Neurosphere formation is an independent predictor of clinical outcome in malignant glioma

Abstract

Renewable neurosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. This retrospective study was designed to assess the relationship among neurosphere formation in cultured human glioma, tumorigenic capacity, and patient clinical outcome. Tumor samples were cultured in neurosphere conditions from 32 patients with glioma, including a subpopulation of 15 patients with primary glioblastoma. A subsample of renewable neurosphere cultures was xenografted into mouse brain to determine if they were tumorigenic. Our study shows that both renewable neurosphere formation and tumorigenic capacity are significantly associated with clinical outcome measures. Renewable neurosphere formation in cultured human glioma significantly predicted an increased hazard of patient death and more rapid tumor progression. These results pertained to both the full population of glioma and the subpopulation of primary glioblastoma. Similarly, there was a significant hazard of progression for patients whose glioma had tumorigenic capacity. Multivariate analysis demonstrated that neurosphere formation remained a significant predictor of clinical outcome independent of Ki67 proliferation index. In addition, multivariate analysis of neurosphere formation, tumor grade and patient age, demonstrated that neurosphere formation was a robust, independent predictor of glioma tumor progression. Although the lengthy duration of this assay may preclude direct clinical application, these results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma. Furthermore, this study suggests that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome.

Figure 1

Human cultured glioblastoma cells. (A) & (B) Nestin staining (blue) of undifferentiated neurospheres. (C) TUJ-1 staining (green) of differentiated cells. (D) GFAP staining (red) of differentiated cells. (E) Coronal section through the striatum shows normal cellular composition in the hemisphere contralateral to the injection site for xenotransplant into the brain of immunodeficient mice (EC=external capsule, Ctx=Cortex, CPu= caudate putamen (striatum)). (F) Hemisphere of injection site with dense nuclei of infiltrating tumor cells. (G) Coronal section through the striatum shows EGFP expressing human glioblastoma cells forming a tumor mass (arrow) in the injected (right) hemisphere. The xenograft here exhibits a highly infiltrative character that is typical of high grade glioma. Major routes of infiltration from the injected hemisphere to the contralateral side are the corpus callosum (cc) and the anterior commissure (ac). Section was pre-treated with RNAse and counterstained with Ethidium Bromide (red). Typhoon Scanner image, at 10μm resolution. The scale bar in F represents 200 microns in A, 32 microns in B, 50 microns in C and D, 133 microns in E and F, and 800 microns in G.

Figure 2

Kaplan-Meier curves display the proportion of patients surviving as a function of time since surgery. (A) In the full cohort of glioma (n=32), the group with renewable neurosphere formation has a significantly higher hazard of death (Cox Regression, P=0.032). (B) In the subpopulation of primary glioblastoma (n=15), the group with renewable neurosphere formation has a significantly higher hazard of (Cox Regression, P<0.001).

Figure 3

Kaplan-Meier curves display the proportion of patients surviving without tumor progression as a function of time since surgery. (A) In the full cohort of glioma (n=32), the group with renewable neurosphere formation has a significantly higher hazard of progression (Cox Regression, P=0.002). (B) In the population of glioma (n=26), the group with tumorigenic capacity in culture has a significantly higher hazard of progression (P<0.021).