Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway

Abstract

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

The mTOR pathway is active in growing, but not differentiated glioblastoma stem-like cells. (A) Upper panel: confocal analysis of four GSCs (TG1, TG10, TG16 and OB1) grown as neurosphere (NS) and labelled with Nestin (green), Sox2 (red) and nucleus (blue). Lower panel: confocal analysis of TG1 engaged in differentiation for 72 h and processed for immunostaining with Nestin and Tubulin βIII (green) together with nuclear counterstaining (blue). Scale bars, 10 μm. (B) Akt and S6 phosphorylations (p) were tested on total cell lysates from differentiated cells (#) and growing NS. (C) Total cell lysates from LN229 and U87 glioma cells were analysed for PTEN expression together with the four GSCs. Tubulin was used as a loading control. (D) Phosphatase activity was measured from PTEN immunoprecipitate fractions by colorimetric assay. LN229 and primary rat astrocytes (astro) were used as positive controls and U87 was used as a negative control. Graph represents mean+s.e.m. of three independent experiments. DAPI, 4,6-diamidino-2-phenylindole; GSC, glioblastoma stem-like cell; mTOR, mammalian target of rapamycin; NS, neurosphere.

Figure 2

Inhibition of the mTOR pathway prevents glioblastoma stem-like cell expansion. (A–E) GSCs were treated with vehicle (DMSO), rapamycin (RP 50 nM), PP242 (1 μM) and PI103 (10 μM). (F–I) GSCs were transfected with siRNA against mTor, Rictor and Raptor or a control siRNA (sic) for 72 h. (A,F) Protein extracts were tested by western blot analysis with the indicated antibodies. (B,C,G) Confocal analysis on growing TG1 neurospheres (NS) stained with Sox2 (red) and nucleus (blue) in B and G, and on differentiated TG1 (≠) stained with Tubulin βIII (green) in C. Scale bars, 10 μm. (D,H) The number of secondary neurospheres per field of view (FOV) was counted. (E,I) Cell viability was analysed by MTT assay and normalized to the optical density (OD) obtained with DMSO and sic controls. All graphs represent mean+s.e.m. of three independent experiments. Analysis of variance test: ***P<0.001, **P<0.01, *P<0.05. DAPI, 4,6-diamidino-2-phenylindole; GSC, glioblastoma stem-like cell; mTOR, mammalian target of rapamycin; p, phosphorylated; siRNA, small-interfering RNA.

Figure 3

Brain endothelial cells preserve glioblastoma stem-like cell properties. (A) Human brain endothelial cells (EC) were cultured for 24 h without mitogens, alone or cocultured with TG1. Confocal analysis of VE-cadherin (green), Sox2 (red) and nucleus (blue) staining. Scale bars, 10 μm. (B–F) GSCs were treated for 72 h with EC-conditioned medium (EC-CM) and compared with control medium (Ctl) and epithelial CM (293T-CM). (B) Confocal analysis of TG1 stained with Nestin (green), Sox2 (red) and nucleus (blue). Scale bars, 10 μm. (C) The number of secondary neurospheres per field of view (FOV) was counted. (D) Cell viability was analysed by MTT and normalized to the optical density (OD) obtained with control (Ctl). (E) Cell-cycle rate was assessed by carboxyfluorescein succinimidyl ester dilution and analysed by flow cytometry. (F) Cell death was measured on propidium iodide-stained cells by flow cytometry. All graphs represent mean+s.e.m. of three independent experiments. Analysis of variance test: ***P<0.001, **P<0.01, *P<0.05. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4,6-diamidino-2-phenylindole; GSC, glioblastoma stem-like cell; mTOR, mammalian target of rapamycin.

Figure 4

Brain endothelial cell secretome can restore mTOR activation in glioblastoma stem-like cells. (A) GSCs were treated with human brain endothelial cell-conditioned medium (EC-CM) for 72 h and compared with control medium (Ctl) and 293T-CM. Protein extracts were tested by western blot with the indicated antibodies. (B,C) GSCs were treated with EC-CM alone or in the presence of rapamycin (RP 50 nM), PP242 (1 μM) and PI103 (10 μM) for 24 h. (D,E) GSCs were transfected with siRNA against mTor, Rictor and Raptor or a control siRNA (sic) for 72 h. (B,D) The number of secondary neurospheres per field of view (FOV) was counted. (C,E) Cell viability was analysed by MTT and normalized to optical density (OD) obtained with DMSO and sic, respectively. All graphs represent mean+s.e.m. of three independent experiments. Analysis of variance test: ***P<0.001, **P<0.01, *P<0.05. DAPI, 4,6-diamidino-2-phenylindole; GSC, glioblastoma stem-like cell; mTOR, mammalian target of rapamycin; siRNA, small-interfering RNA.