Exploring tumour heterogeneity and responses to therapy using single-cell resolved microscopy
File(s)
Author(s)
Valand, Karishma
Type
Thesis
Abstract
Intra-tumour heterogeneity is key feature of many tumours and plays vital role in therapy resistance. Benefits of targeted treatments are often short-lived. The evolution of resistant cell clones during therapy remains poorly understood. Genetic variability, i.e. aneuploidy, and spatial variations in the tumour microenvironment (TME) largely influence signalling inputs individual tumour cells receive, directly modulating responses to treatments. This PhD explores factors of heterogeneity in non-small cell lung cancer (NSCLC) at the single-cell level, focusing on the intrinsic cell signalling pathways that are highly upregulated in cancer, specifically the MAPK and PI3K pathways which are vital for normal cell survival, migration and cell death.
A highlight of this thesis was the development of a novel combined fluorescence and phase microscopy technique and image analysis workflow for single-cell resolved, high-content, multichannel time-lapse imaging. This approach integrated fluorescent biosensors, ratiometric FRET, and quantitative phase imaging to follow the fate of individual tumour cells during therapy. I established a proof-of-principle for these methodologies to study the temporal dynamics of ERK signalling and cellular dry mass, and discussed how this approach could be used to monitor cell-cycle dynamics non-invasively at the single-cell level. Using this platform, I investigated single-cell responses in KRAS-G12C-mutant NSCLC cells. I demonstrated that dynamic ERK signalling, dry mass, and cell motility were reduced following inhibition with clinically relevant KRAS-G12C inhibitors.
Furthermore, I investigated whether aneuploidy influences cell growth, proliferation, and survival in EGFR-mutant NSCLC cells, however no significant differences were observed at baseline or upon treatment with a clinically relevant EGFR inhibitor.
Finally, I found that EGFR-mutant cells exhibited reduced sensitivity to EGFR inhibition in the presence of cancer associated fibroblasts (CAFs). These findings suggest that CAFs modulate ERK signalling in tumour cells upon treatment, establishing a foundation for future research into the mechanisms driving CAF-mediated therapy resistance.
A highlight of this thesis was the development of a novel combined fluorescence and phase microscopy technique and image analysis workflow for single-cell resolved, high-content, multichannel time-lapse imaging. This approach integrated fluorescent biosensors, ratiometric FRET, and quantitative phase imaging to follow the fate of individual tumour cells during therapy. I established a proof-of-principle for these methodologies to study the temporal dynamics of ERK signalling and cellular dry mass, and discussed how this approach could be used to monitor cell-cycle dynamics non-invasively at the single-cell level. Using this platform, I investigated single-cell responses in KRAS-G12C-mutant NSCLC cells. I demonstrated that dynamic ERK signalling, dry mass, and cell motility were reduced following inhibition with clinically relevant KRAS-G12C inhibitors.
Furthermore, I investigated whether aneuploidy influences cell growth, proliferation, and survival in EGFR-mutant NSCLC cells, however no significant differences were observed at baseline or upon treatment with a clinically relevant EGFR inhibitor.
Finally, I found that EGFR-mutant cells exhibited reduced sensitivity to EGFR inhibition in the presence of cancer associated fibroblasts (CAFs). These findings suggest that CAFs modulate ERK signalling in tumour cells upon treatment, establishing a foundation for future research into the mechanisms driving CAF-mediated therapy resistance.
Version
Open Access
Date Issued
2025-03-18
Date Awarded
2026-05-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
French, Paul
Sahai, Erik
Dunsby, Christopher
Sponsor
Cancer Research UK (CRUK) Accelerator Award
Publisher Department
Department of Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)