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中华介入放射学电子杂志

中华介入放射学电子杂志 ›› 2021, Vol. 09 ›› Issue (04) : 407 -414. doi: 10.3877/cma.j.issn.2095-5782.2021.04.011

基础研究

基于c-Met抗体的近红外荧光探针用于肝癌的活体成像研究
易柳彤1, 杨美林2, 宫娜娜3, 余美红1, 曹婉维1, 李展宇1,†(), 李丹2   
  1. 1. 519000　广东珠海，中山大学附属第五医院病理科
    2. 519000　广东珠海，中山大学附属第五医院广东省生物医学影像重点实验室；519000　广东珠海，中山大学附属第五医院广东省分子影像技术工程研究中心
    3. 519000　广东珠海，中山大学附属第五医院检验科
  • 收稿日期:2021-09-18 出版日期:2021-11-25
  • 通信作者: 李展宇
  • 基金资助:
    中山大学附属第五医院"五个五"工程青年人才项目(2016972024656)

c-Met antibody-conjugated near-infrared fluorescent probe for in vivo imaging of hepatocellular carcinoma xenograft models

Liutong Yi1, Meilin Yang2, Nana Gong3, Meihong Yu1, Wanwei Cao1, Zhanyu Li1,†(), Dan Li2   

  1. 1. Department of Pathology, the Fifth Affiliated Hospital of Sun Yat-sen University, Guangdong Zhuhai 519000, China
    2. Guangdong Provincial Key Laboratory of Biomedical Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Guangdong Zhuhai 519000, China; Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Guangdong Zhuhai 519000, China
    3. Department of Clinical Laboratory, the Fifth Affiliated Hospital of Sun Yat-sen University, Guangdong Zhuhai 519000, China
  • Received:2021-09-18 Published:2021-11-25
  • Corresponding author: Zhanyu Li
  • About author:
    Co-first authors: Yi Liutong, Yang Meilin
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易柳彤, 杨美林, 宫娜娜, 余美红, 曹婉维, 李展宇, 李丹. 基于c-Met抗体的近红外荧光探针用于肝癌的活体成像研究[J]. 中华介入放射学电子杂志, 2021, 09(04): 407-414.

Liutong Yi, Meilin Yang, Nana Gong, Meihong Yu, Wanwei Cao, Zhanyu Li, Dan Li. c-Met antibody-conjugated near-infrared fluorescent probe for in vivo imaging of hepatocellular carcinoma xenograft models[J]. Chinese Journal of Interventional Radiology(Electronic Edition), 2021, 09(04): 407-414.

目的

探靶向近红外荧光成像为肝癌的精准诊疗带来了重要的契机，而且细胞间质表皮转化因子（c-Met）在肝癌中的表达显著高于正常肝组织。因此，本研究拟采用靶向c-Met的近红外荧光探针SHRmAb-IR800进行肝癌的活体成像研究。

方法

从TCGA和HPA数据库中获取并分析c-Met mRNA和蛋白的表达信息。通过Western Blot分析人肝癌细胞系中c-Met蛋白的表达情况。采用流式细胞技术和共聚焦成像实验评估SHRmAb-IR800与肝癌细胞的体外特异结合能力。构建肝癌皮下瘤模型，进行活体成像分析，并量化SHRmAb-IR800的成像肿瘤背景比。

结果

通过数据库分析发现肝癌中的c-Met mRNA和蛋白表达增加。Western Blot分析也显示人肝癌细胞系表达c-Met蛋白。流式细胞术和共聚焦成像实验均显示探针能特异性靶向c-Met阳性的人源肝癌细胞株；肝癌皮下瘤模型的近红外荧光成像显示靶向探针在皮下瘤的聚集显著高于对照探针（P < 0.05），并且探针在96 h的成像肿瘤背景比达到（2.01 ± 0.18）。

结论

本研究发现靶向c-Met近红外探针能够特异识别c-Met阳性的肝癌细胞和组织，有助于肝癌的诊断，在肝癌的手术导航方面具有潜在应用价值。

关键词: c-Met（细胞间质表皮转化因子）, 近红外荧光探针, 肝癌, 近红外荧光成像
Objective

Targeting near-infrared fluorescent imaging has facilitated the precise diagnosis and treatment of hepatocellular carcinoma (HCC), and cellular mesenchymal-epithelial transition factor (c-Met) has been reported as a highly expressed marker in HCC tissues compared with normal liver tissues. Thus, we intend to use the c-Met targeted near-infrared fluorescent probe SHRmAb-IR800 for imaging in HCC xenograft models.

