Fibroblast heterogeneity in non-small cell lung cancer and optical imaging targeting fibroblast activation protein
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Date
19/05/2023Item status
Restricted AccessEmbargo end date
19/05/2024Author
Mathieson, Layla
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Abstract
Cancer associated fibroblasts (CAFs) are one of the predominant cell types of non-small cell
lung cancer (NSCLC) tumour stroma. This thesis will investigate fibroblast heterogeneity in
NSCLC; defining their phenotype by expression of CAF markers, phenotypic changes in culture
and develop optical imaging probes for imaging fibroblasts in-situ.
In the first results chapter, CAFs have been isolated from NSCLC patient resections and their
phenotype characterised using known CAF markers by flow cytometry. This data led to the
identification of five CAF subsets, CAFS1-S5. By comparing CAFs isolated from tumour tissue
and fibroblasts isolated from tumour adjacent non-cancerous lung (NCL), it was identified that
two of these subsets, CAF-S2 and CAF-S3, were phenotypically similar to NCL fibroblasts,
whereas the three subsets, CAF-S1, CAF-S4 and CAF-S5, were predominant in cancerous
tissue. The presence of these subsets was then investigated by multiplex
immunofluorescence staining of a tumour microarray, which contained a cohort of 163 NSCLC
patient tumours. It was found that the presence of CAF-S1 and CAF-S5, both subsets where
CAFs expressed fibroblast activation protein (FAP) and podoplanin, with CAF-S1 also
expressing alpha-smooth muscle actin, were indicative of poorer recurrence free survival
outcome.
Then in the second results chapter, the effects on cell phenotype of culturing CAFs and NCL
fibroblasts was investigated. This revealed that when cultured by standard tissue culture
methods, fibroblast phenotype converged to a CAF-S3 phenotype, regardless of cancerous or
non-cancerous origin. To try and overcome the limitations of standard tissue culture methods,
a NSCLC tumour organoid model was then developed, which allowed for the culture of other
cell types, predominantly epithelial cells, alongside fibroblasts.
Fibroblast activation protein (FAP) was expressed on the CAF subsets present predominantly
in tumour tissue, and expression of FAP was low on NCL fibroblasts. In the final results
chapter, FAP was considered as a target for optical imaging probes. The iterative development
of a FAP probe which is specific over similar peptidases to FAP is described. These optical
imaging probes could be used to highlight the tumour stroma and allow disease-monitoring,
prognostication and potentially stratify treatment therapies. It is shown that a FAP probe can
be designed to be selective over closely related peptidases, compatible with physiological
levels of FAP expressed on primary CAFs, and the probe can be used to image NSCLC samples
on a clinically compatible fibre-based imaging system.
In conclusion, this thesis describes the CAF signatures in human NSCLC, changes over time in
culture, the development of an organoid model and an optical imaging strategy for FAP
imaging.
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