Innes, James Alexander; (2022) Analysis of glioma cell heterogeneity by lineage-tracing in murine and human model systems. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Intra-tumour heterogeneity and plasticity are key factors in treatment resistance and the recurrence of Glioblastoma (GBM), which is invariably fatal. Genetics, epigenetics, cell metabolism and plastic cancer stem cell (CSC) hierarchies interact with volatile micro-environmental forces to promote and shape cell identity. Improved understanding of these factors will inform more precise and effective GBM therapies. Here, we aim to develop a fluorescent tracking approach for patient derived GBM cells to investigate the relationship between clones, environment and CSC marker expression. Using a murine GBM model combined with Rosa26-confetti fluorescent labelling, we trialled suitable techniques for detection of labelled tumour clones and concluded fluorescent imaging and flow cytometry were the most effective. For patient-derived cells, we modified LeGO-vector fluorescent labelling with the aim of tracking a greater number of clones. We further optimised this technique for simultaneous flow cytometry detection of clones and their CSC marker expression. In the final chapter, we address the hypothesis that whole population CSC surface marker plasticity is a result of emergent clonal predominance. In two patient derived GBM lines, under steady-state environmental conditions, serial passaging and assessment of clonal marker expression detected distinct marker expression patterns between clones in the same culture dish. For both cell lines, transfer and culture of clonal mixtures to Matrigel® spheroids produced an expected plastic transition in population marker expression but also considerable predominance of certain clones. While the clonalsurface marker dynamics of the two cell lines were markedly distinct, divergent surface marker plasticity between clones of the same cell line was a consistent observation. Taken together these results supported our hypothesis that population marker plasticity is in part a result of emergent clonal predominance. We propose our developed techniques are suitable for rapid and economic characterisation of patient specific gene disruption, therapeutic vulnerabilities and resistance mechanisms.

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