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. 2016 Dec 27;7(52):86406-86419.
doi: 10.18632/oncotarget.13415.

Patient-derived Glioblastoma Stem Cells Respond Differentially to Targeted Therapies

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Free PMC article

Patient-derived Glioblastoma Stem Cells Respond Differentially to Targeted Therapies

Pratik Kanabur et al. Oncotarget. .
Free PMC article

Abstract

The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.

Keywords: glioblastoma; glioblastoma stem cells; patient-derived glioblastoma stem cells; targeted therapies.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. GSCs cultured in plates with or without polyHEMA coating
(A) Spheres of U251/GSC and GBM10/GSC. GSCs were maintained in stem cell media as spheres. Pictures were taken using a Zeiss inverted microscope with a 10X lens. (B) LN229/GSCs. Cells were grown as monolayer (left panel) in stem cell media or as spheres (right panel) in poly-HEMA-coated plates. (C) Schematic diagram illustrating the steps of GSC isolation from freshly dissected tumor tissues. (D) Spheres of GSCs derived from patient tissues. (E) Patient-derived GSCs cultured in plates with or without polyHEMA coating. These GSCs were similar to LNC229/GSCs and highlighted in bold. Scale bar is 50 μm.
Figure 2
Figure 2. The propensity of GSCs to self-renew in vitro or to form a tumor in vivo
(A) Sphere formation of GSCs. Single GSCs at different cell densities were plated. The percentages of wells with no spheres were plotted against the numbers of cells plated. A linear regression model was applied. (B) Sphere formation of GSCs in polyHEMA-coated plates. (C) Sphere formation numbers. The numbers that are required for sphere formation (abbreviated as sphere formation number) were determined based on the linear regression models (A and B). GBM10/GSCs were tested in plates with or without polyHEMA coating. The sphere formation numbers of patient-derived GSC lines (highlighted in bold) in polyHEMA-coated plates were adjusted based on the results from GBM10/GSCs. (D) Hematoxylin & Eosin staining of the subcutaneous tumor from VTC-036/GSCs.
Figure 3
Figure 3. Dividing of single GSCs
Single VTC-034/GSCs (A–B), VTC-064/GSCs (C–D), GBM10/GSCs (E–F), adherent LN229/GSCs (G–H) were plated and imaged using a 40X lens of an inverted microscope every day for seven consecutive days. Cropped images are shown. Images of adherent LN229/GSCs were in a different scale in order to show more cells. White arrows indicate the two-cell stages.
Figure 4
Figure 4. Expression of nestin and GFAP in GSCs
(A) The protein levels of nestin (NES) and GFAP. β-actin (ACTB) was used as the loading control. Band intensities were quantified using Image J software. The ratios of NES/ACTB are shown. Correlations between sphere formation numbers and the protein levels of nestin (B) or GFAP (A–C) were determined using the linear regression model. Coefficients of determination (R2) are shown.
Figure 5
Figure 5. Expression of NOTCH1 and PDGFR in GBMs and GSCs
(A) mRNA levels of NOTCH1, PDGFRA, and PDGFRB in GBMs. Gene expression data (RNAseq) were retrieved from the TCGA database and analyzed using the GlioVis program. The mRNA levels were compared between the non-tumor control and GBM samples. (B) Protein levels of NOTCH1 and PDGFR in GSCs determined by immunoblotting. Band intensities were measured using Image J software. The legend for categorizing protein levels is shown.
Figure 6
Figure 6. The effect of imatinib and γ-secretase inhibitor IX on U251/GSCs and GBM10/GSCs
U251/GSCs and GBM10/GSCs were plated at different cell densities and treated with vehicle DMSO, imatinib (10 μM), or γ-secretase inhibitor IX (20 μM). The cells were imaged (A and B) using a 40X lens of an inverted microscope. The sphere formation numbers were calculated as described in Methods section (C). Scale bar is 10 μm.
Figure 7
Figure 7. The effect of imatinib and γ-secretase inhibitor IX on patient-derived GSCs
GSCs were plated at different cell densities and treated with vehicle DMSO, imatinib (10 μM), or γ-secretase inhibitor IX (20 μM). Cells were imaged using a 40 X lens of an inverted microscope (A). The sphere formation numbers were determined based on numbers of cells plated and the percentages of wells with no spheres (B). The responses of VTC-037/GSC to these drugs were determined using the MTS viability assay (C). Scale bar is 10 μm. Error bars represent standard deviations from three independent experiments.
Figure 8
Figure 8. The effect of αCT1 and TMZ in GBM10/GSCs and VTC-036/GSCs
(A) GBM10/GSCs treated with αCT1 and/or TMZ were imaged using a 40 X lens of an inverted microscope. Scale bar is 10 μm. (B) The viability of GBM10/GSCs determined by the MTS viability assay. (C) The sphere formation of VTC-036/GSCs. Error bars represent standard deviations from three independent experiments.

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