基于IRDye800CW靶向荧光探针的胶质母细胞瘤术中导航研究

Intraoperative navigation of glioblastoma based on IRDye800CW-targeted fluorescent probe

目的制备靶向表皮生长因子受体(EGFR)的荧光探针西妥昔单克隆抗体(Cetuximab)-IRDye800CW,探讨其在胶质母细胞瘤(GBM)手术导航中的应用价值。方法测定Cetuximab-IRDye800CW荧光性能;通过免疫印迹法(Western blot)验证Cetuximab-IRDye800CW与GBM细胞结合的特异性;通过酶联免疫吸附测定(ELISA)竞争结合法验证探针是否通过结合EGFR来实现肿瘤靶向性。将GBM裸鼠皮下模型按完全随机法分为实验组和对照组(各3只),分别注射荧光材料Cetuximab-IRDye800CW和IRDye800CW,并于注射后5 min、24 h、48 h、72 h成像,比较2组肿瘤平均荧光强度(MFI)和肿瘤与周围背景的信噪比(TBR)差异。构建GBM裸鼠原位模型(6只),行MRI及术中荧光导航,并与病理学分布对比。采用两独立样本t检验处理数据。结果Cetuximab-IRDye800CW的最大发射波长为820 nm,可被近红外荧光成像设备接收。Western blot证实Cetuximab-IRDye800CW仅与GBM细胞相结合。ELISA竞争结合法证实Cetuximab-IRDye800CW通过与EGFR结合实现肿瘤靶向性。注射荧光材料后5 min、24 h、48 h、72 h,实验组肿瘤MFI分别为109.00±3.81、73.36±9.93、55.24±8.82、37.71±6.11,对照组肿瘤MFI分别为91.32±4.17、42.91±5.39、25.08±6.05、8.33±1.00,实验组肿瘤MFI较高(t值:4.36~9.40,P值:0.011~0.049);注射后24 h及48 h,实验组肿瘤TBR高于对照组(24 h,2.40±0.28与1.57±0.07,t=4.94,P=0.039;48 h,2.07±0.12与1.22±0.08,t=9.85,P=0.010)。MRI证实GBM原位模型构建成功,在荧光设备导航下肿瘤可视化,HE染色肿瘤病理学分布与荧光成像相符。结论Cetuximab-IRDye800CW具备荧光显像能力,在GBM术中导航中可识别肿瘤边界,具有潜在的临床应用价值。

Objective To prepare a fluorescent probe Cetuximab-IRDye800CW targeting epidermal growth factor receptor(EGFR)and investigate its application value in surgical navigation of glioblastoma(GBM).Methods The fluorescence properties of Cetuximab-IRDye800CW were determined by fluorescence spectrophotometer.The specificity of Cetuximab-IRDye800CW bound to GBM cells was verified by Western blot.The competitive binding method of enzyme-linked immunosorbent assay(ELISA)was used to prove whether the probe could achieve tumor targeting by binding to EGFR.Subcutaneous models of 6 nude mice of GBM were divided into experimental group(n=3;injected with Cetuximab-IRDye800CW)and control group(n=3;injected with IRDye800CW),and images were obtained at 5 min,24 h,48 h and 72 h after injection.Differences of mean fluorescence intensity(MFI)and tumor to background ratio(TBR)between experimental group and control group were compared.In situ models of GBM nude mice were established(n=6),and MRI and intraoperative navigation were conducted,which were compared with pathological distribution.Independent-sample t test was used to analyze the data.Results The maximum emission wavelength of Cetuximab-IRDye800CW was 820 nm,which could be received by near infrared fluorescence imaging equipment.Western blot showed that Cetuximab-IRDye800CW was only bound to GBM cells.The competitive binding of ELISA showed that Cetuximab-IRdye800CW could achieve tumor targeting by binding with EGFR.At 5 min,24 h,48 h and 72 h after injection of fluorescent materials,the MFI values of experimental group were 109.00±3.81,73.36±9.93,55.24±8.82,37.71±6.11,which were higher than those of control group(91.32±4.17,42.91±5.39,25.08±6.05,8.33±1.00;t values:4.36-9.40,P values:0.011-0.049).The TBR of experimental group was higher than that of control group at 24 h and 48 h after injection(24 h:2.40±0.28 vs 1.57±0.07,t=4.94,P=0.039;48 h:2.07±0.12 vs 1.22±0.08,t=9.85,P=0.010).GBM in situ model was successfully constructed and verified by MRI,and the tumor was visualized under the fluorescence device navigation.Pathological distribution of the tumor with HE staining was consistent with fluorescence imaging.Conclusion Cetuximab-IRDye800CW has fluorescence imaging capability and can identify tumor boundaries in intraoperative navigation of GBM,which has potential clinical application value.