Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM.

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease.

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can be challenging and is often associated with a lack of reproducibility due to high batch-to-batch variation in animal-derived 3D culture matrices. Similarly, handling issues can limit the usefulness of synthetic hydrogels. In response to this need, we have optimized a simple self-assembling peptide gel, to enable the culture of relevant cell line models of cancer and disease, as well as patient-derived tissue/cells. The gel itself is free from matrix components, apart from those added during encapsulation or deposited into the gel by the encapsulated cells. The mechanical properties of the hydrogels can also be altered independent of matrix addition. It, therefore, acts as a 'blank slate' allowing researchers to build a 3D culture environment that reflects the target tissue of interest and to dissect the influences of mechanical forces and/or biochemical control of cell behavior independently.