Anti-CXCR4纳米抗体抑制胰腺癌新生血管的机制探索

Mechanism of anti-CXCR4 nanobody inhibiting angiogenesis in pancreatic cancer

肿瘤新生血管为肿瘤的发生发展提供足够氧气和营养物质,支持肿瘤的生长和转移。胰腺癌微环境中基质细胞衍生因子-1(stromal cell derived factor 1,SDF-1,又称CXCL12)及其受体C-X-C趋化因子受体4(C-X-C motif chemokine receptor 4,CXCR4)参与了肿瘤细胞的增殖、迁移和新血管生成等多个生理过程,可作为胰腺癌治疗的靶点。本研究利用大肠杆菌系统表达了靶向CXCR4分子的纳米抗体,镍柱亲和层析纯化获得anti-CXCR4纳米抗体(CXCR4 Nb)和anti-PD-L1(programmed cell death ligand 1)&CXCR4双特异性纳米抗体(PX4 BsNb),通过体外实验探究CXCR4 Nb拮抗肿瘤新生血管的作用及机制,体内验证CXCR4 Nb和PX4 BsNb对胰腺癌荷瘤小鼠的抗肿瘤活性。人外周血单个核细胞分离实验获得上海交通大学地方伦理委员会批准,动物福利和实验过程均遵循上海交通大学动物伦理委员会的规定。体外实验结果表明,0.1μmol·L^(-1)CXCR4 Nb能有效抑制SDF-1诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)迁移,拮抗胰腺星状细胞对HUVEC增殖和迁移的促进作用。在NOD/SCID小鼠皮下胰腺癌移植瘤模型中,腹腔给予0.3 mg·kg^(-1)CXCR4 Nb,抑瘤率为28.8%,0.3 mg·kg^(-1)PX4BsNb抑瘤率为36.1%,且免疫荧光显示治疗组肿瘤部位血管生成均减少。CXCR4 Nb具有良好的安全性和有效性,为胰腺癌的抗血管生成治疗和纳米抗体的应用提供了理论基础。

Tumor angiogenesis provides adequate oxygen and nutrition for tumor development and supports tumor growth and metastasis.Stromal cell derived factor 1(SDF-1)and its receptor C-X-C motif chemokine receptor4(CXCR4)in pancreatic cancer microenvironment are involved in tumor growth such as promoting tumor cell proliferation,migration,and angiogenesis.In this study,anti-CXCR4 nanobody(CXCR4 Nb)and anti-programmed cell death ligand 1(PD-L1)&CXCR4 bispecific nanobody(PX4 BsNb)were expressed in Escherichia coli system and purified by nickel column affinity chromatography.We investigated the anti-angiogenesis activity and mechanism of CXCR4 Nb by in vivo and in vitro experiments.Ethical approval was obtained for collection of human peripheral blood mononuclear cell(hPBMC)samples from the Local Ethics Committee of Shanghai Jiao Tong University.All animal experiments were approved by the Animal Ethic Committee of Shanghai Jiao Tong University.The results showed that CXCR4 Nb at 0.1μmol·L^(-1)could effectively inhibit the proliferation and migration of human umbilical vein endothelial cells(HUVEC)promoted by pancreatic stellate cells in vitro.CXCR4 Nb and PX4 BsNb at 0.3 mg·kg^(-1)obviously decreased tumor angiogenesis and inhibited the tumor growth in NOD/SCID mice,the inhibitory rates were 28.8% and 36.1%,respectively.CXCR4 Nb significantly inhibited tumor growth and angiogenesis with great safety,which provides support for application of CXCR4 Nb and anti-angiogenesis therapy of pancreatic cancer.