Fig. 5: MBRT regulates tumor angiogenesis through the expression levels of HIF-1α and VEGFR.

RNA-seq analysis results (n = 3): A The volcano plot shows the distribution of gene expression between the MBRT 0.6 group and the CRT 7 Gy group. B, C GSEA enrichment and heat map of angiogenesis-related gene sets showed that the MBRT 0.6 group negatively regulated tumor angiogenesis. D KEGG pathway analysis of downregulated genes (signal transduction related) in the MBRT 0.6 group compared with the CRT 7 Gy group. E, F HIF-1α immunohistochemical analysis revealed differential responses to radiotherapy regimens; scale bars: 50 μm, 63×, value = mean ± SD, n = 3. G–I CD31/α-SMA double immunofluorescence staining showed that the vascular structure of the MBRT group was significantly improved. The white dotted line outlines the vascular lumen, the CD31 positive signal defines the endothelial cell layer, and the white arrow indicates α-SMA positive pericytes; Scale: 100 μm, 40×; 20 μm, 150×; value = mean ± SD, n = 3, MFI (Mean fluorescence intensity). J The transmission electron microscope images show the effects of treatment groups on tumor vessels. EC endothelial cells, P pericytes, BM basement membrane, RBC red blood cells, LYM lymphocytes, L lumen, J tight junctions, AJ abnormal tight junctions, M mitochondria, SM swollen mitochondria, ELP endothelial luminal projections. TEM magnification: 3000×; scale bar: 5 μm for full view, 1 μm for inset. K Western blot assay of HIF-1α, VEGFR2, eNOS3, Erk, and pErk expression in different groups. Statistical significance for multiple comparisons was determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).