Fig. 4

Evaluation of treatment efficacy on PyMT mammary tumors’ proliferation and angiogenesis. Immunostaining of a, b Ki67 and c, d CD31 expression in PyMT mammary tumors, treated with B20-4.1.1/PTX combination. The whole tumor cross-sections (a, c) and representative magnified tumor areas (b, d) shown for VEH (upper panel) and B20-4.1.1/PTX combinational treatment (lower panel). Ki67-positive signals within the tumors are shown in brown. The boxed areas indicate tumors “hotspots” with the highest density of microvessels, selected for quantification. Scale bars: blue = 20 mm; black = 100 μm. e Quantitative assessment of mean Ki67-positive signal per micrometer of tumor viable area in each sample (n = 5 in each treatment group). The Ki67 proliferation index was considerably low in B20-4.1.1/PTX combination group (the mean value, 0.0035) in comparison to the VEH (0.0049), B20-4.1.1 (0.0048), or PTX (0.0045) monotherapy groups; however, no statistically significant difference was observed (Kruskal-Wallis test, p > 0.05). A box plot displays the range of variation of the Ki67 proliferation index for each treatment group (the horizontal lines indicate the median value). f Tumor vessel density, in vessels/μm². A significant difference was found between the VEH and B20-4.1.1/PTX groups (the mean values 296.2 and 127.2 vessels/μm², respectively), but not between the VEH and B20-4.1.1 or PTX groups (the mean values 236 and 220.7 vessels/μm², respectively; Dunn’s multiple comparisons test, *p < 0.05). Error bars represent standard deviations