Li M, Xiao X, Zhang W, Liu L, Xi N, Wang Y
State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, China.
University of Chinese Academy of Sciences, Beijing, China.
J Microsc. 2014 Apr;254(1):19-30. doi: 10.1111/jmi.12112. Epub 2014 Feb 5.
Rituximab is an exciting monoclonal antibody drug approved for treating B-cell lymphomas and its target is the CD20 antigen which is expressed on the surface of B cells. In recent years, the variable efficacies of rituximab among different lymphoma patients have become an important clinical issue and urgently need to be solved for further development of antibodies with enhanced efficacies. In this work, atomic force microscopy (AFM) was used to investigate the nanoscale distribution of CD20 on the surface of tumour B cells from lymphoma patients to examine its potential role in the clinical therapeutic effects of rituximab. By performing ROR1 fluorescence labelling (ROR1 is a specific tumour cell surface marker) on the bone marrow cells prepared from B-cell lymphoma patients, the tumour B cells were recognized, and then AFM tips carrying rituximabs via polyethylene glycol crosslinkers were moved to the tumour cells to probe the specific CD20-rituximab interactions. By applying AFM single-molecule force spectroscopy (SMFS) at the local areas (500×500 nm²) on the surface of tumour B cells, the nanoscale distributions of CD20 on the surface of tumour B cells were mapped, visually showing that CD20 distributed heterogeneously on the cell surface. Bone marrow cell samples from three clinical B-cell lymphoma cases were collected to analyze the binding affinity and nanoscale distribution of CD20 on tumour cells. The experimental results showed that CD20 distribution on tumour cells were to some extent related to the clinical therapeutic outcomes while the CD20-rituximab binding forces did not have distinct effects to the clinical outcomes. These results can provide novel insights in understanding the rituximab's clinical efficacies from the nanoscale distribution of CD20 on the tumour cells at single-cell and single-molecule levels.
利妥昔单抗是一种获批用于治疗B细胞淋巴瘤的令人瞩目的单克隆抗体药物，其靶点是B细胞表面表达的CD20抗原。近年来，利妥昔单抗在不同淋巴瘤患者中的疗效差异已成为一个重要的临床问题，对于进一步开发疗效增强的抗体而言亟待解决。在这项工作中，原子力显微镜（AFM）被用于研究淋巴瘤患者肿瘤B细胞表面CD20的纳米级分布，以检验其在利妥昔单抗临床治疗效果中的潜在作用。通过对从B细胞淋巴瘤患者制备的骨髓细胞进行ROR1荧光标记（ROR1是一种特异性肿瘤细胞表面标志物）来识别肿瘤B细胞，然后将通过聚乙二醇交联剂携带利妥昔单抗的AFM探针移至肿瘤细胞以探测特异性的CD20-利妥昔单抗相互作用。通过在肿瘤B细胞表面的局部区域（500×500 nm²）应用AFM单分子力谱（SMFS），绘制了肿瘤B细胞表面CD20的纳米级分布图，直观显示CD20在细胞表面呈异质性分布。收集了三例临床B细胞淋巴瘤病例的骨髓细胞样本，以分析CD20在肿瘤细胞上的结合亲和力和纳米级分布。实验结果表明，肿瘤细胞上CD20的分布在一定程度上与临床治疗结果相关，而CD20-利妥昔单抗的结合力对临床结果没有明显影响。这些结果能够从单细胞和单分子水平上肿瘤细胞表面CD20的纳米级分布为理解利妥昔单抗的临床疗效提供新的见解。
