Thayer School of Engineering at Dartmouth College, Hanover, New Hampshire, USA.
PLoS One. 2013 Apr 12;8(4):e60390. doi: 10.1371/journal.pone.0060390. Print 2013.
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).
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.
Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 49±6% 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 up 72±15% of injected protein in the outer 50 um of the tumor). Further ex vivo analysis of detection studies showed that the Affibody provided superior discrimination for differentiation of tumor from surrounding normal brain.
The present study indicates that fluorescently labeled anti-EGFR Affibody can provide significantly better delineation of tumor margins than a fluorescently labeled anti-EGFR antibody and shows considerable potential for guiding margin detection during neurosurgery.
荧光成像是一种有潜力的方法，通过提高肿瘤边界正常组织和癌变组织之间的区分度，从而显著改善神经外科的肿瘤切除术。为了成功标记神经胶质瘤组织，荧光示踪剂必须能够穿透血脑屏障（BBB），并在肿瘤周边提供清晰的描绘，因为肿瘤周边的 BBB 可能是异质完整的。在这项研究中，我们假设由于其较小的尺寸，荧光标记的抗 EGFR Affibody 蛋白（约 7 kDa）将比表皮生长因子受体（EGFR）的全抗体（约 150 kDa）荧光标记的 cetuximab 更清晰地描绘肿瘤边界。
cetuximab 和针对 EGFR 的靶向 Affibody 与两种不同的荧光染料（均在近红外区发射）缀合，并静脉内注射到 6 只荷 GFP 表达的人 U251 神经胶质瘤细胞的同源接种的无胸腺小鼠中。每只小鼠在注射后 1 小时处死，此时取出大脑，迅速冷冻，切片并进行定量荧光分析。
离体分析显示，肿瘤内的 cetuximab 和 Affibody 浓度大致相等（Affibody 平均占注射蛋白的 49±6%），然而，cetuximab 更局限于肿瘤中心，Affibody 在肿瘤周边的浓度明显更高（Affibody 平均占肿瘤外 50 μm 内注射蛋白的 72±15%）。进一步的离体检测研究显示，Affibody 提供了更好的肿瘤与周围正常脑组织的区分度。
本研究表明，荧光标记的抗 EGFR Affibody 比荧光标记的抗 EGFR 抗体能更好地描绘肿瘤边界，并显示出在神经外科中指导边缘检测的巨大潜力。
