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(2024)ThesisAnti-GD2 immunotherapy has significantly enhanced survival of high-risk neuroblastoma patients however efficacy is strongly hampered by the immunosuppressive tumour microenvironment. Given the emerging link between copper and immune evasion, we assessed the impact of copper chelation therapy on neuroblastoma using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA; marketed as Cuprior®). Using the preclinical TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling to assess cellular and molecular changes occurring with TEPA treatment in the neuroblastoma tumour microenvironment. Copper chelation was observed to successfully deplete intratumoural copper to reinvigorate anti-tumour immunity as signalled by increased infiltration and activity of pro-inflammatory immune cells, specifically N1 neutrophils. Mechanistic in vitro assays reveal sequestration of copper by neuroblastoma cells causing dysregulated neutrophil function, with successful reversal upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Copper chelation is further shown to increase GD2 expression, while also enhancing neutrophil antibody-dependent cellular cytotoxicity in vitro. Using the TH-MYCN model, TEPA was found to potentiate anti-GD2 therapy to achieve durable tumour control. This was associated with increased Fc-receptor-bearing natural killer and CD11B+ myeloid cells capable of performing antibody-dependent cellular cytotoxicity. Finally, we evaluated TETA for repurposing as a novel immunomodulatory agent. Comparative in vitro studies with TEPA confirm the ability of TETA to deplete intratumoural copper and downregulate immune checkpoint proteins Programmed Death-Ligand 1 (PD-L1) and indoleamine-pyrrole 2,3-dioxygenase (IDO). Using the preclinical NXS2 model, TETA exhibited an exceptional safety profile without altering copper levels or GD2 expression in healthy nerve tissue. Combination with anti-GD2 therapy achieved durable tumour control with no relapses occurring after treatment cessation and was similarly associated with infiltration of pro-inflammatory immune cells. Collectively, study findings credential copper chelation as a non-toxic strategy to overcome the immunosuppressive tumour microenvironment and improve neuroblastoma patient outcomes.
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(2024)ThesisDNA sequencing reads the order in which the four nucleotide bases – adenine (A), thymine (T), cytosine (C) and guanine (G) – arrange into the long biopolymer that encodes the genes of all living organisms. The ability to sequence DNA has revolutionised the biological sciences and is having a rapidly expanding impact on multiple industries including medicine, agriculture, and biotechnology. The DNA sequencing market is predicted to reach $60 billion by 2030. Nonetheless, there exist serious challenges for the current wave of industry-leading high-throughput sequencing methods including the need for large quantities of DNA, limitations on the maximum DNA length that can be continuously read, and the computational resources needed to build the sequence from raw data. A highly coveted goal is to accurately read the sequence of a single DNA molecule in real time. Recently, significant progress has been made with single-molecule sequencing technologies and two emerging technologies – Oxford Nanopore’s ‘current blockade’ and PacBio’s ‘zero mode waveguide’ sequencing – now lead the market. Both technologies use methods subject to fundamental limits in our ability to precisely manipulate a single DNA molecule and confine the observation volume to a single nucleotide. These limitations result in substantial error rates, and thus accurate sequencing still requires multiple repeated sequencing reactions to attain accuracy statistically. True single-molecule DNA sequencing remains an unsolved problem. Here, we describe a new method for single-molecule DNA sequencing, by enhancing the fluorescent signal from single DNA bases as they are incorporated in the correct sequence by the enzyme DNA polymerase. Fluorescence enhancement is achieved by the use of a plasmonic nanoantenna. Chapter 1 provides an overview of existing state of the art technologies and underlying theory behind plasmonic nanoantenna sequencing. Chapter 2 provides the materials and methods. Chapter 3 describes the embodiment of the plasmonic nanoantenna DNA sequencer. Chapter 4 characterises the fluorescence enhancement of the plasmonic nanoantenna. Chapter 5 provides proof of concept experiments with examples of reactions that yield correct DNA sequences. This method has accurately identified mostly the first 12 coloured bases compared to the expected 27-color sequence in the alignment, which holds great promise for advancing the field of DNA sequencing to achieve sequencing of individual DNA molecules.