Figure 3

Autophagy inhibitors can selectively prune dysfunctional tumor vessels, optimizing the structure and function of the tumor's vascular network. A: Experimental timeline for evaluating the effects of autophagy inhibitors on zebrafish CT26 tumor vasculature. Fluorescently labelled tumor cells were injected into zebrafish 72 h post-fertilization (hpf), followed by screening 12 h post-injection (hpi). Treatment with autophagy inhibitors (chloroquine, 50 μmol/L; ULK-101, 4 μmol/L; MRT68921, 50 μmol/L) was administered from 1 d post-injection (dpi) to 5 dpi, with subsequent assessment of tumor vessels using confocal microscopy. B and C: Confocal 3D projection images of CT26 tumor vessels in the control and autophagy inhibitor-treated groups, with vessels highlighted in green. Blood vessel diameters were quantified using ImageJ software. Quantitative graph showing blood vessel diameter comparisons among the different groups (C). Quantitative data are shown as the mean ± SD. D and E: Confocal images of transgenic zebrafish expressing the endothelial marker flk:GFP and erythrocyte marker Gata1:dsRed in CT26 tumor vessels for the control and autophagy inhibitor-treated groups. Boxplot graph showing the persistence of blood flow quantified as a percentage of the luminated area. Boxplot showing the maximum and minimum values, medians, and 25/75 percentiles. F and G: Confocal images showing injection of low-molecular-weight FITC-dextran (20 kDa) in CT26 tumor vessels for the control and autophagy inhibitor-treated groups, with tumor vessels labelled in red and dextran in green. Bar graph depicting FITC signal intensity within tumor vessels. Quantitative data are shown as the mean ± SD. P values (versus control) were generated using one-way analysis of variance, followed by Tukey's post-hoc tests for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars = 50 μm.