Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia - PubMed (original) (raw)

Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia

Anastasia Murat et al. PLoS One. 2009.

Abstract

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy.Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The hypoxia gene cluster.

Gene dendrogram and heat map of the hypoxia cluster, showing two main gene groups; G2, enriched for inflammatory genes (yellow bar) and G4, containing angiogenesis-related genes (blue bar). G3 (green bar) is a subcluster of G4 (grey bars mark genes not organized in stable subclusters as defined by CTWC), see Table 1 for detailed cluster information.

Figure 2

Figure 2. Components of the hypoxia gene cluster.

A, Loading plots, representing the coefficients of the linear combination of the 52 common probe-sets of the hypoxia cluster used to define the first two Principal Components (PC) (Table 1), are shown for our data-set and 3 published data-sets , , . In all data-sets genes with the highest positive coefficients in the linear combination defining the 2nd PC include VEGF, ZNF395 and KISS1R. Genes with the highest negative contribution to the 2nd PC comprise TREM1, TNFAIP3, BIRC3 and IL8. Probe-sets with the most extreme contributions to the 2nd PC in all data-sets are labeled. B, Pair wise scatter plots and Pearson correlations of the loadings of the 52 probe-sets in the 2nd PC across our data-set (n = 69), and the external data-sets Freije (n = 48), Phillips (n = 54), and Sun (n = 71).

Figure 3

Figure 3. Meta-analysis using four gene expression data-sets of glioblastoma.

The Forest plot visualizes the prognostic value of the meta analysis using the 2nd PC of the hypoxia cluster in a Cox model of four glioblastoma data-sets (n = 242, p = 0.010, HR, 1.09, 95% CI, 1.02 to 1.16) , , , . When combining the three external data-sets formal statistical significance (alpha level of 5%) was not reached (HR = 1.06 95% CI: 0.98, 1.14, p-value: 0.15).

Figure 4

Figure 4. Hypoxia induction of TREM1, ZNF395 and KISS1R.

A, TREM1 expression in primary isolated monocytes under normoxia or 18 hours hypoxia (1% O2); B, ZNF395; C, KISS1R and D, TNFAIP3 expression in four different glioblastoma cell-lines (LN229, LNZ308, LN319, and U87) under normoxia and after 8, and 12 hours of hypoxia (1% O2). All results are normalized to expression of the RNA polymerase II (POLR2A) gene. Error bars representing standard deviation of triplicate qRT-PCR measurements. Histograms are representative of three independent experiments.

Figure 5

Figure 5. Expression patterns of hypoxia inducible genes in glioblastoma.

A, In-situ hybridization (ISH) using TREM1 and ZNF395 anti-sense probes on sequential frozen sections. Pictures were taken from the same region in proximity to a multilayered blood vessel, and an intermittent region, respectively. A respective sense probe (s_probe) was used as negative control. The perinuclear signal for the probes is black-purple (BCIP/NBT), the nuclei are counterstained with methyl green (light blue/green). KISS1R expression was determined by immunohistochemistry (IHC) on paraffin sections: the top panel displays immunoreactivity of the multilayered blood vessel and adjacent cells situated next to a necrosis (original magnification ×40), (middle panel, lower magnification). The bottom panel shows KISSR1 expression in pseudopalisading cells lining a necrosis (original magnification ×10). B, IHC against CD11b, a marker for macrophages; CD45, a marker for leukocytes, and KDR that is predominantly expressed by endothelial cells, was performed on sequential sections used for ISH of TREM1 and ZNF395 and pictures were taken from the same region close to a multilayered aberrant blood vessel. Original magnifications, ×40.

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