Fig 1.
EGFRvIII expression maintains stemness of GSCs.
(A) Semiquantitative RT-PCR of EGFR-WT and EGFRvIII in various GSCs (EGFRvIII positive cells; CSC2, X01, X03, X04, X06, X08, X09, MD30, 1123NS, 83NS, and EGFRvIII negative cells; X02, Ex Vivo, and 528NS) and EGFRvIII-overexpressing Astrocyte (Astrocyte-EGFRvIII is used as EGFRvIII positive control). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Immunoblot (IB) analysis of EGFR, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 and X01 cells (day 0) and 10% serum-cultured CSC2 and X01 cells. Serum-cultured CSC2 and X01 cells were harvested after indicated time (days 4, 7, and 9). α-tubulin was used as a loading control. (C, D) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 cells (day 0) and 10% serum-cultured CSC2 cells (day 7) (C) and in serum-free GSC cultured X01 cells (day 0) and 10% serum-cultured X01 cells (day 7) (D). (E) Immunocytochemistry (ICC) of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 and X01 cells that were incubated in serum-free (day 0) or serum medium for 7 d (day 7). Nuclei were counterstained with DAPI (blue). (F) IB analysis of phosphorylated EGFR (p-EGFR), EGFR, Sox2, Nestin, and GFAP in CSC2 cells transfected with EGFRvIII small interfering RNA (siRNA) or its control. α-tubulin was used as a loading control. (G) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. GAPDH was used as a loading control. (H) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. Nuclei were counterstained with DAPI (blue). (I) Limiting dilution assay (LDA) was performed in CSC2 cells transfected with EGFRvIII siRNA or its control. p = 0.00966. (J) IB analysis of p-EGFR, EGFR, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. α-tubulin was used as a loading control. (K) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. GAPDH was used as a loading control. (L) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. Nuclei were counterstained with DAPI (blue). (M) LDA was performed in X02 infected with EGFRvIII-expressing lentiviral or control construct. p = 0.0000266.
Fig 2.
Identification of PEDF as a novel autocrine factor regulated by EGFRvIII through STAT3 signaling.
(A) Sphere formation assay of CSC2 cell cultured in control medium (serum-free GSC medium), serum-free GSC-conditioned medium (CM), or serum-CM. Prior to the harvest of CMs, the cells were washed with phosphate buffered saline (PBS) and changed with F12 medium without serum or other supplement for 24 h. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± standard error of the mean (SEM) (n = 3). * p < 0.05; ** p < 0.01. (B) Schematic representation of mass spectrometry analysis. CMs from CSC2 cultured in serum-free GSC or serum-cultured medium were respectively harvested after 2 wk. Also, Serum-free GSC CM from CSC2 (EGFRvIII+ GSC) or Ex Vivo (EGFRvIII- GSC) were respectively harvested after 2 wk. Prior to the harvest of CMs, the cells were washed with PBS and changed with F12 medium without serum or other supplement for 24 h. All of them were analyzed by liquid chromatography–mass spectrometry–mass spectrometry (LC-MS/MS). (C) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2, X01, and X02 cells) incubated in serum-free GSC or serum-cultured medium. α-tubulin was used as a loading control. (D) Sphere formation assay was performed in CSC2 cells incubated in serum-free CSC2-Con or CSC2-shPEDF2 CM. Two CMs respectively obtained from CSC2 cells infected with shPEDF2-expressing lentiviral or control construct, cultured in serum-free GSC medium. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). ** p < 0.01. (E) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, and PEDF (in medium) in GSCs (CSC2 and X01cells) transfected with EGFRvIII siRNA or its control. β-actin was used as a loading control. (F) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2 and X01) transfected with siEGFR-WT or siControl. α-tubulin was used as a loading control. (G) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in GSCs (CSC2 and X01) infected with EGFR WT-expressing lentiviral or control construct. α-tubulin was used as a loading control. (H) IB analysis of p-EGFR, EGFR, p-STAT3, STAT3, PEDF (in medium), Sox2, Nestin, and GFAP in X02 cells infected with EGFR-WT, EGFRvIII-expressing lentiviral or their control construct. β-actin was used as a loading control. (I) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in CSC2 and X01 cells treated with a small-molecule inhibitor of STAT3 (Stattic, 5 uM) or vehicle for 6 h. α-tubulin was used as a loading control. (J) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in CSC2 and X01 cells transfected with siSTAT3 or its control. α-tubulin was used as a loading control. (K) IB analysis of PEDF (in medium), Nestin, Sox2, GFAP, p-STAT3, and STAT3 in X02 cells infected with EGFRvIII-expressing lentiviral or their control construct. Also, these cells were transfected with siSTAT3 or its control. GAPDH was used as a loading control.
