IL-8 associates with a pro-angiogenic and mesenchymal subtype in glioblastoma

Abstract

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by a high rate of vascularization. However, therapeutic targeting of the vasculature through anti-vascular endothelial growth factor (VEGF) treatment has been disappointing, for which Angiopoietin-2 (Ang-2) upregulation has partly been held accountable. In this study we therefore explored the interplay of Ang-2 and VEGFA and their effect on angiogenesis in GBM, especially in the context of molecular subclasses. In a large patient cohort we identified that especially combined high expression of Ang-2 and VEGFA predicted poor overall survival of GBM patients. The high expression of both factors was also associated with increased IL-8 expression in GBM tissues, but in vitro stimulation with Ang-2 and/or VEGFA did not indicate tumor or endothelial cell-specific IL-8 responses. Glioblastoma stem cells (GSCs) of the mesenchymal (MES) subtype showed dramatically higher expression of IL8 when compared to proneural (PN) GSCs. Secreted IL-8 derived from MES GSCs induced endothelial proliferation and tube formation, and the MES GBMs had increased counts of proliferating endothelial cells. Our results highlight a critical pro-angiogenic role of IL-8 in MES GBMs.

Keywords: IL-8; angiogenesis; glioblastoma; subclasses.

Figure 1. Combined high expression of Ang-2 and VEGFA associates with survival and increased IL-8 expression

The expression level of ANGPT2 (A), VEGFA (B) and the combined high expression of ANGPT2 and VEGFA (C) associated with poorer survival of GBM patients (TCGA cohort). The differential expression of these factors (above or below median) did not associate with microvascular density (D), vessel size (E) or vessel perimeter (F) (Groningen cohort). Individual values per patient are displayed and horizontal lines represent median scores. (G) Profiling of several angiogenic mRNAs identified IL8 and MMP9 as the targets upregulated most in the tumors with combined high expression of ANGPT2 and VEGFA; ^*P < 0.05, ^**P < 0.01.

Figure 2. Ang-2 and VEGFA expression associate with increased IL-8 expression

In the Groningen cohort tumors with higher than median ANGPT2 expression (A) and tumors with higher than median expression of ANGPT2 and VEGFA (C) expressed significantly higher levels of IL-8 mRNA as well. A similar but non-significant trend was observed for tumors with above median expression of VEGFA (B). Box-and-whiskers were generated according to Tukey’s method with the box representing the 25th and 75th percentile, and whiskers represent 1.5 × IQR. Values outside these intervals are plotted as individual points. The expression levels of ANGPT2 (D), VEGFA (E) and IL-8 (F), explored in the larger TCGA cohort, were all positively correlated; ^*P < 0.05, ^***P < 0.001.

Figure 3. Effect of Ang-2 and/or VEGFA stimulation on U87 GBM cells and HMEC-1 endothelial cells

The proliferation (A) and rate of CC3-mediated apoptosis (B) in HMEC-1 was unaltered following stimulation with Ang-2 and/or VEGFA after 72 and 24 hours, respectively. The transcription of IL8 was upregulated mainly following VEGFA stimulation at different time points (C), but the 24-hour secretion level of IL-8 was unaltered in HMEC-1 cells (D). The proliferation of U87 cells was slightly inhibited after dual stimulation with Ang-2 and VEGFA (E), but CC3-mediated apoptosis levels were unaltered (F). VEGFA induced upregulation of IL-8 transcription in U87 only at the 1-hour time point (G), but 24-hour secretion levels of IL-8 were indifferent between the conditions (H). Assays were repeated three times and mean values ± standard deviations are displayed; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 all relative to control.

Figure 4. IL-8 is upregulated in MES GSCs and mediates in vitro angiogenesis

Expression levels of ANGPT2, VEGFA, and IL8 mRNA are upregulated in MES versus PN GBMs (TCGA cohort, A–C). MES GSCs (GSC 20, 28 and 267) display higher levels of the mRNA transcript in comparison to PN GSCs (GSC 8-11, 11 and 23) (D). The secretion level of IL-8 also indicated increased IL-8 secretion by MES GSCs in comparison to PN GSCs (E). CM of GSCs only had minor effects on HMEC-1 proliferation, with a trend for inhibition of HMEC-1 proliferation when an IL-8 was neutralized in MES GSC28 CM (F). Representative images of tube formation assays are displayed (G) that illustrate enhanced tube formation by the addition of MES CM and significant inhibition of tube formation when IL-8 is neutralized in GSC28 CM (H). The variation in individual assays is also shown (I, J). Assays were repeated five (proliferation) or six (tube formation) times and mean values with standard error of the mean (SEM) are plotted; ^*P < 0.05, ^***P < 0.001.

Figure 5. MES GBMs have more proliferating endothelial cells

A photomicrograph of a double stained GBM sample for CD34 in red and Ki67 in brown is depicted (A). The black arrowheads indicate CD34⁺Ki67⁺ cells. Scale bar is 100 µm. Quantification of the double stained cells indicated that MES GBM tissue had a significantly higher percentage of PECs in comparison to the PN GBM tissues (B). (C) Proposed model of vessel expansion due to increased IL-8 secretion by MES GSCs ; Scale bar = 100 µm; ^**P < 0.01.