesenchymal transition, angiogenesis and migration. In the SCC tumour microenvironment, OSMR overexpression was found to sensitise tumour cells to OSM secreted from macrophages and other immune cells leading to improved tumour growth, angiogenesis and STAT3 activation at the tumour site. Removal of OSMR from either tumour cells or tumour microenvironment led to reduced tumour growth and angiogenesis, along with increased tumour necrosis. I conclude that OSMR overexpression is an important driver of SCC tumour progression and malignancy via STAT3- and HIF-driven signalling and removal of it from either tumour cells or tumour microenvironment drastically hampers tumour growth in vivo. Based on the results of this study, OSMR blockade is a potential novel therapeutic option in advanced SCC.