Diagnosing cancer one cell at a time with single molecule spectroscopy
File(s)
Author(s)
Magness, Alastair
Type
Thesis
Abstract
Mapping protein expression heterogeneity in cancer at single cell resolution is essential for the understanding of disease progression, emergent drug resistance, and metastasis, but is a great technical challenge. Longitudinal monitoring of heterogeneity pertaining to biomarker expression may provide the necessary medical cues for the administering of personalised therapeutics. Studying molecular heterogeneity in the ultra-rare circulating tumour cells [CTCs] found in the blood of cancer patients is a greater challenge still, but success may yield deep insight into the nature of the metastatic cascade, and also provide the technologi-cal means of a non-invasive ‘liquid biopsy’.
The MAC chip is a quantitative single molecule sensitive protein assay for the evalua-tion of protein copy number in single cells. In this thesis, we attempt the development of a new biomarker-targeting MAC chip assay for the breast cancer oncoprotein estrogen recep-tor alpha. We describe a series of improvements to the MAC chip assay architecture allow-ing multiplexed measurement of several proteins simultaneously, and improvements to analysis methods allowing for superior molecule counting. Building on the work of others in the field of circulating tumour cell isolation, we attempt the integration of the MAC chip analysis method into a multiple-stage device for the isolation and proteomic analysis of cir-culating tumour cells. Finally, using a multiplexed MAC chip device for the tumour sup-pressor protein p53 and its activated form phosphorylated at serine-15, we demonstrate for the first time that the MAC chip can be used to study protein expression heterogeneity in quasi-clinical samples. The patient-derived xenografts we use to perform this work are a key resource of clinically-relevant tumour material, and a model system directly analogous to primary patient biopsies, thus demonstrating the feasibility of translational single cell pro-teomics with the MAC chip system.
The MAC chip is a quantitative single molecule sensitive protein assay for the evalua-tion of protein copy number in single cells. In this thesis, we attempt the development of a new biomarker-targeting MAC chip assay for the breast cancer oncoprotein estrogen recep-tor alpha. We describe a series of improvements to the MAC chip assay architecture allow-ing multiplexed measurement of several proteins simultaneously, and improvements to analysis methods allowing for superior molecule counting. Building on the work of others in the field of circulating tumour cell isolation, we attempt the integration of the MAC chip analysis method into a multiple-stage device for the isolation and proteomic analysis of cir-culating tumour cells. Finally, using a multiplexed MAC chip device for the tumour sup-pressor protein p53 and its activated form phosphorylated at serine-15, we demonstrate for the first time that the MAC chip can be used to study protein expression heterogeneity in quasi-clinical samples. The patient-derived xenografts we use to perform this work are a key resource of clinically-relevant tumour material, and a model system directly analogous to primary patient biopsies, thus demonstrating the feasibility of translational single cell pro-teomics with the MAC chip system.
Version
Open Access
Date Issued
2017-08
Date Awarded
2018-02
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Klug, David
Willison, Keith
Coombes, Charles
Ali, Simak
Sponsor
Engineering and Physical Sciences Research Council
Cancer Research UK
Institute of Chemical Biology
Grant Number
C37990/A12991
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)