Fig 1.
Microarray analysis of gene expression in GBM6 cells and tumor xenografts.
RNA was prepared from GBM6 bulk tumor cells, Ad-GSC, and Sp-GSC cultures as well as from subcutaneous tumor xenografts of these injected cells. Biological duplicates were run for each sample and data were collected and analyzed. A. Gene expression was measured by Illumina array and genes reported in the TCGA database were analyzed. B. The PCA plot represents the comparison of gene signatures from each condition.
Fig 2.
Molecular classification of gene expression in GBM6 cells and tumor xenografts.
A. Array analysis was performed and compared to glioblastoma molecular subclasses; PCA map depicts the classical, mesenchymal, neural, and proneural gene signatures and the gene signature of GBM6 cells and tumor tissue derived from bulk tumor cells, Ad-GSCs and Sp-GSCs. B. and C. RNA was isolated from three individual tumors derived from GBM6 bulk tumor cells, Ad-GSCs and Sp-GSCs (B), and from these cells grown in vitro (C.) to determine gene expression of molecular markers of the Mesenchymal (CHI3L1, TRADD, NF1, RelB and CASP4) and Classical (FGFR3, PDGFA, EGFR, AKT2 and Nestin) subclass of glioblastoma by qPCR and normalized to actin expression (n = 3). Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3.
Pathologic analysis of subcutaneous GBM6 tumor xenografts.
GBM6 bulk tumor cells, Ad-GSCs, and Sp-GSCs were injected subcutaneously and allowed to grow to a diameter of ~400mm3. Tumors were harvested and paraffin embedded for histology. A. H&E staining and B. Immunoreactivity for GFAP, S100, OLIG2 and MAP2. (All photomicrographs taken at 200x).
Fig 4.
Characterization of GBM6 tumor xenografts.
A. CB17 SCID mice were injected with 1x106 luciferase-tagged GBM6 cells grown as bulk tumor cells, Ad-GSCs, or Sp-GSCs, and tumor burden was measured by bioluminescence twice a week (n = 10 per group), and (B) Kaplan-Meier analysis of survival data (n = 8) was performed. C. RNA isolated from intracranial tumors was subjected to qPCR as indicated and normalized to actin expression (n = 3). Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5.
Pathologic analysis of orthotopic GBM6 tumor xenografts.
GBM6 bulk tumor cells, Ad-GSCs and Sp-GSCs were orthotopically injected as described in Fig 4, and tumor-bearing brains were paraffin embedded for histology. A. H&E staining and B. Immunoreactivity for GFAP, S100, OLIG2 and MAP2. (All photomicrographs taken at 200x).
Fig 6.
Enrichment of pro-survival and pro-angiogenic genes in Ad-GSC tumor xenografts.
A. RNA was prepared from three separate tumors generated from GBM6 bulk tumor cells and Ad-GSCs, and Nanostring analysis was performed. B. Volcano plot depicting the genes that passed the Bonferroni test. C. The RNA was used to validate the gene expression of SPP1, ETV1, CCND2, CDH1, NQO1, and LYN by qPCR (normalized to actin expression). D. Ingenuity pathway analysis showing that genes upregulated in Ad-GSC tumor xenografts are involved in angiogenesis. E. qPCR validation of pro-angiogenic genes (ANGPTL4, IL8, CDKN2A and CXCL1) upregulated in Ad-GSC tumor xenografts normalized to actin expression (n = 3). Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 7.
Expression of STAT3 and ANGPTL4 in human GBM samples.
A. Gene expression of STAT3 and ANGPTL4 in the TCGA database for normal brain tissue (n = 10), low-grade glioma samples (n = 265) and high-grade (GBM) samples (n = 328). B. and C. RNA was extracted from 24 GBM patient biopsies, and STAT3 and ANGPTL4 expression was measured by qPCR and normalized to actin expression (n = 3), and expression was correlated to short-term and long-term survival (B) and (C) as well as to each other. Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 8.
The STAT3/ANGPTL4 pathway in GSCs.
A. RNA was prepared from GBM6 Ad-GSCs treated with WP1066 (50 μM) for 6 hrs, and the expression of ANGPTL4, VEGF-R1, VEGF-R2, CD133, SOX2 and Nestin was quantified by qPCR and normalized to actin expression (n = 3). B. ChIP-enriched STAT3 binding to the ANPTL4 promoter was analyzed by qPCR and normalized to input DNA, followed by subtraction of nonspecific binding determined by control IgG. C. Single cells isolated from bulk GBM6 tumor cells were incubated with anti-CD133 antibody, and data acquisition and analysis was performed on a three-laser LSR (Becton-Dickinson) flow cytometer using CellQuest software. RNA isolated from CD133+ and CD133- cells was subjected to qPCR as indicated and normalized to actin expression. Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 9.
The antiglioma effects of the STAT3 inhibitor WP1066.
Mice were subcutaneously injected with 1x106 luciferase-tagged Ad-GSCs, and upon initial detection of tumor, mice were treated with WP1066 (40 mg/kg) every other day. A. Tumor volume was determined with calipers (n = 5). B. Representative bioluminescence images at Day 31. C. Lysates of tumor tissue at one week following WP1066 treatment were immunoblotted for STAT3 and phospho-STAT3 as indicated. Error bars, S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 10.
Constitutive activation of STAT3 in Ad-GSCs isolated from various GBM PDXs.
The expression of STAT3 and pSTAT3 in nuclear extracts prepared from Ad-GSCs isolated from GBM6, GBMX12, GBMX16 and GBMX39 PDXs was determined by immunoblotting. Lamin expression served as a loading control.