Saudi Cultural Missions Theses & Dissertations
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Item Restricted The impact of NDRG1 overexpression on the immunological and metabolic reprogramming of the pancreatic tumour microenvironment(The University of Sydney, 2024-07) Alenizi, Shafi; Kovacevic, ZaklinaPancreatic ductal adenocarcinoma (PDAC) is highly aggressive, with no effective treatments for the 80% of patients that are diagnosed at an advanced stage. A major hurdle in treating PDAC is the extensive tumour microenvironment (TME) which facilitates resistance to all current therapies. N-myc downstream regulated 1 (NDRG1) is a metastasis suppressor that was found to inhibit tumour progression and metastasis in PDAC. Recent studies also suggest that NDRG1 reduced PDAC-mediated activation of pancreatic stellate cells (PSCs), although the mechanisms behind this remain to be established. Our studies investigated the effect of NDRG1 on PDAC metabolism and how this influences key TME elements including tumour-associated macrophages (TAMs) and PSCs. We generated PDAC cells (MIAPaCa-2 and PANC-1) that stably overexpress NDRG1 and performed extensive metabolomic, proteomic and secretome analysis under normoxia and hypoxia. Using conditioned media or direct 3D spheroid cocultures, we assessed the effect of PDAC cells on THP-1 and U937 monocytes and primary PSCs using flow cytometry, Seahorse metabolic analysis, western blot and immunofluorescence analysis. The findings indicated that NDRG1 expression profoundly affected the metabolism of cancer cells, which led to significant changes in both the immune and fibroblast components of the TME. In cancer cells, NDRG1 reduced the uptake of branched‐chain amino acids (BCAA) leading to inhibition of the mTOR pathway. The secretome of PDAC cells, including exosomes, cytokines and chemokines was also altered by NDRG1. Specifically, NDRG1 increased secretion of TNF-α, while reducing CCL2 and TGF-β production by PDAC cells. This led to re-programming of TAMs from an anti-inflammatory M2 phenotype to a pro-inflammatory M1 phenotype and altered TAM metabolism. NDRG1 expression in PDAC cells also markedly influenced the metabolic cross-talk with PSCs, leading to increased infiltration of M1 polarized TAMs into PDAC/PSC co-culture spheroids. We demonstrate that NDRG1 is highly involved in regulating PDAC metabolism, significantly altering metabolic cross-talk with PSCs and leading to extensive “re-programming” of TAMs into the M1 phenotype. Hence, NDRG1 has the potential to disrupt the oncogenic interactions between PDAC cells and the TME, and promoting the expression of this protein may enhance PDAC vulnerability to current chemo/immunotherapies.15 0Item Restricted Studying the inhibitory effect of oxysterols on NK-92 metabolism and cytotoxicity(Saudi Digital Library, 2023-12-05) Alharbi, Mona Shujaa; Finlay, David KNK cell-based immunotherapy has become a promising cancer treatment for cancer. One approach is the use of NK cell lines such as NK-92 as this overcomes many of the technical challenges associated with the use of human NK cells isolated from the blood. One of the major restrictions for the activity of NK cells against solid tumours are the conditions within the tumour microenvironment (TME) including adverse metabolic conditions. The Finlay lab has discovered that cellular metabolism is closely linked to NK cell anti-tumour functions, including NK cell cytotoxicity. However, little is known about the metabolic requirements for NK-92 to mediate anti-tumour cytotoxicity. This study investigated the metabolism of NK-92 cells and the importance for metabolic regulators, including mTORC1 and SREBP, in sustaining NK-92 cytotoxicity. Direct inhibition of metabolic pathways inhibited NK-92 cytotoxicity. While IL-2 signaling is required for NK92 cytotoxicity, the activity of the downstream signaling through mTORC1 and Srebp is dispensable. Interestingly, the oxidized cholesterol molecule 25-hydroxycholesterol (25-HC), a known inhibitor of SREBP activation, potentially inhibited NK-92 cytotoxicity, mitochondrial metabolism, along with observed distribution in membrane structure, seemed to occur independently of SREBP and LXR activation. 25-HC can be made by tumour associated macrophages (TAMs) that express cholesterol-25-hydroxylase. These data argue that 25-HC in the TME would inhibit NK-92 anti-tumour activity through modifying the membrane lipid order. Understanding NK92 cell metabolism will support designing better cell-based cancer therapeutic strategies in future. The data emerging from this project suggest that one strategy would be to engineer NK-92 cells to be resistant to the actions of 25-HC.12 0