Tumor angiogenesis after heated lipiodol infusion via the hepatic artery in a rabbit model of VX2 liver cancer

Abstract

Objectives: This study aimed to observe the changes in tumor angiogenesis after heated lipiodol (60°C) infusion via the hepatic artery in a rabbit model of VX2 liver cancer.

Materials and methods: Twenty rabbits with VX2 hepatic tumors were randomly divided into 2 groups (10 rabbits in each group). Under anesthesia, a trans-catheter hepatic arterial infusion was performed, and lipiodol (37°C; control group) or heated lipiodol (60°C; treated group) was injected into the hepatic arteries of the animals. Then, changes in tumor angiogenesis were assessed using the following markers and methods. 1. Vascular endothelial growth factor receptor (VEGFR) and vascular endothelial growth factor (VEGF) expression levels in the tumor were assessed using western blotting and real-time quantitative polymerase chain reaction (PCR). 2. Proliferating cell nuclear antigen (PCNA) expression in the tumor was assessed through immunohistochemical staining. 3. The morphological changes in tumor vascular endothelial cells were observed using transmission electron microscopy (TEM).

Results: VEGFR and VEGF mRNA and protein expression levels were reduced in the treated group compared to the control group. PCNA protein showed reduced expression levels in the treated group compared to the control group. TEM indicated that the endothelial cell endoplasmic reticulum expanded, the chondriosome was swollen, and the endothelial cell microvilli were decreased after heated lipiodol infusion.

Conclusions: The tumor angiogenesis of rabbits with VX2 cancer was inhibited after arterial heated lipiodol infusion compared to lipiodol infusion.

Figure 1. DSA imaging of treated tumors.

The tumors showed hypervascularity in the liver as determined with DSA imaging (A, black arrow). After lipiodol (60°C) injection, the tumors are completely or largely de-vascularized and show on DSA as a lipiodol-filling defect (B, black arrow).

Figure 2. Expression of PCNA protein.

As detected through immunohistochemistry, PCNA protein expression was detected mainly in viable VX2 tumor cells (brown; A, control 400×; B, treated 400×).

Figure 3. Expression of VEGFR and VEGF protein and mRNA levels.

The relative changes in VEGFR or VEGF protein and mRNA levels in tumor tissue were detected after treatment in each group (n = 10). A and B. VEGFR and VEGF mRNA expression levels were evaluated using real-time quantitative PCR as described in the Materials and Methods section. C and D. VEGFR and VEGF protein levels were detected using Western blot analysis (upper panel). β-actin was detected as a loading control. VEGFR and VEGF expression levels were quantified through densitometry and plotted as the fold change (lower panel). The values are presented as the mean±SD of 3 independent experiments (* P<0.05 vs. control group).

Figure 4. TEM results of treated tumors.

Stimulated by heated (60°C) lipiodol perfusion, TEM results revealed that the tumor endothelial cell microvilli decreased (A, 5000×), the vascular endothelial cell endoplasmic reticulum expanded, and the chondriosome was swollen (B, 10000×).