Microfluidic methods for single cell analysis in clinically relevant samples
File(s)
Author(s)
Katsanovskaja, Ksenia
Type
Thesis
Abstract
Single cell protein analysis has the potential to comprehensively profile cellular
heterogeneity advancing the understanding of cell function, disease progression and drug
development. Although it is a possible contributor to the discovery of novel therapy, the protein
mapping on the individual cell basis is complex and poses challenges. For example, cancer is
a heterogeneous disease which analysis using conventional bulk methods can potentially
conceal a dangerous population like Circulating Tumour Cells (CTCs). Analysis of CTCs can
only be reliably attained using single cell technologies. The Microfluidic Affinity Capture
(MAC) chip is a tool that was developed in our group to study cellular heterogeneity by
measuring protein abundance in single cells. Another problem in analysing cancer cells is the
difficulty in obtaining samples in a non-invasive manner. This thesis reports on attempts to
obtain, concentrate and analyse CTCs from blood using a composite MAC-based device.
The ability to analyse single cells has advantages beyond the potential for analysing
heterogeneity. Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous illness that
is characterised by a chronic inflammation. Although the immune response in COPD requires
investigation, it is complicated by a lack of biomarkers and methods to non-invasively collect
samples. The ability to obtain and effectively analyse precious and scarce biomaterial from
lungs, is greatly advantageous for both diagnosis and tracking of COPD. Here, we report on a
workflow to achieve this using sputum from negative control volunteers and COPD+ patients.
Also, we describe the establishment of a novel MAC chip assay targeting FOXO3 protein
which is involved in regulation of processes like tumour suppression, inflammation and
senescence. Therefore, it has a biomarker potential to monitor and study diseases like cancer
and COPD. We developed the protocol to analyse Forkhead box 3 (FOXO3) protein expression
in nasal cells from healthy donor samples that were retrieved with a non-invasive tool
(NASAM, nasal synthetic absorptive matrix). Nasal cavity is a front line of exposure to inhaled
pernicious substances and, it is speculated to reflect anomalous bioprocesses in lungs preceding
respiratory disease development and progression. This study provides the first-time
quantification of FOXO3 protein in single cells from nasal samples. This work shows the
analytical capacity of the MAC chip to study cellular heterogeneity and quantify important
biomarkers in single cells of clinically relevant material.
heterogeneity advancing the understanding of cell function, disease progression and drug
development. Although it is a possible contributor to the discovery of novel therapy, the protein
mapping on the individual cell basis is complex and poses challenges. For example, cancer is
a heterogeneous disease which analysis using conventional bulk methods can potentially
conceal a dangerous population like Circulating Tumour Cells (CTCs). Analysis of CTCs can
only be reliably attained using single cell technologies. The Microfluidic Affinity Capture
(MAC) chip is a tool that was developed in our group to study cellular heterogeneity by
measuring protein abundance in single cells. Another problem in analysing cancer cells is the
difficulty in obtaining samples in a non-invasive manner. This thesis reports on attempts to
obtain, concentrate and analyse CTCs from blood using a composite MAC-based device.
The ability to analyse single cells has advantages beyond the potential for analysing
heterogeneity. Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous illness that
is characterised by a chronic inflammation. Although the immune response in COPD requires
investigation, it is complicated by a lack of biomarkers and methods to non-invasively collect
samples. The ability to obtain and effectively analyse precious and scarce biomaterial from
lungs, is greatly advantageous for both diagnosis and tracking of COPD. Here, we report on a
workflow to achieve this using sputum from negative control volunteers and COPD+ patients.
Also, we describe the establishment of a novel MAC chip assay targeting FOXO3 protein
which is involved in regulation of processes like tumour suppression, inflammation and
senescence. Therefore, it has a biomarker potential to monitor and study diseases like cancer
and COPD. We developed the protocol to analyse Forkhead box 3 (FOXO3) protein expression
in nasal cells from healthy donor samples that were retrieved with a non-invasive tool
(NASAM, nasal synthetic absorptive matrix). Nasal cavity is a front line of exposure to inhaled
pernicious substances and, it is speculated to reflect anomalous bioprocesses in lungs preceding
respiratory disease development and progression. This study provides the first-time
quantification of FOXO3 protein in single cells from nasal samples. This work shows the
analytical capacity of the MAC chip to study cellular heterogeneity and quantify important
biomarkers in single cells of clinically relevant material.
Version
Open Access
Date Issued
2020-04
Date Awarded
2020-12
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Klug, David
Willison, Keith
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)