Patient-Derived Glioma Models: From Patients to Dish to Animals

Abstract

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults associated with a poor survival. Current standard of care consists of surgical resection followed by radiation and chemotherapy. GBMs are highly heterogeneous, having a complex interaction among different cells within the tumor as well as the tumor microenvironment. One of the main challenges in the neuro-oncology field in general, and GBM in particular, is to find an optimum culture condition that maintains the molecular genotype and phenotype as well as heterogeneity of the original tumor in vitro and in vivo. Established cell lines were shown to be a poor model of the disease, failing to recapitulate the phenotype and harboring non-parental genotypic mutations. Given the growing understanding of GBM biology, the discovery of glioma cancer stem-like cells (GSCs), and their role in tumor formation and therapeutic resistance, scientists are turning more towards patient-derived cells and xenografts as a more representative model. In this review, we will discuss the current state of patient-derived GSCs and their xenografts; and provide an overview of different established models to study GBM biology and to identify novel therapeutics in the pre-clinical phase.

Keywords: 3D cultures; cancer stem cells; glioblastoma; organoids; patient-derived xenograft model.

Figure 1

Glioblastoma model from patient to dish to animal. Typical workflow of establishing patient-derived glioblastoma (GBM) models. (Top) Tumor sample obtained from resective surgery is dissociated and single tumor cells are selected. Culturing with adequate growth conditions will facilitate the formation of GBM neurospheres. (Bottom Right) Obtained neurospheres can be tested for various properties, such as stemness, clonogenic potential, and viability; or can be engineered to express different proteins of interest or reporters for cell tracking. (Bottom Left) GBM neurospheres can be used to establish patient-derived xenografts (PDX) to study tumor biology and to test novel therapeutics by non-invasive monitoring of tumor volume with in vivo bioluminescence imaging or ex vivo fluorescence and histological analysis with Haematoxylin and Eosin staining. GSC: glioma stem cell.

Figure 2

In vitro analysis of GSC self-renewal using the limiting dilution assay. GSCs are dissociated into single cells and different amounts are plated in different wells. The number of wells containing spheres is then evaluated and micrographs are obtained to visualize sphere morphology. In the examples provided, sphere formation assays are performed on different GSC populations to evaluate their stemness and clonogenic potential. (A) Micrographs of GSC population A (top) and GSC population B (bottom); scale bar, 200µm. (B) The amount of initially seeded cells (x-axis) is plotted against the log fraction of non-responders corresponding to wells without any detected spheres (y-axis). The slope of the line represents the log-active cell fraction. (C) Data obtained using the limiting dilution assay entered into the extreme limited dilution assay (ELDA) tool. (D) Overall test for differences in stem cell frequencies between the two groups. (E) Confidence intervals for 1/(stem cell frequency). (F) Goodness of fit tests. (G) Representative images of neurospheres formed by 3565 GSCs following 10 days of Bone Morphogenetic Protein 2 (BMP2) and/or Gremlin1 treatment. (H) In vitro limiting dilution assay and (I) quantification following 10 days of BMP2 and/or Gremlin1 treatments. (G–I) Reproduced and adapted with permission from Yan et al., Genes & Development; published by Cold Spring Harbor Laboratory Press, 2014 [51].

Figure 3

Tumor xenograft analysis workflow in vitro, in vivo, and ex vivo. (A) Patient-derived MGG23 GSCs were treated with different concentrations of temozolomide (TMZ; 100 nM to 1 mM) in the presence or absence of 30 μM hydroxyurea (HU), and cell viability was measured 3 days post-treatment. Results are shown as the mean ± SD where the vehicle control was set at 100%. (B,C) MGG23 neurospheres were treated with either DMSO, HU, TMZ, or HU + TMZ for 4 days. Spheres were counted, washed, and left without treatment for another 5 days to allow recovery. Recovered spheres were dissociated and plated in a new 48-well plate to measure secondary sphere formation 5 days later. Representative image from each treatment group is shown; scale bar, 200 μm (B). Total sphere numbers in the well were recorded at each event (C). (D,E) Mice-bearing MGG23 tumors were injected with DMSO, HU, TMZ, or TMZ + HU. Representative Fluc bioluminescence image of a single mouse from each group is shown over time (D) with corresponding Kaplan–Meier survival analysis comparing the two groups; # p < 0.05 vs. control, ** p < 0.01 TMZ + HU vs. TMZ (E). (F) Ex vivo histological analysis with Haematoxylin and Eosin staining 42 days after tumor injection. Reproduced and adapted with permission from Teng et al., Neuro-Oncology; published by Oxford University Press, 2018 [53].