Approaches to the Development of In Vivo Propagation Systems for Hepatitis C Virus (HCV)

Hepatitis C virus (HCV) is a recently discovered major human pathogen infecting 170 million individuals worldwide. It was the first hepatotropic virus to be isolated by molecular biology techniques. Since its discovery in 1989 considerable progress has been made in the identification of the viral functional regions, the interactions between viral products, and viral pathogenesis. However, little is known about its replication cycle and the only experimental in vivo HCV infection model is limited to the chimpanzee. Thus, the development of a cheap and accessible in vitro or in vivo culture system has become a priority. The work in this thesis explores a variety of different approaches to an in vivo culture system using the severely combined immunodeficient (SCID) mouse. These animals lack functional T and B cells and will accept xenografts from a range of species including humans. Hepatocyte derived cell lines and primary human hepatocytes (PHHC) were transplanted intrasplenically and under the renal capsules after performing a partial hepatectomy. The rationale rests on the hypothesis that these cells would attain a biologically functional state of differentiation in vivo, increasing their sensitivity to HCV infection and acting as targets for HCV replication in an in vivo culture system. HCV positive serum was used to infect the cells prior to transplantation or 3 days post transplantation. PHHCs were also mixed with anti- met antibody and interleukin 6 (IL6) prior to transplantation to increase their survivability and proliferation in vivo; silica was administered intraperitoneally to deplete host macrophage cells. All the animals transplanted with hepatocyte derived cell lines developed extremely large metastatic tumours which were hemorrhagic, additionally these cells were not susceptible to HCV infection, irrespective of the route of infection and source of the HCV positive serum. PHHCs failed to engraft irrespective of treatment and were HCV reverse transcription polymerase chain reaction (RTPCR) negative

Genomic and molecular analyses identify molecular subtypes of pancreatic cancer recurrence

Pancreatic cancer (PC) remains a highly lethal malignancy, and most patients with localized disease that undergo surgical resection still succumb to recurrent disease. Pattern of recurrence after pancreatectomy is heterogenous, with some studies illustrating that site of recurrence can be associated with prognosis.1 Another study suggested that tumors that develop local and distant recurrence can be regarded as a homogenous disease with similar outcomes.2 Here we investigate novel molecular determinants of recurrence pattern after pancreatectomy for PC

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Spatial expression of IKK-alpha is associated with a differential mutational landscape and survival in primary colorectal cancer

Background: To understand the relationship between key non-canonical NF-κB kinase IKK-alpha(α), tumour mutational profile and survival in primary colorectal cancer. Methods: Immunohistochemical expression of IKKα was assessed in a cohort of 1030 patients who had undergone surgery for colorectal cancer using immunohistochemistry. Mutational tumour profile was examined using a customised gene panel. Immunofluorescence was used to identify the cellular location of punctate IKKα expression. Results: Two patterns of IKKα expression were observed; firstly, in the tumour cell cytoplasm and secondly as discrete ‘punctate’ areas in a juxtanuclear position. Although cytoplasmic expression of IKKα was not associated with survival, high ‘punctate’ IKKα expression was associated with significantly reduced cancer-specific survival on multivariate analysis. High punctate expression of IKKα was associated with mutations in KRAS and PDGFRA. Dual immunofluorescence suggested punctate IKKα expression was co-located with the Golgi apparatus. Conclusions: These results suggest the spatial expression of IKKα is a potential biomarker in colorectal cancer. This is associated with a differential mutational profile highlighting possible distinct signalling roles for IKKα in the context of colorectal cancer as well as potential implications for future treatment strategies using IKKα inhibitors

Inducible cytokine gene expression in the brain in the ME7/CV mouse model of scrapie is highly restricted, is at a strikingly low level relative to the degree of gliosis and occurs only late in disease

The temporal course of cerebral cytokine gene expression was investigated in the ME7/CV murine scrapie model to determine any association with neuropathological events. Analysis by RNase protection assay (RPA) demonstrated no transcripts for ILs 2, 3, 4, 5, 6, 7, 10, 12p40 and 13, granulocyte macrophage colony-stimulating factor, IFN-γ or lymphotoxin-α at any time during the course of this disease. Transcripts for transforming growth factor-β1 were constitutively expressed in both control and scrapie-infected brain and were elevated at terminal disease. RPA and quantitative real-time RT-PCR detected low levels of transcripts for IL-1α, IL-1β and TNFα in scrapie-infected brain but only IL-1β was elevated consistently in all mice studied. Although glial cell activation within the hippocampus was evident from 100 days post-infection (p.i.), elevated IL-1β transcripts (and immunoreactivity) were evident from 180 days p.i., around the time of hippocampal pyramidal neuron loss, and increased steadily thereafter to reach a 3·5-fold increase at terminal disease. Even at their maximum, levels of these transcripts were disproportionately low relative to the degree of glial cell activation. It is concluded that cytokine gene expression in the ME7 scrapie-infected mouse brain, relative to the degree of reactive gliosis, is highly restricted, temporally late and disproportionately low

CXCL8 expression is associated with advanced stage, right sidedness, and distinct histological features of colorectal cancer

CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I–IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope®. Analysis was performed using HALO® digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer-specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right-sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal-rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour-infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right-sided colonic disease and stroma-rich tumours

HNF4A and GATA6 loss reveals therapeutically actionable subtypes in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3b) a key regulator of glycolysis. Pharmacological inhibition of GSK3b results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3b inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC

Genomic and Molecular Analyses Identify Molecular Subtypes of Pancreatic Cancer Recurrence

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