Assessment The Early Changes Of Nude Mice Huh7 Hepatocellular Carcinoma Xenografts Response To Vascular Disrupting Agent A64 With Contrast Enhanced Ultrasound

Objective: Using contrasted-enhanced ultrasound examination to assess the early change of nude mice Huh7 hepatocellular carcinoma xenografts response to vascular disrupting agent A64. Materials and methods: Construct the model of nude mice Huh7 hepatocellular carcinoma xenografts, divided them into VDA A64 experimental group and saline contrast group. Every nude mouse went through regular color Doppler ultrasound examination, and contrast-enhanced ultrasound examination before and up to 24 hours after treatment with 0.2ml A64 or salt solution. The size and the blood flow signals of the xenografts are recorded by regular color Doppler ultrasound examination, while TTP, PI, Wi R were obtained through the contrast-enhanced ultrasound examination. Two groups underwent a pathological examination including routine HE staining and immunohistochemical CD31 staining at same time points corresponding to the ultrasound exams to detect the tumor cells, tumor vessels quantitatively or qualitatively.Result: 1. Forty-two Huh7 hepatocellular carcinoma xenografts in nude mice were established successfully. Regular color Doppler ultrasound shows no significant change in tumor size before and 1h, 2h, 4h, 24 h after treatment both in experimental group(n=8) and contrast group(n=7). No obvious blood flow signals were achieved in two groups pre and post treatment using color Doppler ultrasound. 2. Contrast-enhanced ultrasonography: Using MANOVA of repeated measuring method, A64 induced a significant decrease in TTP(F=5.295,P=0.047) and PI(F=6.303, P=0.029) compared with baseline at different time points, meanwhile a dramatic increase in Wi R(F=15.643, P=0.002). Comparison within A64 group indicate that TTP at baseline had a remarkable change at 1h(P=0.005), 2h(P=0), 4h(P=0) and 24h(P=0) post treatment. There is also a distinct change in TTP between 1h-24h(P=0.06). PI at baseline shows no difference within 2 hours, but a decline at subsequent time points up to 24h(P=0.047, 0.004). 3. Pathology results: 27 nude mice divided into three groups, pre-treatment group(n=3), A64 group(n=12), contrast group(n=12), went through pathology examination using H.E staining and specific endothelial antigen CD31 staining. 1 hour, 2hours, 4hours, 24 hours after injection,A64 group and contrast group were sacrificed(each time point 3 animals) to obtain pathology sections. HE staining indicating that 2 hours after A64 administrated, few scattered tumor cells began to necrosis, up to 4 hours and 24 hours, lesions contain small patchy necrosis were seen intratumoral. Moreover, CD31 shows a vascular damage induced by A64, which presents as a stenosis of tumor blood vessels and breakdown of vascular walls. 24 hours after treatment, central necrosis became expanded, great vessels nearly shut down, comparatively, more peripheral new small blood vessels increased obviously.Conclusion: 1. Regular ultrasound have little effect reflecting early changes induced by A64 in Huh7 hepatocellular carcinoma, meanwhile TTP, PI, Wi R can reflect the tumor perfusion rate and volume associated index precisely. 2. A64 have a transient effect on vascular endothelial cells in Huh7 hepatocellular carcinoma xenografts, causing tumor tissue ischemia, necrosis and hemorrhage. CD31 reduced significantly after the treatment, and increased reactively after tumor necrosis, permeability of tumor vessels increased furthermore. Ultrasonic parameters had a close relationship with pathological changes.