Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

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Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.

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Integrins have become a target for novel therapeutic strategies against malignant gliomas. Cilengitide, a synthetic Arg-Gly-Asp (RGD)-motif peptide, interferes with ligand binding to avb3 and avb5 integrins and is currently investigated in clinical trials. Integrins may also be involved in the activation of transforming growth factor (TGF)-b, a mediator of invasiveness and immune escape of glioma cells. Using flow cytometry, we demonstrate that the target integrins of cilengitide are expressed not only in glioblastoma blood vessels, but also by tumor cells. After exposure of glioma cells to cilengitide, we noticed reduced phosphorylation of Smad2 in most glioma cell lines, including stem-like glioma cells. Phophorylation of Smad2, but not cilengitide-induced detachment, is rescued by addition of recombinant TGF-b. Administration of cilengitide to glioma cells results in reduced TGF-b-mediated reporter gene activity. Furthermore, exposure to cilengitide leads to decreased TGF-b 1 and TGF-b 2 mRNA and protein expression. These effects are mimicked by blocking av, b3 or b5 antibodies or by silencing of integrins av, b3, b5 or b8 using RNA interference. Treatment of mice bearing experimental LN-308 glioma xenografts with cilengitide results in reduced pSmad2 levels. Taken together, cilengitide may exert anti-invasive and immune stimulatory activity in human glioblastoma patients by its anti-TGF-b properties.