by Kong Siu-Kai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 313-340.

Tumour associated macrophages (TAMs) have been associated with prognosis in a wide variety of tumours with most studies showing a high number of macrophages equating with poor prognosis. This is postulated to be due to TAMs providing support to the tumour through a wide variety of mechanisms including suppression of the immune response, promotion of angiogenesis and provision of growth supporting signals. Previous work within the group has characterised some of the mechanisms by which Burkitt lymphoma cells attract macrophages to the tumour and some of the mechanisms by which these macrophages support tumour cell growth. This thesis extends some of the work carried out in Burkitt lymphoma to Diffuse Large B cell lymphoma (DLBCL) and examines TAMs in this tumour type. Diffuse Large B cell lymphoma is the commonest high grade lymphoma in the Western world. Like Burkitt lymphoma it is characterised by diffuse sheets of lymphoid blasts. In contrast to Burkitt lymphoma, it represents a less well-defined entity that encompasses tumours with variable morphology, genetic abnormalities and outcome. Rates of proliferation and apoptosis vary between individual tumours, and unlike Burkitt lymphoma not all cases are characterised by a prominent macrophage infiltrate. Previous work within the group has shown a relationship in Burkitt lymphoma between apoptosis, macrophage infiltration and proliferation suggesting that apoptosis recruits macrophages to provide support to the tumour cells. This relationship was studied here in a large cohort of patients with DLBCL and the same relationship shown to exist in this tumour also. Following this observation, a bioinformatic approach was taken to define a gene expression signature of the TAM in DLBCL in situ in an unbiased way. Using large publicly available human tumour gene expression datasets, a graph clustering approach using the tool Biolayout Express 3D was used to explore the transcriptome of DLCBL and other human tumours. Signatures of immune cells and stromal cells, functional pathways and tumour specific signatures were defined from individual tumour type transcriptomes by study of clusters of co-expressed genes. Further work used a novel graph clustering approach based on mean Pearson correlations to define a ‘core’ transcriptome signature shared across many unrelated tumour types and in which elements of the tumour stroma were prominent. To validate the TAM signature derived from the DLBCL dataset, protein expression of selected elements of the signature were analysed at the protein level by immunohistochemistry in an unrelated cohort of DLBCL. Selected markers from the DLBCL TAM signature were then assessed for relationship to outcome in a cohort of patients treated with CHOP chemotherapy. Of the proteins studied, a significant difference in outcome was demonstrated only for leukocyte associated immunoglobulin receptor 1 (LAIR1) expression by TAMs, where low intensity staining for LAIR1 in TAMs was associated with better overall survival. LAIR1 is a collagen-binding inhibitory receptor expressed only in cells of haemopoetic lineage whose role is little studied in macrophages. The final results chapter presents some preliminary data from co-culture experiments in which the expression of LAIR-1 on the ‘macrophage-like’ cell line THP-1 is studied in various polarisation states and the ability of these cells to support or constrain tumour cell growth studied in the presence or absence of collagen.

