Figure 1.
Tumor-associated Astrocytes within PDGF-driven Glioma.
(A–C) GFAP immunohistochemistry of astrocytes in the normal brain (A, A′), WHO II low-grade glioma (B, B′) and glioblastoma (GBM; C, C′, C″) at 1× (A, B, C) and 40× (A′, B′, C′, C″). Note that tumor-associated astrocytes (TAAs) are morphologically different than normal astrocytes. Moreover, in low grade glioma, TAAs are present within and surrounding the tumor and all of these astrocytes have a ‘reactive’ morphology identified by swollen cell bodies as well as multipolar and hyperextended processes (B′). Within GBM (C), astrocytes are present in two areas: the peri-tumoral area, where the astrocytes have a ‘reactive’ morphology (C′) similar to low grade astrocytes and the perivascular niche, where the astrocytes still have swollen cell bodies but have a more uni-polar or bi-polar morphology (C″). Scale bars: A, B, C = 300 µm, A′, B′, C′, C″ = 15 µm. D) Unbiased hierarchical clustering of astrocytes from normal brain, low-grade glioma and GBM indicates that, when factoring in the mRNA expression levels of approximately 15,000 genes significantly expressed on the array, TAAs are very different from normal astrocytes, however most genes are similarly regulated between low grade-associated and GBM-associated astrocytes and thus, low grade-associated and GBM-associated astrocytes do not segregate.
Table 1.
Antigen Presentation Pathway is Active in Tumor-associated Astrocytes.
Figure 2.
A Gene Signature for GBM Astrocytes.
A) Dot-plot of the expression levels of all genes significantly expressed in low-grade-associated astrocytes and GBM-associated astrocytes. Red lines demarcate genes similarly expressed regardless of grade and dots outside the red lines represent genes expressed at higher levels in GBM-associated astrocytes. Red arrows point to the dots representing Cd44 and Tenascin C. B) Hierarchical clustering of the genes off the plot that are expressed at a higher level in GBM-associated astrocytes relative to low-grade-associated astrocytes indicates that these genes are able to segregate GBM-associated astrocytes from low grade-associated astrocytes. C,D) Validation of CD44 (C) and Tenascin-C (TNC; D) expression at low- and high-magnification. CD44 and TNC are expressed at high levels in perivascular astrocytes but expressed at lower levels or not expressed at all in peri-tumoral astrocytes. Arrows point to perivascular astrocytes and arrowheads point to peri-tumoral astrocytes. Scale bars = 100 µm, 10 µm.
Table 2.
A Small Group of Genes are Expressed Only in GBM-associated Astrocytes.
Figure 3.
Perivascular Astrocytes Can Be Stromal in Origin.
A) Immunofluorescence for PDGF-GFP (green) and GFAP (red) in a PDGF-GFP-driven glioma shows expression of PDGF-GFP and GFAP are mutually exclusive, indicating that TAAs are not derived from the tumor cell of origin. Scale bars = 100 µm. Inset, high-magnification images. Scale bars = 10 µm. B) Immunofluorescence for GFAP (red) and GFP (green) in an orthotopic model of human glioma. Human tumorspheres were injected into GFAP-GFP reporter mice such that tumor derived astrocytes will express GFAP but not GFP and host/stromal astrocytes will co-express GFAP and GFP. While some astrocytes express only GFAP, many astrocytes also express GFP and are thus derived from the host stromal environment. Expression of stromal GFAP-GFP occurs in all areas of GFAP immunoreactivity. Scale bars = 25 µm. Inset, high-magnification images. Scale bars = 10 µm. C) FACS analysis of a PDGF-RFP-driven glioma in an Olig2-rp-GFP mouse, where PDGF-infected tumor cells express RFP and Olig2-expressing cells express GFP. Most cells infected with the PDGF-RFP virus are Olig2-rp-GFP-expressing cells. D) FACS analysis of a PDGF-RFP-driven glioma in a GFAP-GFP mouse, where PDGF-infected cells express RFP and astrocytes express GFP. Most of the TAAs were not infected with RCAS-PDGF-RFP.
Figure 4.
GBM Astrocyte-associated Stromal Genes Can Predict Survival in Human Proneural Glioma.
A) Dot-plot of the expression levels of all genes significantly expressed in whole tumor extracts (tumor plus stroma) versus Olig2-expressing tumor cells only. Red shaded area demarcates genes expressed at a higher level in Olig2-expressing tumor cells, whereas blue shaded area demarcates genes expressed at a higher level in Olig2-negative, stromal cells. Black dots represent genes identified as GBM TAAs signature genes. B) Analysis of the predictive value of the GBM TAA genes. For each gene, patients were divided into high expressers and low expressers, based on whether their expression of the gene was above or below the median. Kaplan Meier curves were then generated for the two groups and the difference in survival between low and high expressers was plotted. Black dots indicate a non-significant difference in survival and colored dots indicate a significant difference in survival, where red is *** (p<0.001), blue is ** (p<0.01), and green is * (p<0.05).