Methods

The expression information of c-Met mRNA and protein was obtained and analyzed from TCGA and HPA databases, respectively. The expression of c-Met protein in HCC cell lines was analyzed by Western Blot. Flow cytometry and confocal imaging experiments were used to evaluate the specific binding ability of SHRmAb-IR800 with HCC cells in vitro. HCC subcutaneous xenograft models were constructed to perform in vivo imaging analysis and quantify the imaging tumor background ratio (TBR) of SHRmAb-IR800.

Results

The data extracted and analyzed from TCGA and HPA showed c-Met mRNA and protein were highly expressed in HCC tissues. Western Blot analysis also displayed that HCC cell lines expressed c-Met protein. Flow cytometry and confocal imaging were performed to verify the high specificity of SHRmAb-IR800 toward human HCC cell lines. Then the in vivo imaging of the probe in HCC xenograft models showed that the aggregation of SHRmAb-IR800 in tumor was higher than the control probe (P < 0.05). Moreover, the tumor-to-background ratio (TBR) of the probe in 96 h reached to 2.01 ± 0.18.

Conclusions

Our study demonstrated that c-Met-targeted probe had highly specific binding ability to HCC cells and tissues, which could be used to detect the HCC and have profound application potential in surgical navigation of HCC.

Key words: cellular mesenchymal-epithelial transition factor (c-Met), Near-infrared fluorescent probe, Hepatocellular carcinoma, Near-infrared fluorescent imaging
图1 数据库中c-Met的表达分析1A：在TCGA数据库中MET的表达分析；1B：MET在肝癌和正常肝组织中的表达情况；1C：蛋白数据库中c-Met的表达分析。***：P < 0.001；BLCA：膀胱尿路上皮癌；BRCA：乳腺癌浸润性癌；CESC：宫颈鳞状细胞癌；CHOL：胆管癌；COAD：结肠腺癌；ESCA：食管癌；GBM：多形性胶质母细胞瘤；HNSC：头颈部鳞状细胞癌；KICH：肾嫌色细胞癌；KIRC：肾透明细胞癌；KIRP：肾乳头状细胞癌；LIHC：肝细胞癌；LUAD：肺腺癌；LUSC：肺鳞状细胞癌；PAAD：胰腺癌；PRAD：前列腺癌；PCPG：嗜铬细胞瘤和副神经节瘤；READ：直肠腺癌；SARC：肉瘤；SKCM：皮肤黑色素瘤；THCA：甲状腺癌；THYM：胸腺瘤；STAD：胃腺癌；UCEC：子宫内膜癌。
图2 人肝癌细胞系中c-Met的表达分析2A：肝癌细胞系中c-Met的表达情况；2B：c-Met表达的量化分析。
图3 SHRmAb-IR800的体外结合能力分析3A~3B：探针与肝癌细胞孵育后的流式细胞术分析；3C：SHRmAb-IR800与SK-Hep1孵育后的共聚焦成像实验；比例尺：10 μm。
图4 SHRmAb-IR800在SK-Hep1肝癌皮下模型中的活体成像分析4A：SHRmAb-IR800在肝癌下瘤模型中的活体成像；4B：活体成像的MFI分析；*：P < 0.05。
图5 SHRmAb-IR800在SK-Hep1肝癌皮下瘤模型中的离体成像分析5A：皮下瘤和主要脏器的离体成像；5B：离体成像的定量分析；*：P < 0.05。
图6 SK-Hep1肝癌皮下瘤的c-Met免疫组化染色分析，比例尺：200 μm & 100 μm
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