State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, 110016, China.
J Mol Recognit. 2013 Sep;26(9):432-8. doi: 10.1002/jmr.2287.
Knowledge of drug-target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single-molecule techniques, the information of individual interactions between ligand-receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand-receptor forces under near-physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single-molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug-target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti-CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B-cell lymphomas but not on normal cells. First, B-cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20-rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20-rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab-conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20-Rituximab binding force was measured by single-molecule force spectroscopy.
药物-靶标相互作用的知识对于我们理解药物作用至关重要，并有助于设计更好的药物。由于缺乏足够的单分子技术，直到原子力显微镜（AFM）的出现，我们才获得了配体-受体之间单个相互作用的信息，该技术可以通过将配体连接到 AFM 尖端的表面，并在细胞上获得力曲线，从而在接近生理条件下直接测量单个配体-受体的力。目前大多数 AFM 单分子力谱实验都是在体外培养的细胞（细胞系）上进行的，这些细胞与体内的人类细胞有很大的不同。从临床实践的角度来看，直接在患者的癌细胞上研究药物-靶标相互作用将带来更有价值的知识，这些知识可能成为个性化治疗的重要参数。在这里，我们展示了 AFM 在 ROR1 荧光识别的辅助下，直接在淋巴瘤患者的癌细胞上测量靶标（CD20）和药物（利妥昔单抗，一种靶向 CD20 的单克隆抗体）之间结合力的能力。ROR1 是在一些 B 细胞淋巴瘤上表达的受体，但不在正常细胞上表达。首先，我们使用 B 细胞淋巴瘤 Raji 细胞（细胞系）进行 ROR1 荧光标记，然后测量 CD20-利妥昔单抗的结合力。结果表明，Raji 细胞表达了 ROR1，并且 ROR1 的标记并不影响 CD20-利妥昔单抗结合力的测量。然后，我们在滤泡性淋巴瘤患者的骨髓制备的病理样本上进行了建立的实验程序。癌细胞通过 ROR1 荧光识别。在荧光的引导下，使用与利妥昔单抗偶联的尖端，通过 AFM 成像可视化细胞形貌，并通过单分子力谱测量 CD20-利妥昔单抗的结合力。
