Object: Fluorescence imaging has the potential to significantly improve neurosurgical resection of oncologic lesions through improved differentiation between normal and cancerous tissue at the tumor margins. In order to successfully mark glioma tissue a fluorescent tracer must have the ability to penetrate through the blood brain barrier (BBB) and provide delineation in the tumor periphery where heterogeneously intact BBB may exist. In this study it was hypothesized that, due to its smaller size, fluorescently labeled anti-EGFR Affibody protein (,7 kDa) would provide a more clear delineation of the tumor margin than would fluorescently labeled cetuximab, a full antibody (,150 kDa) to the epidermal growth factor receptor (EGFR). Methods: Cetuximab and anti-EGFR targeted Affibody were conjugated to two different fluorescent dyes (both emitting in the near-infrared) and injected intravenously into 6 athymic mice which were inoculated orthotopically with green fluorescent protein (GFP) expressing human U251 glioma cells. Each mouse was sacrificed at 1-h post injection, at which time brains were removed, snap frozen, sectioned and quantitatively analyzed for fluorescence distribution. Results: Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 4966 % of injected protein), however, the cetuximab was more confined to the center of the tumor with Affibody showing significantly higher concentrations at the tumor periphery (on average Affibody made u
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