Luo Haiming, Hong Hao, Slater Michael R, Graves Stephen A, Shi Sixiang, Yang Yunan, Nickles Robert J, Fan Frank, Cai Weibo
Department of Radiology, University of Wisconsin-Madison, Madison, Wisconsin.
Promega Corp., Madison, Wisconsin.
J Nucl Med. 2015 May;56(5):758-63. doi: 10.2967/jnumed.115.154690. Epub 2015 Apr 3.
The hepatocyte growth factor (HGF) and its receptor, c-Met, are actively involved in tumor progression and metastasis and are closely associated with a poor prognostic outcome for cancer patients. Thus, the development of PET agents that can assess c-Met expression would be extremely useful for diagnosing cancer and subsequently monitoring response to c-Met-targeted therapies. Here, we report the characterization of recombinant human HGF (rh-HGF) as a PET tracer for detection of c-Met expression in vivo.
rh-HGF was expressed in human embryonic kidney 293 cells and purified by nickel-nitrilotriacetic acid affinity chromatography. The concentrated rh-HGF was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid and labeled with (64)Cu. c-Met binding evaluation by flow cytometry was performed on both U87MG and MDA-MB-231 cell lines, which have a high level and a low level, respectively, of c-Met. PET imaging and biodistribution studies were performed on nude mice bearing U87MG and MDA-MB-231 xenografted tumors.
The rh-HGF expression yield was 150-200 μg of protein per 5 × 10(6) cells after a 48-h transfection, with purity of approximately 85%-90%. Flow cytometry examination confirmed that rh-HGF had a strong and specific capacity to bind to c-Met. After (64)Cu labeling, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-rh-HGF in c-Met-positive U87MG tumors (percentage injected dose per gram, 6.8 ± 1.8 at 9 h after injection) and significantly lower uptake in c-Met-negative MDA-MB-231 tumors (percentage injected dose per gram, 1.8 ± 0.6 at 9 h after injection). The fact that sonication-denatured rh-HGF had significantly lower uptake in U87MG tumors, along with histology analysis, confirmed the c-Met specificity of (64)Cu-NOTA-rh-HGF.
This study provided initial evidence that (64)Cu-NOTA-rh-HGF visualizes c-Met expression in vivo, an application that may prove useful for c-Met-targeted cancer therapy.
肝细胞生长因子（HGF）及其受体c-Met积极参与肿瘤进展和转移，并且与癌症患者的不良预后密切相关。因此，开发能够评估c-Met表达的PET试剂对于癌症诊断以及随后监测对c-Met靶向治疗的反应将极为有用。在此，我们报告了重组人HGF（rh-HGF）作为用于体内检测c-Met表达的PET示踪剂的特性。
rh-HGF在人胚肾293细胞中表达，并通过镍-次氮基三乙酸亲和色谱法纯化。将浓缩的rh-HGF与2-S-（4-异硫氰酸苄基）-1,4,7-三氮杂环壬烷-1,4,7-三乙酸偶联，并用（64）Cu标记。通过流式细胞术对c-Met水平分别较高和较低的U87MG和MDA-MB-231细胞系进行c-Met结合评估。对携带U87MG和MDA-MB-231异种移植瘤的裸鼠进行PET成像和生物分布研究。
转染48小时后，rh-HGF的表达产量为每5×10（6）个细胞150 - 200μg蛋白质，纯度约为85% - 90%。流式细胞术检查证实rh-HGF具有与c-Met结合的强大且特异性能力。用（64）Cu标记后，PET成像显示（64）Cu-NOTA-rh-HGF在c-Met阳性的U87MG肿瘤中具有特异性且显著的摄取（注射后9小时每克注射剂量百分比，6.8±1.8），而在c-Met阴性的MDA-MB-231肿瘤中的摄取显著较低（注射后9小时每克注射剂量百分比，1.8±0.6）。超声处理变性的rh-HGF在U87MG肿瘤中的摄取显著降低，以及组织学分析，证实了（64）Cu-NOTA-rh-HGF的c-Met特异性。
本研究提供了初步证据，表明（64）Cu-NOTA-rh-HGF可在体内显示c-Met表达，这一应用可能对c-Met靶向癌症治疗有用。
