Innate immuno-oncology of Malignant Pleural Mesothelioma (MPM)

Abstract

Malignant pleural mesothelioma is a type of cancer that develops in the pleural cavity. The pleural cavity is the lining covering the outer surface of the body’s most vital organs, such as the lung and heart. Asbestos is the only scientifically proven cause of the disease. My thesis involves ascertaining the role of human pulmonary surfactant protein D (SP-D) in the pathogenesis or protection against malignant pleural mesothelioma (MPM), a chronic cancer that is linked to long-term exposure to asbestos. The overall aim of my research is to determine whether our generated recombinant form of human SP-D (rfhSP-D) induces apoptosis in MPM, as it does in leukaemia, prostate and pancreatic cancers via a range of signalling pathways. An alternative hypothesis is that since SP-D can induce a potent pro-inflammatory immune response while interacting with asbestos-like particles, it creates a perfect chronic microenvironment for the development of MPM. Thus, rfhSP-D may negate the intrinsic protumorigenic consequences of pulmonary SP-D-asbestos needle interaction. Our team has established apoptotic activity of rfhSP-D in pancreatic cancer, breast cancer and lung cancer. So far, I have performed immunohistochemical staining on mesothelioma biopsies sections to detect the presence of human SP-D, and I have found within these mesothelioma subtypes that human SP-D is present and saturated throughout the tissue histotypes. This raises various questions, such as what is the function of human SP-D in MPM? Is human SP-D pro- or antitumorigenesis, and essentially why is human SP-D being profoundly present in MPM biopsy sections received from patients? This suggest a crucial role for SP-D in mesothelioma, as its presence marks its significance in tumour growth. I have generated a recombinant fragment of human SP-D (rfhSP-D) to mimic the functional effects of human SP-D. Recombinant fragment of human SP-D (rfhSP-D) is composed of homotrimeric neck and carbohydrate recognition domain, purposely to analyse the apoptotic activity of rfhSP-D in MPM using in vitro assays and ultimately to provide a potential therapeutic intervention to improve upon new strategies that will potentially reduce tumour growth and in future to increase patient survival and prognosis. Firstly, the recombinant form of SP-D (rfhSP-D) is prepared using a plasmid pUKD1 that contains collagen triple-helix consisting of cDNA sequences for repeating 8 Gly-X-Y triplets, the a-helical coiled-coil neck and CRD region of human SP-D, for which under bacteriophage T7 promoter is used to express the recombinant form of human SP-D (rfhSP-D) (177 residues: Gly179 to Phe355) that is transformed into Escherichia coli BL21 (lDE3) pLysS. Once rfhSP-D is produced an purified in large batches, it then undergoes endotoxin removal, as toxins produced from E. coli is potent to all cells and creates an inflammatory response that will overpower rfhSP-D when carrying out in vitro assays, potentially causing false positive results. Here, I have provided data and an insight into a new area of research, that was not carried out before. This includes the ability of rfhSP-D to bind to malignant pleural mesothelioma (MPM) cell line (MSTO) in a calcium dependent manner on its carbohydrate moiety structure, and rfhSP-D induces apoptosis in MPM cell lines (MSTO and Met-5a), which are confirmed using a number of valid in vitro experimental techniques.