Development of Bioelectronic Technologies for the Investigation of Extracellular Vesicle Function and Anti-Cancer Drug Discovery


Type
Thesis
Change log
Authors
Traberg, Walther 
Abstract

Extracellular vesicles (EVs) are cell-secreted phospholipid bilayer-delimited particles of varying size and composition that facilitate intercellular communication. EVs play a critical role in a wide range of diseases by mediating the transfer of active biomolecules between cells, both in the vicinity of the source cell and at distant sites, to elicit a variety of phenotypic responses. Tumour-derived (T)EVs facilitate the transfer of information between tumour and non-malignant cells to initiate and drive metastasis through a variety of processes, including the epithelial-to-mesenchymal transition (EMT) and angiogenesis. As such, TEVs represent a novel therapeutic target in a field severely lacking in efficacious anti-metastasis treatments. Research into EV biology and function has seen a boom over the last decade owing to their unique features and potential clinical utility, and the demand for new and robust tools to study their functional activity has risen commensurately. However, conventional characterisation methods fail to comprehensively capture the dynamic nature of EV function, and this has obscured the true role of EVs in pathophysiological processes despite their pleiotropic functions in vitro. Furthermore, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are missing.

Here, bioelectronic technologies are utilised as a promising, scalable technology for investigating the dynamics of EV function in a truly quantitative manner using both cell-free and cell-based models. Furthermore, proof of concept drug screening studies demonstrate the applications of these bioelectronic platforms to preclinical drug discovery, addressing the current lack of robust, scalable sensing technologies that facilitate facile translation to in vivo outcomes. In this work, organic bioelectronic devices were integrated with cell-free, supported lipid bilayer (SLB) models and a cell-based breast cancer metastasis model. In the case of the former, electronic devices were used to detect virus-membrane fusion, which laid the groundwork for the subsequent study on EV-membrane binding, as viruses and EVs share many similarities in form and function. SLB integration with conducting polymer coated substrates and electronic devices was assessed using optical techniques. Virus hemifusion to both synthetic and natively derived SLBs was detected electrically using electrodes and organic electrochemical transistors (OECTs). Next, a supported biomimetic stem cell membrane incorporating membrane components from human primary adipose-derived stem cells (ADSCs) was formed to monitor the binding of cancer-derived exosomes (cEXOs) to the plasma membrane and show that this binding can be blocked when an antibody to integrin β1, a component of ADSC surface, is exposed to the membrane surface prior to cEXOs. These SLB platforms used a label-free electronic readout as well as a dual capability of optical (fluorescence) readout.

Lastly, the development of a functional phenotypic screening platform based on OECTs for real-time, non-invasive monitoring of TEV-induced EMT and screening of anti-metastatic drugs is reported. TEVs derived from the triple-negative breast cancer (TNBC) cell line MDA-MB-231 induced EMT in non-malignant breast epithelial cells (MCF10A) over a 9-day period, recapitulating a model of invasive ductal carcinoma metastasis. Epigenetics and immunoblot analysis showed that TEVs modulate TWIST1 protein amount but not DNA methylation level, and dual optical and electrical readouts of cell phenotype using OECTs were obtained. Further, heparin, a competitive inhibitor of cell surface receptors, was identified as an effective blocker of TEV-induced EMT, providing proof of principle that inhibitors of TEV function can be potential anti-metastatic drug candidates.

Together, these results demonstrate the utility of bioelectronic platforms presented here for studying TEV function, both in a cell-based and cell-free manner, and identifying inhibitors of TEV function for drug discovery applications. In additional to being readily scalable, organic electronics facilitate facile modelling of the transient drug response using electrical measurements and are amenable to integration with highly tuneable models systems.

Description
Date
2022-08-12
Advisors
Owens, Roisin
Keywords
Bioelectronics, Cancer, Drug screening, Exosomes, Extracellular vesicles, Materials, Metastasis
Qualification
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Sponsorship
Cambridge Trust