The tumor microenvironment strongly impacts master transcriptional regulators and gene expression class of glioblastoma

Abstract

The Cancer Genome Atlas (TCGA) project has generated gene expression data that divides glioblastoma (GBM) into four transcriptional classes: proneural, neural, classical, and mesenchymal. Because transcriptional class is only partially explained by underlying genomic alterations, we hypothesize that the tumor microenvironment may also have an impact. In this study, we focused on necrosis and angiogenesis because their presence is both prognostically and biologically significant. These features were quantified in digitized histological images of TCGA GBM frozen section slides that were immediately adjacent to samples used for molecular analysis. Correlating these features with transcriptional data, we found that the mesenchymal transcriptional class was significantly enriched with GBM samples that contained a high degree of necrosis. Furthermore, among 2422 genes that correlated with the degree of necrosis in GBMs, transcription factors known to drive the mesenchymal expression class were most closely related, including C/EBP-β, C/EBP-δ, STAT3, FOSL2, bHLHE40, and RUNX1. Non-mesenchymal GBMs in the TCGA data set were found to become more transcriptionally similar to the mesenchymal class with increasing levels of necrosis. In addition, high expression levels of the master mesenchymal factors C/EBP-β, C/EBP-δ, and STAT3 were associated with a poor prognosis. Strong, specific expression of C/EBP-β and C/EBP-δ by hypoxic, perinecrotic cells in GBM likely account for their tight association with necrosis and may be related to their poor prognosis.

Figure 1

Representative examples of The Cancer Genome Atlas (TCGA) glioblastoma (GBM) frozen sections marked up for (A, B) necrosis and (D, E) angiogenesis and the distribution of cases within TCGA gene expression classes based on their percent necrosis (C) and percent angiogenic vessels (F). The blue line surrounds regions of necrosis in (A, B) and the pink line surrounds the entire tissue and represents the total tissue area. The red line surrounds angiogenic vessels in (D, E) and the green line represents the lumen, which is subtracted from the vessel area. Each dot in (C, F) represents a GBM sample. Solid horizontal lines within each category represent the mean value. The mean value of necrosis was larger in the mesenchymal class than in the other three classes (one-way analysis of variance, P = 8.7e-4). There was no statistically significant difference in mean angiogenic areas among expression classes.

Figure 2

Distribution of CEBP-β and STAT3 gene expression levels within The Cancer Genome Atlas gene expression classes. Each dot represents one glioblastoma (GBM) sample. Compared to the neural, proneural, and classical gene expression classes combined, the mesenchymal class showed enrichment for tumors with high CEBP-β (A) and STAT3 (B). All six activators and the repressor (ZNF238) are significantly differentially expressed when comparing mesenchymal to non-mesenchymal GBMs (two-way t-test, unequal variances; see Table 3).

Figure 3

Ingenuity pathway analysis (IPA) identified critical expression networks containing genes that are significantly correlated with the extent of necrosis in The Cancer Genome Atlas (TCGA) glioblastoma (GBM) samples from all subclasses (A and B). The STAT3 network was among the most closely associated with necrosis (A). The C/EBP gene network was also highly correlated with the extent of necrosis and demonstrates the central importance of IL-6. (B). Gene networks that were differentially expressed between mesenchymal GBMs with high (>15%) and low extent of necrosis (≤15%) included those that were centered around CEBP-β and were strongly associated with upregulated IL-8 signaling (C).

Figure 4

Glioblastomas (GBMs) with high expression of C/EBP-β (upper quartile) were associated with a significantly shorter survival (50% survival, 11 months) than those with low expression (50% survival, 14 months) (log rank test P = 0.0071), and high co-expression of C/EBP-β, C/EBP-δ, and STAT3 was associated with a shorter survival (50% survival, 12 months) than those that had low expression of all three (50% survival, 21 months) (log rank test P = 0.0021).

Figure 5

Influence of necrosis on transcriptional distance to mesenchymal class signature. Euclidean distance to the mesenchymal class signature was computed for all nonmesenchymal samples, and the Spearman correlations between transcriptional distance and extent of necrosis were computed. The median distance between mesenchymal samples and the mesenchymal signature (indicated in green) is 14.8. There is an inverse relation between the extent of necrosis and the distance to the mesenchymal gene signature.

Figure 6

Double immunohistochemistry for macrophages (CD163, red) and C/EBP-β (brown) in human glioblastoma (GBM) samples demonstrates that macrophages represent a minority of the cells in GBM and are scattered throughout the tumor stroma (arrow) but do not accumulate around necrosis (A, asterisk). C/EBP-β (A–C) and C/EBP-δ (D) expression was increased in perinecrotic pseudopalisading cells directly surrounding necrosis (asterisk) of GBM, whereas expression of pSTAT3 (E) and STAT 3 was uniform in tumor cells and did not show microregional variation. Expression of CEBP-β was restricted to the nucleus of tumor cells within 4 to 6 cell layers from necrosis (A, arrowhead). Only rare cells away from necrosis showed expression (B, C, arrowhead). Hypoxia causes increased expression of C/EBP-β in GBM cells (F). U87MG cells grown for 24 hours under hypoxia (H) (1% O₂) show increased C/EBP-β protein in the nuclear compartment by Western blot, with low protein expression under normoxia (N) (21% O₂). Protein expression of C/EBP-δ and STAT3 was seen in both the nuclear and cytoplasmic compartments under normoxia and hypoxia. pSTAT3 was expressed exclusively in the nucleus and did not show any hypoxic upregulation. Increased nuclear HIF-1α expression under hypoxia serves as a positive control for hypoxic conditions, with histone H1 serving as a loading control for the nuclear compartment.