Fig. 5. Antibody–dye has more homogenous spatial distribution across tumor when co-administered with the unlabeled antibody.

a Each tumor was gridded into small square regions and antibody penetration within each square region was quantified as the ratio of fluorescent positive area to EGFR-positive area. b The ratio of fluorescence to EGFR over the entire tumor area was not significantly different between the LD and non-LD group (Scale bar: 5 mm). c–e Comparison of three metrics, including interquartile range (IQR), Pearson’s correlation coefficient between fluorescence and EGFR expression and CLCM correlation, all reflected less heterogeneity in antibody penetration across tumors in patients with loading dose (*p < 0.05; ns: p > 0.05). f, g Co-administration of parent antibody could overcome binding site barrier and improve antibody–dye distribution within the tumors. f As fluorescent antibody extravasates from blood vessels (red tube), it immediately binds EGFR in the tissue (represented by cells in the cube). g By co-administering non-fluorescent antibody, the same amount of fluorescent antibody reaches the tissue, but these antibodies compete for binding sites locally in the tissue. This results in improved distribution of fluorescent antibody at the microscopic scale (Scale bar: 250 μm). Mann–Whitney U test (two-tailed) was used in b–e. p value = 0.025 c, 0.025 d, 0.036 e. The number of independent patient samples available for b–e: n = 12 patients in the LD group and n = 10 in the non-LD group. Graphs plotted mean with standard deviation.