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Title: Investigating the anti-tumour effects of macrophages in the zebrafish brain
Author: Hamilton, Lloyd Liang Ming
ISNI:       0000 0004 8510 2419
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Glioblastoma (GBM) remains an incurable tumour fraught with a high probability of death. One of the key characteristic of GBM is the development of local immunosuppression that promotes immune evasion and lays a solid foundation for the tumour to progress. Breakthroughs in our understanding in cancer biology have shown that GBM have evolved unique mechanisms that influence infiltrating macrophages, a key immune cell type, to facilitate tumour progression. Macrophages have been identified in many studies to promote angiogenesis, extra cellular matrix reorganisation, establishment of local immunosuppression and tumour growth. Thus, there is great need to improve the clinical development of immunotherapeutics that can address tumour-specific immune responses. Herein, we tested the capability of solidly supported Gold and Palladium nanoparticles as biorthogonal catalytic converters of prodrugs in a zebrafish U87 glioblastoma xenograft model. Intriguingly, we report that the implantation of Palladium and Gold bead into the zebrafish brain causes a potent anti-tumour responses that leads to U87 cell clearance, fragmentation and increased macrophage number. Further investigation revealed that Gold and Palladium beads did not cause aberrant necrosis when implanted in the zebrafish brain and that macrophages played a key role in mediating the associated anti-tumour response of Palladium and Gold bead implantation. The role of macrophages was investigated further using Next Generation RNA sequencing of macrophages isolated from Palladium bead implanted zebrafish. RNA sequencing results revealed differentially expressed genes in Palladium bead implanted zebrafish with 389 genes upregulated and 361 genes downregulated. Enrichment analysis of these genes showed significant enrichment of oxidation-reduction processes as a result of Palladium bead implantation. In addition, confirmatory RT-qPCR highlighted two key TLR signalling inflammatory genes, Cxcl8b.1 and TNF-α, to be overexpressed in macrophages of Palladium and Gold bead implanted zebrafish. Since Cxcl8b.1 is a potent attractant of neutrophils, we studied the dynamics of macrophages and neutrophil number in the zebrafish brain. Indeed, we detected an accumulation of neutrophils upon gold bead transplantation. Thus, we analysed the role of Cxcl8b.1 and TNF-α in the initiation of the anti-tumour effect. This was achieved by com- bining CRISPR-Cas9 knock out and genetic overexpression transgenesis techniques of Cxcl8b.1 and TNF-α. The results here conclude that TNF-α were not key genetic mediators of the associated bead induced anti-tumour phenotype. Finally, this study opens new ave- nues for the development of novel cancer immunotherapeutics. RNA sequencing results showed high number of other candidate genes that exploit the intrinsic capabilities of transitions metals to initiate an anti-tumour response.
Supervisor: Sieger, Dirk ; Brennan, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Glioblastoma ; macrophages ; zebrafish ; Palladium beads ; gold beads ; Cxcl8b.1 ; TNF-a ; immune system