Fig 3.
Recombinant PEDF promotes stemness and self-renewal of GSCs.
(A) Sphere formation assay was performed in X02 cells treated with recombinant PEDF (rPEDF) (100 ng/ml) or control vehicle. Images are representative of three independent experiments. The graph represents average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). * p < 0.05. (B) IB analysis of Sox2, Nestin, and GFAP in X02 cells treated or not treated with rPEDF (100 ng/ml). β-actin was used as a loading control. (C) Sphere formation assay was performed in X01 cells cultured in serum-free GSC medium (containing EGF and bFGF) or serum-free GSC medium without EGF and bFGF. X01 cells cultured in serum-free GSC medium without EGF and bFGF were treated with rPEDF (100 ng/ml) or control vehicle. Images are representative of three independent experiments. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). ** p < 0.01. (D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X01 cells cultured in three different conditions (C). α-tubulin was used as a loading control. All images were taken at 5x magnification.
Fig 4.
PEDF expression maintains stemness and self-renewal of GSCs.
(A, C) LDA was performed in GSCs (CSC2 and X01) infected with shPEDF1-expressing lentiviral, shPEDF1 with PEDF-overexpressing lentiviral, or control construct. CSC2-Con or CSC-shPEDF1; p = 0.000201, CSC2-Con or CSC-shPEDF1-PEDF; p = 0.576, CSC2-shPEDF1 or CSC-shPEDF1-PEDF; p = 2.23e-05 (A) and X01-Con or X01-shPEDF1; p = 0.000265, X01-Con or X01-shPEDF1-PEDF; p = 0.589, X01-shPEDF1 or X01-shPEDF1-PEDF; p = 2.99e-05 (C). (B, D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in CSC2-Con, CSC2-shPEDF1, or CSC2-shPEDF1-PEDF (B) and X01-Con, X01-shPEDF1, or X01-shPEDF1-PEDF (D). β-actin was used as a loading control. (E) LDA was performed in X02 infected with PEDF-expressing lentiviral or control construct. X02-Con or X02-PEDF; p = 0.00936. (F) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X02-Con or X02-PEDF cells. β-actin was used as a loading control. (G) IB analysis of PEDF (in medium), p-EGFR, EGFR, p-STAT3, STAT3, Nestin, Sox2, and GFAP in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. (H) Sphere formation assay was performed in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). * p < 0.05.
Fig 5.
PEDF maintains stemness and self-renewal of GSCs by activating Notch-Sox2 pathway.
(A) IB analysis of PEDF (in medium) and NICD in X02 cells infected with PEDF-expressing lentiviral or control construct (upper panel) and Ex Vivo cells infected with PEDF-expressing lentiviral or control construct (lower panel). α-tubulin was used as a loading control. (B) IB analysis of PEDF (in medium) and NICD in X01 cells infected with shPEDF2-expressing lentiviral or control construct (upper panel) and CSC2 cells infected with shPEDF2-expressing lentiviral or control construct (lower panel). α-tubulin was used as a loading control. (C) IB analysis of NICD, Sox2, and Nestin in X02-Con, X02-PEDF, or X02-PEDF cells treated with 1 μM of DAPT (γ-secretase inhibitor). (D) Sphere formation assay was performed in X02-Con, X02-PEDF, or X02-PEDF treated with 1 uM of DAPT. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). ** p < 0.01. (E) Semiquantitative RT-PCR of PEDF and Sox2 in X02-Con or X02-PEDF (upper panels) and X01-Con or X01-shPEDF2 (lower panels). GAPDH was used as a loading control. (F) Chromatin immunoprecipitation (ChIP assay) was performed in X02-Con or X02-PEDF cells with NICD-specific or control antibodies. The strategy for the ChIP assay is represented in the upper panel. Consensus CBF1 binding site (TGGGAA) located in Sox2 at -1007 and -889. (G) IB analysis of PEDF (in medium), Sox2, Nestin, GFAP, and NICD in X01-Con, X01-shPEDF2, X01-Sox2, or X01-shPEDF2 coinfected with Sox2-expressing lentiviral construct. α-tubulin was used as a loading control. (H) Sphere formation assay was performed in X01-Con, X01-shPEDF2, X01-Sox2, or X01-shPEDF2 coinfected with Sox2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM (n = 3). ** p < 0.01.