Pancreatic ductal adenocarcinoma is a highly desmoplastic tumour, and non-malignant stromal cells contribute to progression and treatment resistance. Inflammatory cells in particular are known drivers of carcinogenesis, and macrophages are one of the most abundant inflammatory leucocytes. Therefore, exploring how macrophages drive tumour progression in pancreatic cancer would not only aid in understanding disease biology but could also offer insight to novel treatment strategies. Results presented in this thesis show macrophages secrete factors that drive epithelial-to-mesenchymal transition, promote invasion and lead to expression of checkpoint inhibitors. To determine what factors were driving this phenotype, the serine protease inhibitor SerpinB3 was initially explored, as it was highly upregulated in cancer cells cultured with conditioned media from macrophages. However, SerpinB3 gene overexpression and knockdown did not confirm a direct role for this gene in mediating migration and invasion. Further investigation revealed macrophages were secreting the cytokine oncostatin M, which was driving a metastatic phenotype through activation of the STAT3 pathway. Expression of oncostatin M receptor was upregulated in cancer cells following culture with macrophage conditioned media and conferred a worse prognosis in patient samples. STAT3 pathway activation by oncostatin M led to increased invasion in vitro, particularly of the highly tumourigenic cancer stem cell population, and increased metastasis in vivo. Moreover, oncostatin M mediated expression of the immune 'checkpoint' inhibitors on the surface of pancreatic cancer cells. Using antibody and small molecule inhibitors, reversion of these signalling pathway effects were seen and preliminary data from in vivo assays showed decreased metastasis formation with cytokine receptor antibody inhibition. Overall, the findings in this thesis contribute to emerging knowledge of how tumour associated macrophages drive tumour progression in pancreatic adenocarcinoma. Not only do they promote invasion and metastatic potential through oncostatin M secretion, but also potentiate inherent biological properties of cancer stem cells and assist in immune tolerance. In addition, results provide preliminary data to support a rationale for clinical targeting of macrophage-derived oncostatin M in pancreatic cancer.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Immunity and metabolism have been viewed as separate fields, however recent evidence show that these two systems are intimately integrated, share resources and cross-regulate each other. Activated immune cells have to alter their metabolism in order to support effector functions. On the other hand, viruses are obligatory parasites that counter and exploit host pathways, including metabolism, to effectively propagate. Like immune cells, viruses have to alter the metabolic profile of infected cells in order to propagate. The regulation of metabolism in immune cells or virally infected cells has been well studied. However, the precise metabolic regulation that ensues when both immune system and viral infection in immune cells interact and compete for the limited resources and metabolic pathways available is not clear. In this thesis, I have sought to investigate the integrative process by studying the metabolic programming of macrophages infected with murine cytomegalovirus (MCMV) The central hypothesis of this thesis is that productive infection of macrophages by MCMV takes advantage of the early inflammatory metabolomic reprogramming of activated macrophages to establish infection, and modulates metabolism at late stages of infection towards fatty acid (FA) production to promote viral progeny. To study this interaction, I have analysed the temporal profile of the transcriptome and metabolome of bone marrow derived macrophages (BMDM) infected with productive (WT) and non-productive (attenuated) (MCMV) strains. This aimed to unravel the host-directed versus virus-driven metabolic alterations. I show evidence indicating that during early times of productive and non-productive MCMV infection glycolysis is, in infected BMDM, markedly increased. Furthermore, pharmacological and siRNA mediated inhibition of glycolysis resulted in attenuation of viral growth demonstrating the dependency of MCMV on this pathway. Additionally, using interferon receptor A (IFNAR) and interferon receptor A (IFNB) deficient BMDM showed that type-I interferon (IFN) signalling is essential for the early upregulation of glycolysis that was observed. In addition to the changes in glycolysis, MCMV infection alters the tricarboxylic acid (TCA) cycle in infected BMDM. Metabolomic and transcriptomic data revealed a shift from catabolic to anabolic function for the TCA to promote production of TCA intermediates. Finally, the urea cycle is also altered both on transcriptional and metabolomic level, consistent with the support of Nitric oxide (NO) production which is a hallmark metabolite in classically activated macrophages. These changes observed in the TCA cycle and glycolysis are consistent with supporting the FA elongation pathway during late time points of productive infection. Only productive MCMV infection upregulates this pathway. At the same time, pharmacological and siRNA mediated inhibition of FA elongation pathway greatly attenuates viral growth. This indicates that MCMV growth is dependenton FA elongation. The effect was very specific for the elongation and not the de novo synthesis pathway indicating that MCMV remodels FA that already in the cells. It is argued, that in agreement to known literature, MCMV uses these FA for the formation of its lipid membrane. To further investigate the dependency of MCMV on FA elongation pathways I studied additional lipids pathway associated with the former. I found that MCMV infection also upregulates the triacylglycerol formation and membrane remodelling pathways, which are dependent on FA biosynthesis and elongation. The inhibition of triacylglycerol formation and membrane remodelling pathway also attenuated MCMV growth. This indicates that apart from the formation of its lipid membrane MCMV requires FA to remodel the cellular environment. I have also explored the effects of infection on regulating lipid mediators, in particular eicosanoids. Eicosanoids are lipid signalling molecules that can act as potent inflammation modulators. Here I demonstrated that productive MCMV infection specifically increases PGE2 production in infected BMDM. Moreover, addition of PGE2 increased viral replication in infected fibroblasts in comparison to non-treated cells, while pharmacological blocking of EP4 (PGE2 receptor) rescued the phenotype. These studies reveal how MCMV advantageously use inflammatory lipid pathways to promote growth In conclusion, the data presented in this thesis support my hypothesis and provide an insight in the role of metabolism during viral infection. Evidence is provided to show that MCMV co-ops the early alterations that metabolic pathways undergo in activated macrophage, including but not limited to glycolysis, TCA cycle and urea cycle. These early changes in metabolism appear to be coupled with upregulation of FA elongation pathways and remodeling of lipids in infected cells. Finally, MCMV co-ops the function of regulatory lipids, in particular PGE2, to promote viral growth. It is further argued that MCMV productive infection dictates these fatty acid metabolism alterations in order to remodel the host cell's environment, regulate the immune system response and provide resources for its lipid membrane.

Abstract not available

Thèse de doctorat : Sciences de la vie et de la santé. Biologie cellulaire et moléculaire : Paris 12 : 2004. / Titre provenant de l'écran-titre. Bibliogr. f. 102-114.

Thèse de doctorat : Mécanique. Biophysique-Biomécanique : Paris 12 : 2005. / Titre provenant de l'écran-titre. Pagination : 151 f. Bibliogr. f. 142-151.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 232-255).

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.