THREE-DIMENSIONAL MULTICOLOUR LINEAGE TRACING OF INTRINSIC BRAIN TUMOUR MODELS

Abstract

Multicolour lineage tracing has provided striking demonstrations of stem cell tissue maintenance and development. Our objective is to gain insight into histogenesis, clonal expansion and migration of intrinsic brain tumours. We aim to investigate intratumoral heterogeneity through the identification of differently labelled clones but also the impact of different genetic lesions on tumour propagation. Combining multicolour tracing with advanced tissue clearing and image analysis we will interrogate these parameters in three-dimensions (3D). Rosa26-Confetti and Brainbow mice were crossed into lines containing different combinations of floxed tumour suppressor alleles. In this model, recombination is targeted to neural stem cells by intraventricular injection of a retrovirus expressing Cre recombinase and PDGFβ. Using this method multicolour tumours with histological hallmarks of human gliomas can be induced within 4 – 6 weeks. This rapid induction model will be used for the development of the methodology, including 3D image acquisition and image processing. Traditional sectioning and passive clarity (PACT) are used to prepare tissue for 2D and 3D imaging. This model of primary murine brain tumours will be complemented by a xenogafting approach, using genetically characterised multiple human glioma initiating cell lines, genetically labelled with lineage tracing vectors. Tumour formation will be assessed with methods established using the retroviral model. Retroviral injections induce tumour formation currently at an efficiency of ~50%. On frozen sections multiple colours are visible within all tumours representing the identification of different genetic clones. Tissue clearing has been successfully employed to image 3D volumes. The presence of differentially labelled cells in our tumour model opens the potential for assessing clonal expansion and tumour heterogeneity.