靶向超声造影优化肝细胞癌抗血管生成治疗策略的研究

With the increasement of anti-angiogenic therapeutic approaches in the clinic, it emerged an urgent need for new imaging techniques capable of providing earlier indications of treatment efficacy. Our previous studies have demonstrated contrast-enhanced ultrasound (CEUS) played a fundamental role in monitoring the efficacy of anti-angiogenic drugs. However, the evaluation of therapeutic response is commonly based on traditional non-targeted contrast enhanced ultrasound. The researches concerning anti-angiogenic therapy for hepatocellular carcinoma (HCC) focus on targeted contrast enhanced ultrasound are limited. In this study, we will apply endothelial cell as target to create targeted ultrasonic contrast agent by adding biotinylated anti-vascular endothelial growth factor 2 (VEGFR2) and anti-αvβ3 integrin antibodies to streptavidin coated microbubbles. Multiple techniques including immunohistochemistry, polymerase chain reaction (PCR), western-blotting and targeted contrast enhanced ultrasound will be used to monitor the angiogenesis of tumors and change of VEGF/VEGFR axises after treatment by bevacizumab and sorafenib on the orthotopic nude mouse HCC-mode. We will investigate the feasibility of using targeted ultrasound contrast agent for assess tumor response to anti-angiogenic treatment by analyzing correlation between functional parameters and angiogenesis, for the purpose of optimizing molecular targeted treatment on HCC. This study will provide a new scientific thought for the anti-angiogenic therapy from the perspective of targeted contrast enhanced ultrasound, and promote application of molecular ultrasound imaging.

肿瘤抗血管生成治疗已在临床逐步开展，我们的前期研究证实超声造影能反映肿瘤内血管血流灌注变化，有助于检测肿瘤抗血管生成治疗后反应。目前对疗效的评价多基于非靶向超声造影，尚缺乏针对肝细胞癌新生血管的靶向超声造影研究。本项目以肿瘤血管内皮细胞为靶点，采用"生物素-链霉亲和素"法制备针对VEGFR2、αvβ3整合素靶点微泡，并通过建立裸鼠人肝癌原位种植瘤模型，应用免疫组化、PCR、Western- blotting、靶向超声造影等技术观察肿瘤血管生成的变化情况，评估贝伐单抗与索拉非尼靶向治疗后肿瘤VEGF/VEGFR轴的变化规律。同时，通过定量软件分析造影参数与血管生成间的相关性，从靶向超声造影角度研究肝细胞癌的抗血管生成治疗的可行性，并阐明其应用机制，从而优化以血管生成为靶点的肝细胞癌分子靶向治疗策略，为肝细胞癌的临床抗血管生成治疗提供循证医学的理论依据，推动靶向超声造影新技术的可持续发展。

本项目以肿瘤血管内皮细胞为靶点，采用“生物素-链霉亲和素”法制备靶向VEGFR2、单靶向整合素、双靶向VEGFR2+整合素靶向微泡，体外实验通过流式细胞法检测了各种微泡的接靶率，并且通过激光共聚焦显微镜证实了靶向微泡的寻靶能力。通过建立裸鼠皮下移植瘤模型，对比了几种靶向微泡在裸鼠皮下移植瘤超声造影定量参数的差异及与肿瘤血管生成相关性，证实双靶向微泡能够更多的结合在裸鼠皮下移植瘤的血管内皮细胞上。随后，建立裸鼠原位肝脏移植瘤模型，应用免疫组化、靶向超声造影等技术观察肿瘤血管生成的变化情况，评估贝伐单抗靶向治疗后肿瘤生长变化规律，通过定量软件分析造影参数与肿瘤血管生成间的相关性，探讨了靶向超声造影评估肝细胞癌的抗血管生成治疗的可行性，并与非靶向微泡的评估能力进行了对比。发现靶向微泡能够更早监测到肿瘤对贝伐单抗治疗的反应性。在项目基础上，进一步将靶向超声造影应用于肿瘤热消融模型，在一定程度上较好的监测了裸鼠皮下种植瘤热消融与抗血管生成治疗后血流灌注变化，显示抗血管生成治疗可抑制不全热消融后残癌的生长。本课题研究建立制备的双靶向微泡对于早期监测肿瘤靶向治疗的反应性，及时调整肿瘤抗血管生成方案，延长患者生存期具有重要的研究意义和应用前景。