Tian Fengyu, Zhou Shurui, Xie Shiyi, Zhang Zhenhua, Peng Ling, Jiang Ling, Wang Zeyuan, Nie Zhou, Huang Yan
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University Changsha 410082 P. R. China
Chem Sci. 2023 Oct 3;14(43):12182-12193. doi: 10.1039/d3sc03972b. eCollection 2023 Nov 8.
Monitoring the spatiotemporal dynamics of cancer biomarkers within the tumor microenvironment (TME) is critical to understanding their roles in tumorigenesis. Here, we reported a multifunctional fusion protein (collagen-binding domain and duck circovirus tag fused to mCherry, CBD-mCherry-DCV) capable of binding collagen with high affinity and covalently binding specific nucleic acids with exceptional efficiency. We then constructed a chimeric protein-nucleic acid nanodevice (CPNN) using CBD-mCherry-DCV and an aptamer-based sensing module to enable spatially controlled ratiometric imaging of cancer biomarkers in the TME. The collagen-anchoring module CBD-mCherry-DCV allowed specific immobilization of CPNN on 3D multicellular tumor spheroids, enabling the sensing module to achieve "off-on" fluorescence imaging of cancer biomarkers upon specific target recognition by an aptamer. Taking advantage of the constant fluorescence signal of mCherry and the activatable fluorescence response of Cy5 to specific cancer biomarkers, the detection sensitivity and reliability of CPNN were improved by self-calibrating the signal intensity. Specifically, CPNN enabled ratiometric fluorescence imaging of varying concentrations of exogenous PDGF-BB and ATP in tumor spheroids with a high signal-to-background ratio. Furthermore, it allowed the visual monitoring of endogenous PDGF-BB and ATP released from cells. Overall, this study demonstrates the potential of the nanodevice as a versatile approach for the visualization and imaging of cancer biomarkers in the TME.
监测肿瘤微环境（TME）中癌症生物标志物的时空动态对于理解它们在肿瘤发生中的作用至关重要。在此，我们报道了一种多功能融合蛋白（胶原结合结构域和与mCherry融合的鸭圆环病毒标签，CBD-mCherry-DCV），它能够以高亲和力结合胶原蛋白，并以极高的效率共价结合特定核酸。然后，我们使用CBD-mCherry-DCV和基于适配体的传感模块构建了一种嵌合蛋白-核酸纳米装置（CPNN），以实现对TME中癌症生物标志物的空间控制比率成像。胶原锚定模块CBD-mCherry-DCV允许CPNN特异性固定在三维多细胞肿瘤球体上，使传感模块在适配体特异性识别靶标后能够实现癌症生物标志物的“关闭-开启”荧光成像。利用mCherry的恒定荧光信号和Cy5对特定癌症生物标志物的可激活荧光响应，通过自校准信号强度提高了CPNN的检测灵敏度和可靠性。具体而言，CPNN能够对肿瘤球体中不同浓度的外源性血小板衍生生长因子-BB（PDGF-BB）和三磷酸腺苷（ATP）进行比率荧光成像，具有高信噪比。此外，它还能够对细胞释放的内源性PDGF-BB和ATP进行可视化监测。总体而言，本研究证明了该纳米装置作为一种通用方法用于可视化和成像TME中癌症生物标志物的潜力。
