Astrocyte-Specific Expression Patterns Associated with the PDGF-Induced Glioma Microenvironment

The tumor microenvironment contains normal, non-neoplastic cells that may contribute to tumor growth and maintenance. Within PDGF-driven murine gliomas, tumor-associated astrocytes (TAAs) are a large component of the tumor microenvironment. The function of non-neoplastic astrocytes in the glioma microenvironment has not been fully elucidated; moreover, the differences between these astrocytes and normal astrocytes are unknown. We therefore sought to identify genes and pathways that are increased in TAAs relative to normal astrocytes and also to determine whether expression of these genes correlates with glioma behavior.We compared the gene expression profiles of TAAs to normal astrocytes and found the Antigen Presentation Pathway to be significantly increased in TAAs. We then identified a gene signature for glioblastoma (GBM) TAAs and validated the expression of some of those genes within the tumor. We also show that TAAs are derived from the non-tumor, stromal environment, in contrast to the Olig2+ tumor cells that constitute the neoplastic elements in our model. Finally, we validate this GBM TAA signature in patients and show that a TAA-derived gene signature predicts survival specifically in the human proneural subtype of glioma.Our data identifies unique gene expression patterns between populations of TAAs and suggests potential roles for stromal astrocytes within the glioma microenvironment. We show that certain stromal astrocytes in the tumor microenvironment express a GBM-specific gene signature and that the majority of these stromal astrocyte genes can predict survival in the human disease

Guiding Brain Tumor Resection Using Surface-Enhanced Raman Scattering Nanoparticles and a Hand-Held Raman Scanner

The current difficulty in visualizing the true extent of malignant brain tumors during surgical resection represents one of the major reasons for the poor prognosis of brain tumor patients. Here, we evaluated the ability of a hand-held Raman scanner, guided by surface-enhanced Raman scattering (SERS) nanoparticles, to identify the microscopic tumor extent in a genetically engineered RCAS/tv-a glioblastoma mouse model. In a simulated intraoperative scenario, we tested both a static Raman imaging device and a mobile, hand-held Raman scanner. We show that SERS image-guided resection is more accurate than resection using white light visualization alone. Both methods complemented each other, and correlation with histology showed that SERS nanoparticles accurately outlined the extent of the tumors. Importantly, the hand-held Raman probe not only allowed near real-time scanning, but also detected additional microscopic foci of cancer in the resection bed that were not seen on static SERS images and would otherwise have been missed. This technology has a strong potential for clinical translation because it uses inert gold-silica SERS nanoparticles and a hand-held Raman scanner that can guide brain tumor resection in the operating room

Tumor-associated Astrocytes within PDGF-driven Glioma.

<p>(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.</p

Antigen Presentation Pathway is Active in Tumor-associated Astrocytes.

<p>© 2000–2010 Ingenuity Systems, Inc. All rights reserved.</p><p>Ingenuity Pathway Analysis of genes significantly increased more than four-fold in low-grade glioma- and glioblastoma-associated astrocytes indicates the pathways that are significantly increased in TAAs relative to normal astrocytes. The top ten canonical pathways represented on the array are shown in the table. The Antigen Presentation Pathway is most represented within this gene set.</p

A Small Group of Genes are Expressed Only in GBM-associated Astrocytes.

<p>A list of the genes expressed at higher levels in GBM-associated astrocytes when compared to low-grade-associated astrocytes. Genes are in order of the difference of expression between GBM-associated and low grade-associated-astrocytes. CD44 and TNC are each represented twice on the list. Osteopontin, which is at the top of the list, is a ligand for the CD44 receptor.</p

<i>In vivo</i> quantification of ribosome-bound and total RNA levels revealed a broad range of ribosome recruitment efficiencies amongst mRNA transcripts.

<p>(A) Distribution of mRNA expression in ribosome-bound and total RNA pools from PDGF-driven glioma identified differential TE (N = 4). (B) TE values for each biological replicate (black points) plotted with the average of the other three replicates (red line) demonstrated reproducibility of measurements. (C) Signal-to-noise ratios of TE measurements (blue bars) identified range of high confidence measurements relative to a normal distribution (red line). (D) GSEA identified statistical overrepresentation of defined gene ontologies amongst efficiently and inefficiently translated genes. Black bars represent distribution of mRNAs from indicated geneset amongst all genes ranked by signal to noise ratio (top panel). Red line represents GSEA output enrichment score. R = Pearson correlation coefficient.</p

Perivascular Astrocytes Can Be Stromal in Origin.

<p>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.</p

A Gene Signature for GBM Astrocytes.

<p>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 <i>Cd44</i> and <i>Tenascin C.</i> 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.</p