Figure 5

L2A5 and anti-STn mAbs binding assessment to STn⁺ and STn⁻ cancer cells using flow cytometry. Representative histograms of cell count (normalized) versus the mean fluorescent intensity (MFI) obtained for L2A5, B72.3 or 3F1 mAbs (anti-STn control antibodies) and control antibody (secondary anti-mouse Ig-FITC mAb – grey profile). For the assessment of STn binding, the breast cancer cell line MDA-MB-231 (A) or the bladder cancer cell line MCR (B) STn positive or negative (STn⁺ or STn⁻) were used (see also Materials and Methods). To assess binding specificity to STn antigen, STn-expressing cell lines were desialylated by sialidase treatment. The control antibody is represented as filled grey area, whereas solid and dashed lines represent the histograms for non-treated and sialidase treated cells, respectively. Numbers represent percentage of positive cells and MFI of total cells for each condition. (C,D) Column plots showing the mean ± standard deviation from 3 independent experiments of the flow cytometric analysis using L2A5 mAb to stain MDA-MB-231 STn⁺ (C) and MCR STn⁺ (D) cells, which were (treated) or not pre-treated (non-treated) with sialidase. MFI of stained samples was normalized to the MFI value of the negative control. Statistical analysis was performed using Student’s t-test (***p < 0.001).