Abstract
Lung cancer is a leading cause of mortality worldwide with non-small cell lung cancer (NSCLC) being a major contributor. NSCLC is strongly associated with an up-regulation of epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1). EGFR activates multiple signalling pathways that promote tumour growth and survival. PD-L1 is an immune checkpoint protein that triggers cancer immune evasion through PD-L1/PD-1 axis, inhibiting the anti-tumour activity mediated by cytotoxic T cells. Small drug molecules or monoclonal antibodies have been developed and exhibited excellent anti-tumour effects upon EGFR and PD-L1 blockade. However, their therapeutic efficacy is limited by drug resistance, poor response rate and undesirable toxicities, rendering the drugs ineffective. Excitingly, small interfering RNA (siRNA) emerges as a new class of therapeutic that inhibit disease-causing proteins via RNA interference. Unfortunately, the lack of an effective delivery system is the major hurdle that hampers its application. This study evaluated a synthetic non-viral PEG12-KL4 peptide as a delivery vector of siRNAs targeting EGFR and PD-L1 in human NSCLC cells. Among the four human NSCLC cell lines investigated, the siRNA-mediated EGFR and PD-L1 knockdown was the most significant and durable in NCI-H1975 cells. In addition, the cellular uptake of siRNA mediated by PEG12-KL4 was superior to a commercial lipid-based transfection agent, highlighting the potentials of PEG12-KL4 as a promising siRNA delivery vector.Somatic EGFR mutation is a common oncogenic driver in NSCLC. The missense secondary T766M mutation of EGFR is a common cause of drug resistance, which is also present in NCI-H1975 cells. The role of T766M mutation in tumour growth was investigated using various EGFR mutant variants in Ba/F3 cell xenografted subcutaneous model. It was found that T766M mutation conferred tumour growth without ligand stimulation, which was partially reversed by H566F mutation and almost completely impaired by K946E mutation. While these mutations are involved in collapsing EGFR conformation and reducing receptor phosphorylation, this work paves the way for the development of novel drugs that can treat the T766M-driven tumours.
For lung cancer therapy development and its preclinical validation, in vivo traceable lung cancer models are highly beneficial as they permit non-invasive serial imaging and enable quantitative tumour burden assessments that are necessary for reliable therapy efficacy determination. Both bioluminescence and radionuclide reporter gene imaging are commonstrategies for tracking cancer. In this study, the NCI-H1975 cells were selected to establish the reporter cell models with firefly luciferase or sodium iodide symporter fused with a red fluorescent protein (NIS-RFP). Importantly, two orthotopic ‘airside’ in vivo traceable NCI-H1975-based xenograft tumour models were established, where the tumour growth was monitored by bioluminescence imaging (BLI) and single photon emission computed tomography coupled with computed tomography (SPECT/CT).
In conclusion, this study demonstrated that PEG12-KL4 is an effective vector for delivering siRNAs in human NSCLC cell lines, whereas pulmonary delivery showed maximal siRNA localisation in the lungs. Furthermore, in vivo traceable lung cancer models were established. They serve as a useful tool to evaluate the anti-tumour efficacy of siRNA therapeutics which a promising strategy to treat NSCLC.
| Date of Award | 1 Mar 2024 |
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| Original language | English |
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| Supervisor | Jenny Lam (Supervisor) & Gilbert Fruhwirth (Supervisor) |