Fig. 3. EphrinB2 knockout HNSCC cells implanted in EFNB2^fl/flTie2-Cre-ERT mice result in tumor growth retardation by normalization of tumor vasculature.

a CD31 immunostaining is performed on EFNB2^fl/flTie2-Cre-ERT (Double KO) tumors and their respective controls followed by quantitative analysis of vascular parameters using ImageJ software. Dual immunofluorescence staining is performed on tumors harvested from these mice to analyze the co-expression of CD31 with alpha-SMA (b), NG2 (c), VE-cadherin (d), PCNA (f), and PARP (g), showing improved vascular function in tumor-bearing mice following the loss of ephrinB2 on cancer cells and vasculature. The immunostaining in Figures a–d, f, g is performed in two sets with following number of captured images: a n = 12; b n = 5; c n = 5; d n = 7; f n = 5; g n = 5 control. Total magnification: x200. e DCE-MRI show increased enhancement of contrast agent in a time-dependent manner in representative ephrinB2 KO tumor-bearing EFNB2^fl/flTie2-Cre-ERT mice (Double KO). h Decrease in the protein levels of key markers responsible for different aspects of angiogenesis is evident in ephrinB2 double knockout tumor tissues. i VEGF ELISA shows decreased levels of circulating VEGF in ephrinB2 double knockout mice (n = 5) compared to the controls (n = 6). The experiments were performed in two except in (e). Color key for histogram plots in Figures b–d, f, g, i: Blue: Control; Red: Double KO. Data are shown as mean ± SEM. Control mice refers to the littermate controls implanted with Moc2 control KO tumors. Comparison between the control and experimental groups was done using two-sided Student’s t-test. p-values are indicated for the figures: a master junctions **p = 0.005; master segments, total segments length **p = 0.001; segments **p = 0.008; total branches *p = 0.03; total master segments length **p = 0.005; vascular density ***p = 0.0002, b *p = 0.01, c ***p = 0.0007, d **p = 0.001, g **p = 0.003, i *p = 0.015.