Fig. 7.

Clioquinol and MK-2206 inhibit TNBC development, as assessed by histology and immunohistochemistry. a Light microscopic images of H&E-stained 4T1 tumors (bordered by dotted line) in control, clioquinol, MK-2206, and combination group. Scale bar: 160 μm. b Tumor size (mm²) in control, clioquinol, MK-2206, and combination group, as assessed by histology (n = 10). c Fluorescence microscopic images of tumor microvessels in control, clioquinol, MK-2206, and combination group. Tumor sections were stained with an anti-CD31 antibody (red) and Hoechst 33342 (blue) for the visualization of ECs and cell nuclei, respectively. Scale bar: 70 μm. d Microvessel density (mm^− 2) of 4T1 tumors in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry (n = 10). e, g Light microscopic images of Ki67- (e) and cleaved caspase-3-positive (g) tumor cells in control, clioquinol, MK-2206, and combination group. Scale bars: 55 μm. f, h Ki67- (f) and cleaved caspase-3-positive tumor cells (h) (% of total cell number) in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry (n = 10). i Fluorescence microscopic images of tumor microvessels in control and clioquinol group. Tumor sections were stained with an anti-VEGFR2 antibody (red), an anti-CD31 antibody (green), and Hoechst 33342 (blue). Scale bar: 60 μm. j Area of VEGFR2 signal normalized to CD31 area (% of control) in tumors of control and clioquinol group. (k) VEGFR2 MFI (% of control) in tumors of control and clioquinol group. Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (b, d, f, h: one-way ANOVA with Tukey’s multiple comparisons test; j, k: unpaired Student’s t-test)