Nanoscale distribution of CD20 on B-cell lymphoma tumour cells and its potential role in the clinical efficacy of rituximab

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.