Fibroblast heterogeneity in non-small cell lung cancer and optical imaging targeting fibroblast activation protein

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.