GEBP11用于胃癌术中近红外荧光实时成像及引导肿瘤切除

Complete removal of tumors by surgery is the one of the most important prognostic markers and a determinant predictor of recurrence and survival in cancer patients. However, Cancer patients with a positive tumor margin is common clinical study. Advantages of NIR fluorescent light include high tissue penetration，low autofluorescence and not altering the surgical field,make it be used to visualise structures and tumor resectionin real-time during surgery. Antibody,peptide and small molecular have been labeled with near infrared fluorescence (NIR) dye molecules as an introperative targeted imaging probes. GEBP11 is a new nine amino acid vascular homing peptide, which was screened and identified using phage display technology. Then 131I labeled GEBP11 peptides, including a novel bifid PEGlylated GEBP11 trimer and its corresponding monomer, were developed and conducted multimodality imaging and therapy. They were associated with perfect tumor uptake and tumor suppression. In this study,we developed gastric cancer vascular homing peptide targeted optical imaging probes using near infrared fluorescence(NIR) dye congugated to GEBP11 or trimeric GEBP11 peptide. A series of cell Immunofluorescence and cell NIR optical imaging as well as tumor imaging and NIR optical imaging of tumor resection were performed to compare the biological activity between near fluorescence dye labeled GEBP11 monomer and trimer. The aim of this study is to screen a near infrared fluorescence labeled GEBP11 probe with higher specificity and binding affinity as well as lower autofluorescence, which could be used as a potentical candidate to guide tumor resection more accurate and totally in gastric cancer.

手术是否可以完全切除肿瘤是决定胃癌患者预后的重要因素。然而，手术切缘肿瘤细胞阳性是一个常见的临床问题。近红外荧光成像因其良好的组织穿透力、较低的自发荧光及不影响手术视野等优点可用于术中实时成像及肿瘤切除。抗体、短肽以及小分子已经成功与NIR进行标记并作为术中成像的靶向探针。前期工作中，我们通过噬菌体肽库筛选得到胃癌血管特异结合短肽 GEBP11，并成功对其单体及三聚体进行放射性131I标记、多模态成像及治疗，显示出良好的肿瘤浓聚度及抑瘤效应。本研究拟通过近红外荧光基团标记GEBP11短肽及其三聚体制备NIR荧光探针，通过细胞水平、动物水平的NIR成像引导的胃癌的实时成像和术中引导肿瘤的切除及切除肿瘤的NIR成像等试验，比较它们的生物学活性的差异，筛选出具有高特异性、高浓聚度及低背景的近红外荧光成像的GEBP11探针，旨在胃癌术中应用近红外荧光靶向探针可更准确、完全的切除胃癌提供候选分子。

本项目进展顺利，达到预期目标。胃癌是世界第四大恶性肿瘤，也是引起死亡的第二大病因。由于早期的临床症状不典型，胃癌经常是在晚期才能被发现及诊断。手术仍是胃癌的主要治疗手段，术后阳性切缘的发生率为 1.8%-5.1%，肿瘤细胞切缘阳性不仅会增加局部肿瘤的复发，也是术后影响预后的重要预测因子。胃癌术后复发与术中肿瘤的不完全切除密切相关，因此发展胃癌或胃癌血管特异性的短肽或抗体并进行胃癌术中实时成像及引导肿瘤切除是十分必要的。GEBP11是我们通过噬菌体肽库筛选得到的胃癌血管特异性短肽。本研究首先合成GEBP11、(GEBP11)2-Acp及对照肽URP，制备Cy5.5标记的GEBP11短肽，应用质谱及高压液相色谱检测近红外荧光探针的标记率。免疫荧光结合特异性试验及竞争抑制试验显示GEBP11单体修饰组及二聚体修饰组与共培养内皮细胞结合效率最高，并可特异性的被GEBP11抑制其结合。接着我们重点进行了皮下荷瘤鼠体内荧光成像、生物学分布试验及原位癌荷瘤鼠体内荧光试验进一步证实了(GEBP11)2-Acp-cy5.5结合的特异性及靶向性。应用二聚体荧光探针引导皮下荷瘤鼠、原位癌及原位癌+腹膜转移的肿瘤切除，(GEBP11)2-Acp-cy5.5探针在肿瘤的定位于边界界定表现出良好的示踪效果，对于未来开展近红外成像引导下术中肿瘤切除方面体现出重要价值。将原位肿瘤切除后进行HE染色及近红外荧光成像，结果显示(GEBP11)2-Acp-cy5.5探针可特异性的浓聚于肿瘤血管，进一步验证了二聚体探针的血管靶向性。以上结果显示 (GEBP11)2-Acp-cy5.5探针可特异性、靶向性的浓聚于肿瘤部位，并引导肿瘤完全切除，为术中可更准确、全面的切除胃癌提供候选分子。本课题在Theranostics（IF=8.766）杂志发表论文1篇，培养2名研究生，1次参加国外会议交流。