Fig 6.
EGFRvIII/STAT3/PEDF signaling in GSCs.
(A) IB analysis of EGFR, EGFRvIII, p-EGFR, p-STAT3, STAT3, and PEDF (in medium) in 13 GSCs. α-tubulin was used as a loading control. EGFRvIII+/PEDFhigh GSCs are CSC2, X01, X03, X04, X06, X08, and X09 cells. EGFRvIII+/PEDFlow GSCs are MD30, 1123NS, and 83NS cells. EGFRvIII-/PEDFlow GSCs are X02, Ex Vivo, and 528NS cells. (B) Histopathology of Balb-c/nu mouse brain tissue was orthotopically injected with three representative types of GSCs (A). Upper panel is hematoxylin and eosin (H&E) staining of the whole brain. Red box indicates a site of corpus callosum far from the injection site. This site was stained by H&E, Nestin, EGFRvIII, p-STAT3, and Sox2.
Fig 7.
PEDF promotes tumor progression of GSCs.
(A) A free-floating assay of mouse brain was injected with X01-Con cells (upper) or X01-shPEDF2 cells (bottom). These cells were labeled by GFP (green). Nuclei were counterstained with Hoechst (blue). All images were taken at 5x magnification. (B, C) Kaplan-Meier survival curves of mouse implanted with 1 x 104 cells (B; p = 0.0013) and 1 x 103 cells (C; p < 0.01) infected with shPEDF2-expressing lentiviral or control construct. (D) Histopathology of Balb-c/nu mouse brain, orthotopically injected with 1 x 104 of X01-Con cells (left) or X01-shPEDF2 cells (right). Upper panel is H&E staining of the whole brain. Red box indicates an injection site and H&E (IS). This site was stained by Sox2, NICD, and HES1. Blue box indicates a site of corpus callosum far from injection site and H&E (CC). All images were taken at 40x magnification. (E) Histopathology of Balb-c/nu mouse brain, orthotopically injected with 1 x 103 X01-Con cells (left) or X01-shPEDF2 cells (right). Upper panel is H&E staining of the whole brain. Red box indicates an injection site and H&E staining (bottom); all images were taken at 40x magnification. (F) H&E staining of mouse brain, orthotopically injected with 1 x 105 Ex Vivo cells infected with PEDF-expressing lentiviral (right) or control construct (left). The upper panel is the whole brain (a, b). The yellow box indicates injection sites, caudate putamen (c, d). The blue box indicates corpus callosum (e, f). Red box indicates another caudate putamen that is a local site far from the injection site (g, h). All images were taken at 20x magnification. (G) Kaplan-Meier survival curves of mouse were implanted with X02-Con, X02-EGFRvIII, or X02-EGFRvIII-shPEDF2 cells (Control versus EGFRvIII; p = 0.0442, Control versus EGFRvIII-shPEDF2; p > 0.05, EGFRvIII versus EGFRvIII-shPEDF2; p = 0.0216).
Fig 8.
PEDF expression correlates with patient survival in human glioma.
(A–C) Kaplan-Meier plot of survival curve for all glioma patients (A; p < 0.0001), GBM patients (B; p = 0.0134), and astrocytoma (C; p = 0.0336). These data are based on the log-rank test. Data were obtained from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) program of the National Cancer Institute. (D) IB analysis of PEDF, p-EGFR, EGFR, NICD, and p-STAT3 using protein extracts from GBM patient tissues. GAPDH was used as a loading control. (E) Table represents correlation coefficients (r) of PEDF, EGFRvIII, NICD, and p-STAT3. ** p < 0.01. (F) A schematic model illustrates the signaling and function of PEDF. Image credit: Eunji Choi.