Zeh Ryan, Sheikh Saad, Xia Leilei, Pierce John, Newton Andrew, Predina Jarrod, Cho Steve, Nasrallah MacLean, Singhal Sunil, Dorsey Jay, Lee John Y K
Department of Neurosurgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Center for Precision Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2017 Jul 24;12(7):e0182034. doi: 10.1371/journal.pone.0182034. eCollection 2017.
Fluorescence-guided surgery has emerged as a powerful tool to detect, localize and resect tumors in the operative setting. Our laboratory has pioneered a novel way to administer an FDA-approved near-infrared (NIR) contrast agent to help surgeons with this task. This technique, coined Second Window ICG, exploits the natural permeability of tumor vasculature and its poor clearance to deliver high doses of indocyanine green (ICG) to tumors. This technique differs substantially from established ICG video angiography techniques that visualize ICG within minutes of injection. We hypothesized that Second Window ICG can provide NIR optical contrast with good signal characteristics in intracranial brain tumors over a longer period of time than previously appreciated with ICG video angiography alone. We tested this hypothesis in an intracranial mouse glioblastoma model, and corroborated this in a human clinical trial.
Intracranial tumors were established in 20 mice using the U251-Luc-GFP cell line. Successful grafts were confirmed with bioluminescence. Intravenous tail vein injections of 5.0 mg/kg (high dose) or 2.5 mg/kg (low dose) ICG were performed. The Perkin Elmer IVIS Spectrum (closed field) was used to visualize NIR fluorescence signal at seven delayed time points following ICG injection. NIR signals were quantified using LivingImage software. Based on the success of our results, human subjects were recruited to a clinical trial and intravenously injected with high dose 5.0 mg/kg. Imaging was performed with the VisionSense Iridium (open field) during surgery one day after ICG injection.
In the murine model, the NIR signal-to-background ratio (SBR) in gliomas peaks at one hour after infusion, then plateaus and remains strong and stable for at least 48 hours. Higher dose 5.0 mg/kg improves NIR signal as compared to lower dose at 2.5 mg/kg (SBR = 3.5 vs. 2.8; P = 0.0624). Although early (≤ 6 hrs) visualization of the Second Window ICG accumulation in gliomas is stronger than late (≥24 hrs) visualization (SBR = 3.94 vs. 2.32; p<0.05) there appears to be a long plateau period of stable ICG NIR signal accumulation within tumors in the murine model. We call this long plateau period the "Second Window" of ICG. In glioblastoma patients, the delayed visualization of intratumoral NIR signal was strong (SBR 7.50 ± 0.74), without any significant difference within the 19 to 30 hour visualization window (R2 = 0.019).
The Second Window ICG technique allows neurosurgeons to deliver NIR optical contrast agent to human glioblastoma patients, thus providing real-time tumor identification in the operating room. This nonspecific tumor accumulation of ICG within the tumor provides strong signal to background contrast, and is not significantly time dependent between 6 hours to 48 hours, providing a broad plateau for stable visualization. This finding suggests that optimal imaging of the "Second Window of ICG" may be within this plateau period, thus providing signal uniformity across subjects.
荧光引导手术已成为在手术环境中检测、定位和切除肿瘤的有力工具。我们实验室开创了一种新方法，即使用美国食品药品监督管理局（FDA）批准的近红外（NIR）造影剂来协助外科医生完成这项任务。这项技术被称为第二窗口吲哚菁绿（ICG），它利用肿瘤血管的天然通透性及其清除缓慢的特点，将高剂量的吲哚菁绿（ICG）输送到肿瘤部位。该技术与已有的ICG视频血管造影技术有很大不同，后者在注射后几分钟内就能可视化ICG。我们推测，与单独使用ICG视频血管造影相比，第二窗口ICG能在更长时间内为颅内脑肿瘤提供具有良好信号特征的近红外光学对比度。我们在颅内小鼠胶质母细胞瘤模型中验证了这一假设，并在一项人体临床试验中得到了证实。
使用U251-Luc-GFP细胞系在2０只小鼠体内建立颅内肿瘤。通过生物发光确认移植成功。经尾静脉注射5.0mg/kg（高剂量）或2.5mg/kg（低剂量）的ICG。在注射ICG后的七个延迟时间点，使用珀金埃尔默IVIS Spectrum（封闭视野）来可视化近红外荧光信号。使用LivingImage软件对近红外信号进行量化。基于我们的实验结果，招募了人类受试者进行临床试验，并静脉注射高剂量5.0mg/kg。在注射ICG一天后的手术过程中，使用VisionSense铱星（开放视野）进行成像。
在小鼠模型中，胶质瘤中的近红外信号与背景比值（SBR）在输注后1小时达到峰值，然后趋于平稳，并在至少48小时内保持强烈且稳定。与2.5mg/kg的低剂量相比，5.0mg/kg的高剂量可改善近红外信号（SBR = 3.5对2.8；P = 0.0624）。虽然早期（≤6小时）可视化显示第二窗口ICG在胶质瘤中的积聚比晚期（≥24小时）更强（SBR = 3.94对2.32；p<0.05），但在小鼠模型中，肿瘤内ICG近红外信号积聚似乎有一个很长的平稳期。我们将这个长平稳期称为ICG的“第二窗口”。在胶质母细胞瘤患者中，肿瘤内近红外信号的延迟可视化很强（SBR 7.50±0.74），在19至30小时的可视化窗口内没有任何显著差异（R2 = 0.019）。
第二窗口ICG技术使神经外科医生能够将近红外光学造影剂输送给人类胶质母细胞瘤患者，从而在手术室中实现实时肿瘤识别。ICG在肿瘤内的这种非特异性积聚提供了很强的信号与背景对比度，并且在6小时至48小时之间没有明显的时间依赖性，为稳定可视化提供了一个广阔的平稳期。这一发现表明，“ICG第二窗口”的最佳成像可能在这个平稳期内，从而为不同受试者提供信号均匀性。
