Glioblastoma's Next Top Model: Novel Culture Systems for Brain Cancer Radiotherapy Research

Abstract

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, remains one of the least treatable cancers. Current standard of care-combining surgical resection, radiation, and alkylating chemotherapy-results in a median survival of only 15 months. Despite decades of investment and research into the development of new therapies, most candidate anti-glioma compounds fail to translate into effective treatments in clinical trials. One key issue underlying this failure of therapies that work in pre-clinical models to generate meaningful improvement in human patients is the profound mismatch between drug discovery systems-cell cultures and mouse models-and the actual tumors they are supposed to imitate. Indeed, current strategies that evaluate the effects of novel treatments on GBM cells in vitro fail to account for a wide range of factors known to influence tumor growth. These include secreted factors, the brain's unique extracellular matrix, circulatory structures, the presence of non-tumor brain cells, and nutrient sources available for tumor metabolism. While mouse models provide a more realistic testing ground for potential therapies, they still fail to account for the full complexity of tumor-microenvironment interactions, as well as the role of the immune system. Based on the limitations of current models, researchers have begun to develop and implement novel culture systems that better recapitulate the complex reality of brain tumors growing in situ. A rise in the use of patient derived cells, creative combinations of added growth factors and supplements, may provide a more effective proving ground for the development of novel therapies. This review will summarize and analyze these exciting developments in 3D culturing systems. Special attention will be paid to how they enhance the design and identification of compounds that increase the efficacy of radiotherapy, a bedrock of GBM treatment.

Keywords: 3D culture systems; glioblastoma; glioma; pre-clinical drug development; radiotherapy.

Figure 1

Schematic comparison of tumor microenvironment and current culture systems. (A) The brain microenvironment provides a multitude of non-tumor cell types, including neurons, astrocytes, microglia, and endothelial cells. These populations are present in the tumor environment and have been shown to influence the progression of GBM tumors, as well as abet their ability to resist current standard of care therapies, including radiation. In addition, tumor cells in the brain grow in a complex 3D structure entangled with a brain-specific extra-cellular matrix (ECM). They also exhibit a marked amount of intratumoral heterogeneity, with a variety of subpopulations within each tumor. Finally, the tumor microenvironment varies widely across different anatomic locations, with differences in oxygen and nutrient availability; this range of milieus contributes to the intratumoral heterogeneity of GBM. (B) In contrast, current culture systems are over-simplified and introduce variables not encountered in the brain. Oxygen concentrations are held constant at 20% by most incubators, while media contains an elevated amount of glucose. Plastic flasks introduce an unnaturally stiff surface, which GBM cells are known to react to. Further, coating flask with matrigel, while better than culturing directly on plastic, exposes tumor cells to elevated levels of collagen and laminin. Mono-cultures mean that in vitro experiment fail to account for non-tumor cells contributions to the microenvironment.

Figure 2

Comparison of erlotinib and phenformin activity on three different cell culture systems. (A) Schematic representation (upper panels) and 3D reconstructions of immunofluorescent images (lower panels) of cells stained for F-actin (red) and nuclei (DAPI, blue) of three cell culture systems: 2D growth on plastic plates (left images, 63× magnification); 3D scaffold growth using 3D-Alvetex system (middle images 63× magnification); and 3D neurospheres (right images, 63× magnification, zoom 2×). 2D plastic and 3D scaffolds were previously coated with diluted Matrigel as described in section Matrigel-coating for 2D growth. (B) Bar charts representing surviving fraction of cells relative to control grown on different culture systems. G7 and E2 patient-derived GBM cell lines treated with vehicle (DMSO, control), phenformin (10 µM), or erlotinib (1 µM). Drug-treated cells were normalised to vehicle (mean ± SD, of three independent experiments). * p < 0.05, relative to DMSO control of their respective culture system.