gms | German Medical Science

Objective: The fibronectin extra domain B (ED-B) is an alternative splice variant of the extracellular matrix protein fibrobection. ED-B is expressed by neonatal and neoplastic cells, but is absent from healthy adult tissue. We investigated the expression of ED-B in human glioma. Also, we explored ED-B as a target for molecular imaging studies in a mouse glioma model. Therefore, we have developed an ED-B directed Anticalin which was applied as a scintigraphy tracer. Anticalins are a novel class of small, robust binding proteins with high target specificity, based on the lipocalin protein scaffold that can be radioactively labelled. Overall, we investigated ED-B as a marker for the localization and pathological grading of gliomas in histology and imaging.

Method: For this study, tissue samples from high-grade glioma (HGG, n=23), low-grade glioma (LGG, n=10) and non-neoplastic brain (NNB, n=10) have been stained immunohistochemically for ED-B positive fibronectin expression using an ED-B specific Anticalin (N7A) and a wild type Lipocalin (Lcn2) as control. Additionally, the Anticalin fused to green fluorescent protein (eGFP-PAS#1(200)-N7A) was used for immunofluorescence. FACS analysis for expression of ED-B has been performed using glioblastoma cell lines as well as GBM derived endothelial cells (gbECs) and HUVECs as control. Imaging was assessed in SCID mice bearing an U87 flank tumor using 123I-labelled N7A.

Results: We found, that the ED-B specific Anticalin N7A can reliably distinguish low-grade from high-grade gliomas in immunohistochemistry and immunofluorescence. Wild type fibronectin could be detected in NNB, LGG as well as HGG, whereas the tumor specific ED-B was only detectable in GBM. ED-B Anticalins specifically stained tumor vessels, but not surrounding parenchyma. FACS analyses confirmed ED-B expression on glioma cells as well as gbECs, but not on corresponding control cells. Using the radioactively labelled Anticalin, U87-derived flank tumors could be detected in SCID mice.

Conclusions: Anticalins comprise a new class of small and highly specific ligand-binding properties with better tissue penetration than antibodies, providing the benefit of facile engineering and labelling. An ED-B-directed Anticalin specifically labels glioblastoma, but not other gliomas or non-neoplastic brain, which makes it an ideal tracer for the localization of GBM and for the discrimination of high-grade from low-grade tumors by molecular imaging.