基于光点击化学筛选双靶标分子探针用于胰腺癌早期诊断的实验研究

Early detection of pancreatic adenocarcinoma is very difficult. Molecular tracers targeting mono-receptor displayed limited values in detecting highly heterogeneous small lesions of early stage pancreatic adenocarcinoma due to the relatively low sensitivity and specificity of tracers. As proved in literatures, dual-targeting strategy has exhibited significant impacts in solving the problem of tumor heterogeneity and increasing the sensitivity and specificity of molecular tracers. Plectin-1 and CXCR4 are found to be highly expressed in pancreatic adenocarcinoma and its distant metastases and even its precursor PanIN III lesions, while none or little of them are expressed in normal pancreatic cells, making them promising specific targets. In this proposal, a Plectin-1/CXCR4 dual-receptor targeted tracer labeled with 68Ga will be screened out by a visualized method and be constructed using a modularized strategy, and used for early detection of pancreatic adenocarcinoma and monitoring its metastases by non-invasive PET imaging. This study proposed two novel techniques for visible screening and facile construction of dual-receptor targeted molecular tracers based on the in-situ fluorophore generation by photoclick chemistry, and the combination of photoclick and string promoted azide–alkyne cycloaddition (SPAAC) click chemistry. Both techniques will have profound impacts on the development of tumor targeted molecular tracers.

胰腺癌早期诊断十分困难，常规分子影像探针往往靶点单一，灵敏度和特异性难以兼顾，很难识别出高异质性的胰腺癌早期病灶。研究证实，双靶标分子影像策略在解决肿瘤异质性、提高探针灵敏度、亲和力和特异性方面具有显著的优势。本研究将以在正常胰腺细胞中不表达或少量表达、在早期胰腺癌及其原位癌PanIN III中高表达的Plectin-1和CXCR4受体为特异性靶标，可视化筛选、模块化构建68Ga标记的双靶标分子探针，提高胰腺癌早期诊断的灵敏度和特异性，并为胰腺癌转移灶的识别提供有效的监测手段。本研究在技术上有两大创新，一是建立基于光点击化学（Photoclick）原位生成荧光可视化筛选双靶标分子探针的新方法，二是建立Photoclick联用叠氮-环辛炔无铜点击化学模块化制备双靶标分子探针的新策略。两种创新技术将对肿瘤分子影像探针的开发产生深远影响。

背景：常规探针靶点单一，灵敏度和特异性难以兼顾，难以诊断高异质性的肿瘤和早期病灶。双靶标策略在解决肿瘤异质性、提高探针灵敏度、亲和力方面具有显著的优势。整合素αvβ3、氨肽酶（CD13）、趋化因子受体 4型（CXCR4）和迟现抗原3（VLA-3）等在很多肿瘤中高表达，在肿瘤的发生、发展、侵袭及转移中起重要作用。本研究筛选合适的靶标，构建双靶标分子探针，评估其在提高胰腺癌、乳腺癌、卵巢癌等诊断灵敏度中的作用。.研究内容：通过无铜催化点击化学构建同时靶向整合素αvβ3和CD13的双靶点分子探针68Ga-NGR-RGD和同时靶向整合素αvβ3和CXCR4的双靶点分子探针68Ga-yG5-RGD。通过标记实验、细胞实验等测试分子探针的标记效率、稳定性、细胞结合亲和力等。构建胰腺癌、乳腺癌、卵巢癌等荷瘤鼠模型，通过小动物PET显像和生物分布实验，评估分子探针对肿瘤的靶向能力。通过单、双阻断实验以及不同受体表达量肿瘤模型、炎症模型评估双靶点分子探针的特异性。.结果：两个探针68Ga均展示出很高的标记率和放化纯，并具有很好的体内外稳定性。小动物PET/CT 显像显示，MCF-7肿瘤高摄取68Ga-NGR-RGD，肿瘤清晰可见，其摄取值高于68Ga-RGD或68Ga-NGR。当给予阻断剂（RGD、NGR、NGR+RGD 及 NGR-RGD）时，肿瘤摄取显著降低。68Ga-NGR-RGD在SKVO3和ES-2卵巢癌中也有较高的摄取，其肿瘤与背景比值显著高于18F-FDG。68Ga-NGR-RGD在炎症模型中摄取较低，在腹腔转移灶中较高，与18F-FDG炎症部位高、转移灶低相比具有很大的优势。胰腺癌BXPC3肿瘤高摄取68Ga-yG5-RGD，高于单靶点的68Ga-yG5 和68Ga-RGD。而注射过量阻断剂 AMD3100、RGD或AMD3100+RGD时，BXPC3 肿瘤摄取显著下降。而阴性对照组的MX-1 肿瘤始终未见显影。生物分布及肿瘤组织免疫组化结果与显像结果一致。.结论：成功构建了两个双靶点探针，均展示出比相应单靶点探针更高的肿瘤摄取，更长的肿瘤滞留时间。在炎症鉴别与微小转移灶评估方面比18F-FDG更有优势，具有很高的临床转化前景。