Arathimou, Samantha A.; (2020) Characterisation of normal human pleural mesothelium to understand malignant pleural mesothelioma. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Malignant pleural mesothelioma (MPM) is an aggressive and fatal malignancy. Gene expression analysis of MPM tumours and use of MPM in vitro cell models are established tools used to investigate the disease and identify potential therapeutic pathways. However, the study of MPM has been hindered by the lack of normal human pleura to utilise as a control for transcriptomic studies and to derive pre-clinical mesothelial cell models from. Obtaining normal pleural tissue can be challenging and not accessible to many research laboratories. I present data to support the use of visceral pleura acquired from two distinct human tissue pipelines; routine lung resection surgery and post-mortem tissue from advanced cancer patients. Using these samples, I provide a full morphological and structural evaluation of human visceral mesothelium in homeostasis (n=18). Importantly, I found that pleural samples from both tissue pipelines offer a viable source of human visceral mesothelial cells (VMCs). These findings will enable the future adoption of these pipelines and the expansion of human-based pleural studies by the research field. In the limited human studies of normal pleura, pleural fluid from effusions remains the main method of mesothelial cell isolation, although this produces a heterogeneous cell population of parietal and visceral mesothelial cells. Here, I developed a method to isolate VMCs using brushings of visceral pleura, which resulted in a VMC population with estimated purity of 92% based on Cytokeratin 5 (CK5) expression (n=4). Subsequently, by implementing negative selection for contaminating cell types, I purified pleural brushing cell populations by Fluorescence-Activated Cell Sorting (FACS). The combination of pleural brushing and FACS-purification enabled the elimination of immune cells, red blood cells, CD31-expressing endothelial cells and CD90-expressing fibroblasts, thus improving purity to an estimated 95% and yielding VMC cell counts of up to 25 x 103 (n=5). VMC populations were RNA Sequenced for the first time, which generated novel insights into this cell type in homeostasis (n=4). Importantly, this normal VMC dataset allowed us to elucidate upregulated pathways in MPM using the MPM RNA sequencing dataset available through The Cancer Genome Atlas (TCGA). Pathway analysis revealed 30 significantly upregulated pathways in MPM compared to normal VMCs, including TGFβ and Hedgehog signalling. Further exploration of the TCGA MPM cohort showed higher expression of GLI2 in a subset of MPM tumours when compared to other cancers and the association of TGFB2 with GLI1. Subsequent stimulation of MPM cells in vitro with the cytokines TGFβ1 and TGFβ2 resulted in a significant increase of GLI1 and GLI2 mRNA levels. These results suggest for the first time that there is an upregulation of GLI2 in MPM which can be partially explained by convergence of TGFβ signalling on Hedgehog transcription factors. Finally, siRNA and shRNA-mediated knockdown of GLI2 in MPM cell models resulted in statistically significant reduction in cell viability and colony-forming capacity respectively. Pharmacological inhibition of Hedgehog signalling with currently available GLI inhibitors showed modest efficacy when tested in a panel of MPM cell lines (n=9). However, pathway analysis of differentially expressed genes identified by microarray of shRNA-mediated GLI2 knockdown in H2803 cells, indicated that GLI2 may play a role in cell cycle regulation. Therefore, the development of more potent and selective pharmacological agents for Hedgehog pathway inhibition at the level of the GLI2 protein is warranted.

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