肿瘤淋巴管特异转运的MRI造影剂的研制与淋巴微转移的靶向成像

Lymphatic metastasis is thought to be responsible for the failure of anti-tumor therapy and cancer deaths in many cases. It has been shown that tumor-induced lymphangiogenesis is associated with metastasis formation in many types of cancers. Tumor-induced lymphangiogenesis may occur both locally and in regional lymph nodes. Therefore, the investigation of the role of lymphangiogenesis in primary tumors and the draining of lymph nodes during lymphatic metastasis are currently popular areas of research. Although lymphatic imaging on metastatic lymph node and nonmalignant lymphatic vessel disorders have been reported, no paper has been published on the imaging of lymphangiogenesis in lymphatic metastasis in vivo of either human nor animal models. Based on our previous research on contrast agent and MR imaging of the lymphatic system, we will use our newly developed nano-particle, HA-Gd-DTPA, as a drug carrier covalently bonded to Gd-DTPA, to image tumor-induced lymphangiogenesis in this project. The HA-Gd-DTPA nanoparticle is prepared to target the tumor-related lymphatic endothelial cell marker: lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). This receptor has been identified as an HA receptor and plays a role in transporting hyaluronic acid across the lymphatic vessel wall. In order to insure that the HA-Gd-DTPA nanoparticle will be correctly targeted, actively transported by LYVE-1 and locally accumulated in lymphatic vessels, we will chemically modify the molecular structure and surface nature of the nanoparitcle. Meanwhile, a VX2 tumor animal model will be set up. Tumor-induced lymphangiogenesis and lymph node micrometastasis will be imaged by our newly developed MRI techniques using LYVE-1-targeted HA-Gd-DTPA nanoparticle as a MRI contrast agent. After MR scanning, the tumor tissue and lymph nodes in the experimental animal will be dissected and fixed in formalin solution. A well-designed and optimized enzyme, immunohistochemical technique (IHC), will be applied to investigate the lymphangiogenesis in the tumor tissue and metastatic lymph nodes. If successful, we will not only create a novel lymphatic-specific targeted nano-particle, but also obtain in vivo images of lymphangiogenesis in lymphatic metastasis for the first time. This will open a new door to understanding tumor-induced lymphatic vessels and elaborate the underlying mechanisms of tumor lymphangiogenesis and lymph node metastasis. Clinically, the information gathered from these results will be helpful for tumor staging, treatment and prognosis assessment.

肿瘤淋巴转移是导致患者治疗失败和死亡的原因之一。研究表明肿瘤发生淋巴转移与肿瘤淋巴管生成密切相关，且肿瘤淋巴管生成不仅见于肿瘤本身，还可见于转移淋巴结，因此，显示肿瘤淋巴管意义重大。但有关肿瘤淋巴管成像目前未见报道。我们在继往研究淋巴系统造影剂及其磁共振成像基础上，选择肿瘤淋巴管特异标识物LYVE-1的配体、生物功能优异的透明质酸分子为载体，制备一种全新的纳米磁共振阳性造影剂HA-Gd-DTPA。通过对HA-Gd-DTPA纳米颗粒的修饰，实现肿瘤淋巴管对HA-Gd-DTPA的靶向识别、局部浓聚和主动转运，结合成熟的动物肿瘤模型、先进的MR成像和免疫组化技术，实现并验证肿瘤淋巴管和淋巴微转移的同时显像。本项目的完成将首次实现对肿瘤淋巴管及淋巴微转移的无创靶向成像，为阐明肿瘤淋巴管生成及淋巴转移机制、肿瘤性病变分期和预后分析及抗淋巴生成的肿瘤治疗示踪提供全新的分子影像手段。

肿瘤淋巴转移是导致患者治疗失败和死亡的原因之一。研究表明肿瘤发生淋巴转移与肿瘤淋巴管生成密切相关，且肿瘤淋巴管生成不仅见于肿瘤本身，还可见于转移淋巴结，因此，显示肿瘤淋巴管意义重大，但有关肿瘤淋巴管成像目前未见报道。目前在临床应用的阴性和小分子阳性MR对比剂对淋巴管/结的显像并不能达到满意效果。本研究以透明质酸（Hyaluronic acid）为载体，制备新型亲淋巴系统MR造影剂HA-Gd-DTPA，探索该造影剂对肿瘤淋巴管及转移淋巴结的显示能力。我们合成的新型造影剂HA-Gd-DTPA分子量为70kDa左右，粒径约125nm，纵向弛豫率R1为21LmM-1s-1。具有良好的生物安全性，体外实验显示HA-Gd-DTPA对淋巴内皮细胞有良好的靶向性。对正常淋巴系统的成像中，HA-Gd-DTPA的强化效果及强化持续时间窗均优于小分子非特异性造影剂。HA-Gd-DTPA可以选择性增强淋巴系统而无静脉影像干扰。兔VX2肿瘤模型HA-Gd-DTPA造影显示，HA-Gd-DTPA能清晰显示肿瘤新生淋巴管。免疫组化分析HA-Gd-DTPA增强的强度与肿瘤淋巴管密度呈正相关。炎症淋巴结及肿瘤转移淋巴结模型HA-Gd-DTPA淋巴造影显示，HA-Gd-DTPA对鉴别炎症淋巴结及肿瘤转移淋巴结敏感性和特性性分别达到85%和90%，明显高于小分子非特异性造影剂。通过研究我们发现制备的新型亲淋巴造影剂HA-Gd-DTPA 稳定、安全，驰豫性能好、可以充分可靠地显示正常及肿瘤淋巴管及正常淋巴结的结构和功能，在鉴别良、恶性淋巴结方面具有较高特异性及敏感性，显示出良好的淋巴系统特异性。本项目的完成将为淋巴系统疾病MR分子影像研究和后续的靶向治疗奠定坚实基础。本课题严格按照实验方案和实验计划稳步实施，实验进展顺利，成果丰富，共发表SCI论文11篇，其中IF8.9分SCI论文1篇；国际会议交流3次，每年均有不同研究成果在国内会议交流；申请专利3项，获得国家教育部科技进步奖二等奖1项，培养硕士研究生1名，博士研究生2名，达到了既定的研究目标。