Proteomic analysis of factors secreted by glioblastoma that induce endothelial tissue factor

Abstract

We have proposed that intravascular thrombosis initiates or propagates necrosis in GBM, leading to hypoxia‐induced angiogenesis and rapid biological progression. We investigated factors secreted by gliomas following PTEN loss and under hypoxia that induce endothelial expression of tissue factor (TF), a potent procoagulant that could promote thrombosis. We used a PTEN null human GBM cell line (U87MG) with an inducible wt PTEN (23.11 cells) to model progression. Conditioned media (CM) from 23.11 +/− PTEN was collected after 48 hours of normoxia (21% O2) or hypoxia (1% O2). CM was tested on human brain derived microvascular endothelial cells (HBMEC) for its effect on proliferation, apoptosis, tube stability on Matrigel, or TF expression. Proteins from CM were precipitated, concentrated and labeled by Isotope Coded Affinity Tags (ICAT). Paired ICAT samples were separated and analyzed by tandem mass spectrometry (MS/MS). Proteins identified by MS/MS were measured in CM by ELISA and tested directly on HBMEC. We found that CM from PTEN− 23.11 cells strongly and consistently induced HBMEC TF expression compared to PTEN+ cells and that hypoxia potentiated this effect. CM from PTEN− and PTEN+ gliomas did not have differential effects on proliferation or apoptosis. ICAT coupled with MS/MS identified MMP‐2 (1.5‐fold) and macrophage migration inhibitory factor (MIF; 1.3‐fold) as proteins secreted by PTEN− compared to PTEN+ gliomas. IL‐8 (3.2‐fold) and insulin‐like growth factor binding protein 3 (IBP‐3; 3.2‐fold) were secreted at much higher levels in hypoxia. Both purified IL‐8 and MIF induced endothelial TF in the concentration range in CM. Thus, MIF and IL‐8 are secreted factors that induce endothelial TF and may be associated with the development of thrombosis in GBM.