Fig. 2. Endothelial ablation is observed in in vivo mouse tumor models (subcutaneous tumor implantation model and genetically engineered mouse model).

(A) Examination of endothelial cells in the ectopic mouse tumors in vivo. YFP PD7591 (in green) was subcutaneously injected into mice for 14 days. Apoptotic endothelial cells were also observed in the in vivo tumor microenvironment, similarly to the observation in the 3D organotypic model. Apoptotic endothelial cells (in red) were marked by cleaved caspase-3 signal (in white). (B) Endothelial ablation in subcutaneous tumor implantation model. Resected tumors at day 14 exhibited partial ablation of endothelial cells by pancreatic cancer cells (red arrows) in hybrid blood vessels. Some small vessels, decorated with collagen IV (in white), a basement membrane protein, demonstrated complete endothelial ablation by YFP PD7591 in the luminal side of the vessels (yellow arrows). Blood vessels were stained with anti-mouse endomucin antibody (in red). (C) Endothelial ablation was observed in GEMM of PDAC. Partial ablation of endothelium in the blood vessel was indicated by red arrows, where the blood vessel displayed the occupation of yellow fluorescent protein (YFP) tumor cells in place of the endothelium. Endothelial cells in all images were stained with either anti-mouse CD31 antibody or anti-mouse endomucin. YFP PD7591 was restained with FITC-conjugated anti-GFP antibody. Cell nuclei were stained with DAPI (in blue).