Figure 2

Endothelial autophagy is preferentially induced in dysfunctional tumor vessels. A: Pathway enrichment analysis based on the DEGs (top 500 highly expressed genes) in endothelial cells with high- versus low-autophagy gene scores. B: Dot plot comparing the expression of autophagy-related genes in normoxic (HIF-1α-negative) versus hypoxic (HIF-1α-positive) TECs. The color scale indicates expression levels, whereas dot size reflects the percentage of samples in which the gene was detected. C and D: Multiplex immunofluorescence analysis of autophagic activity in the vasculature of hypoxic and normoxic regions in ICC tissues. CA-IX (magenta) labels hypoxic regions, CD31 (red) identifies endothelial cells in the tumor vasculature, and LC3B (green) indicates autophagic activity. The zoomed-in section highlights a specific region comparing LC3B density in the tumor vasculature between the hypoxic and normoxic areas. The corresponding mask image shows the precise localization of the LC3B puncta (autophagic vesicles) within the zoomed region. The right panel (D) presents a box plot quantifying LC3B puncta density (puncta/μm²) in TECs from hypoxic and normoxic regions, showing significantly higher autophagic activity in hypoxic areas (*P < 0.05). Scale bar = 100 μm. E and F: In vivo 3D imaging of autophagy in the zebrafish tumor xenograft vasculature. Flk-GFP (green) visualizes the vessel structure, while LC3-mCherry (red) indicates autophagy in normal brain vessels, as well as functional tumor vessels and dysfunctional tumor vessels in CT26 cells at 4 dpi. Quantification of autophagic puncta showed a significant increase in dysfunctional tumor vessels compared to that in normal and functional vessels (***P < 0.001). Scale bar = 50 μm. G: Discrimination between autophagic and perfused endothelia in B16-F10 melanoma mouse tumor xenografts. Double immunostaining for CD31 (red) and LC3B (white) was used to label the endothelial cells and autophagy, respectively, with DAPI (blue) counterstaining of the nuclei. Perfused vessels were detected using fluorescein isothiocyanate (FITC)-dextran angiography (green). Autophagic endothelium (LC3B⁺ CD31⁺) was predominantly observed in non-perfused tumor vessels, with minimal overlap between FITC-dextran-positive (perfused) vessels and autophagic cells. Scale bar = 100 μm.