Tracing clonal cell plasticity in the microenvironment of pancreatic ductal adenocarcinoma

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The pancreas may regenerate without stem cells by induction of cellular plasticity between differentiated cells. Although essential for the remarkable capacity for pancreas regeneration, this property entails a vulnerability to cancer through acinar-to-ductal metaplasia and dedifferentiation by epithelial-mesenchymal transition, which contributes in largely unknown ways to metastasis in pancreatic cancer. As with most solid tumours, pancreatic ductal adenocarcinoma develops at the interface of cellular plasticity, genomic instability, and tumour microenvironments. However, current methodologies of analysing tumour tissue are unable to measure these modalities jointly. Here, by developing a method for epitope barcoding measured by cyclical imaging of antibody binding with confocal microscopes, this thesis explores the utility of multicolour cell labelling to investigate how polyclonal cancer evolution relates to epithelial-mesenchymal transition. The assembled barcodes consist of combinations of epitopes attached to a fluorophore and a nuclear-localisation signal, which enables cell sorting, quantitative image analysis, and effective decoding by statistical analysis.

Using subcutaneous mouse models of pancreatic cancer, this thesis demonstrates that isogenic epitope labelling can detect epithelial clonal expansion events co-localised with fibroblasts in the tumour microenvironment. The epitope barcode constructs were stably expressed in mouse tumours for at least 2-3 weeks, proving feasibility of multicolour lineage tracing with cyclical imaging. By amending the barcodes with 8bp tags sequenced with single-cell RNA-seq, transcriptomic scores for epithelial-mesenchymal transition showed evidence for selectional acquisition of epithelial lineages during subcutaneous colonisation. Such labelled isogenic cell populations were amenable to Luria–Delbrück fluctuation analysis detecting lineages with epigenetic inheritance of cell states. Altogether, epitope barcoding and cyclical imaging form a compelling experimental strategy for investigating the clonality of transdifferentiation in the microenvironment of pancreatic cancer.

Gill, Michael B
Marioni, John
Miller, Martin
clonal expansion, cyclical imaging, epithelial-mesenchymal transition, epitope barcoding, pancreatic cancer, polyclonality, tumour microenvironment
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Anglo-Danish Society. Augustinus Fonden.