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.

The proline-specific serine protease fibroblast activation protein (FAP) can participate in the progression of malignant tumors and represents a potential diagnostic and therapeutic target. Recently, we demonstrated an increased expression of FAP in glioblastomas, particularly those of the mesenchymal subtype. Factors controlling FAP expression in glioblastomas are unknown, but evidence suggests that transforming growth factor beta (TGFbeta) can trigger mesenchymal changes in these tumors. Here, we investigated whether TGFbeta promotes FAP expression in transformed and stromal cells constituting the glioblastoma microenvironment. We found that both FAP and TGFbeta-1 are upregulated in glioblastomas and display a significant positive correlation. We detected TGFbeta-1 immunopositivity broadly in glioblastoma tissues, including tumor parenchyma regions in the immediate vicinity of FAP-immunopositive perivascular stromal cells. Wedemonstrate for the first time that TGFbeta-1 induces expression of FAP in non-stem glioma cells, pericytes, and glioblastoma-derived endothelial and FAP+ mesenchymal cells, but not in glioma stem-like cells. In glioma cells, this effect is mediated by the TGFbeta type I receptor and canonical Smad signaling and involves activation of FAP gene transcription. We further present evidence of FAP regulation by TGFbeta-1 secreted by glioma cells. Our results provide insight into the previously unrecognized regulation of FAP expression by autocrine and paracrine TGFbeta-1 signaling in a broad spectrum of cell types present in the glioblastoma microenvironment.

Background and Aims. Proteolytic enzymes contribute to the progression of various cancers. We previously reported increased expression of the proline specific peptidases dipeptidyl peptidase-IV (DPP-IV) and its closest paralogue fibroblast activation protein (FAP) in human glioblastomas. Here we analyze the molecular heterogeneity of DPP-IV and FAP in glioblastomas. Methods. ELISA, isoelectric focusing, 1D and 2D electrophoresis followed by WB or enzyme overlay assay were utilized to analyze DPP-IV and FAP isoforms. Cell fractionation using a Percoll gradient and deglycosylation with PNGase F were performed to analyze the possible basis of DPP-IV and FAP microheterogeneity. Results. Molecular forms of DPP-IV with an estimated molecular weight of 140-160 kDa and a pI predominantly 5.8 were detected in human glioblastoma; in some tumors additional isoforms with a more acidic (3.5-5.5) as well as alkaline (8.1) pI were revealed. Using 2D electrophoresis, two to three molecular forms of FAP with an alkaline (7.0-8.5) pI and an estimated MW of 120-140 kDa were identified in glioblastoma tissues. In glioma cell lines in vitro, several isoforms of both enzymes were expressed, however the alkalic forms present in glioblastoma tissues were not detected. Removal of N-linked oligosaccharides decreased the estimated molecular weight of both enzymes; the overall pattern of molecular forms nevertheless remained unchanged. Conclusion. Several isoforms of DPP-IV and FAP are present in glioblastoma tissue. The absence of alkaline isoforms of both enzymes in glioma cell lines however suggests that isoforms from other, most likely stromal, cell types contribute to the overall pattern seen in glioblastoma tissues.

Abstracts iv29 NEURO-ONCOLOGY • OCTOBER 2016CONCLUSION: Compared to NB, ATF5 is significantly overexpressed on mRNA level in LGA and GBM independently of tumor growth patterns. Since inhibition of ATF5 might lead to the selective death of glioma cells, but not of non-tumor cells, it might serve as a potential ubiquitous therapeutic target in astrocytic tumors, independently of WHO grading or growth pattern. RESULTS: FAP expression was increased in a subgroup of high-grade gliomas in particular on the protein level, the upregulation was most pronounced in the mesenchymal subtype, but was not associated with survival in glioblastoma. FAP was localized in both transformed and stromal cells that were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. A mouse model recapitulated the variability of FAP expression in glioma cells and the FAP+ tumors were typically GFAP negative. Endogenous FAP was frequently upregulated and part of the FAP+ host cells co-expressed the CXCR4 chemokine receptor. P06.07 FIBROBLAST ACTIVATION PROTEINCONCLUSIONS: 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.