A Correlative Study between CT Perfusion Parameters and Angiogenesis in Rabbit VX2 Liver Tumors

Objective: The purpose of this study was to evaluate the correlation between CT perfusion parameters and the hypoxia-inducible factor-1 alpha (HIF-1α), vascular en-dothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and microvessel density (MVD) marked by CD34 molecular of rabbit VX2 liver tumors and to investigate the value of CT perfusion imaging in evaluating tumor angiogenesis. Material and methods: Twenty-four cases of rabbit VX2 liver tumor were performed by CT perfusion scanning. Hepatic artery perfusion (HAP), portal vein perfusion (PVP), total hepatic blood flow (THBF) and hepatic perfusion index (HPI) were measured by perfusion software. HIF-1α, VEGF and MMP-2 expression and MVD were detected in the 24 rabbit VX2 liver tumor tissue samples using immunohistochemical method. The correlation between the HIF-1α, VEGF, MMP-2 expression and MVD and CT perfusion parameters were analyzed. Results: Correlation analysis revealed that the expression of HIF-1α, MMP-2, MVD were positively related to the HAP, THBF, HPI (p < 0.01), but no relations with PVP (p > 0.05);and correlation analysis revealed that the expression of VEGF was positively related to the HAP, HPI (p 0.05). There was a positive relationship between the expression of HIF-1α, VEGF, MMP-2 and MVD (p < 0.01). Conclusions: CT perfusion imaging can reflect the blood perfusion of the rabbit VX2 liver tumors and evaluate the information of angiogenesis about tumors.

Non-thermal ablation of rabbit liver VX2 tumor by pulsed high intensity focused ultrasound with ultrasound contrast agent:Pathological characteristics

AIM： To investigate the pathological characteristics of non-thermal damage induced by pulsed high intensity focused ultrasound （PHIFU） combined with ultrasound contrast agent （UCA）, SonoVue （Bracco SpA, Milan, Italy） in rabbit liver VX2 tumor. METHODS： Liver VX2 tumor models were established in 20 rabbits, which were divided randomly into PHIFU combined with ultrasound contrast agent group （PHIFU ＋ UCA group） and sham group. In the PHIFU ＋ UCA group, 0.2 mL of SonoVue was injected intravenously into the tumor, followed by ultrasound exposure of Isp 5900 W/cm^2. The rabbits were sacrificed one day after ultrasound exposure. Specimens of the exposed tumor tissues were obtained and observed pathologically under light microscope and transmission electron microscope. The remaining tumor tissues were sent for 2,3,5-Triphenyltetrazolium chloride （TTC） staining. RESULTS： Before Trc staining, tumor tissues in both the sham and the PHIFU ＋ UCA groups resembled gray fish meat, After TIC staining, the tumor tissues were uniformly stained red, with a clear boundary between tumor tissue and normal tissue, Histological examination showed signs of tumor cell injury in PHIFU ＋ UCA group, with cytoplasmic vacuoles of various sizes, chromatin margination and karyopyknosis. Electron microscopic examination revealed tumor cell volume reduction, karyopyknosis, chromatin margination, intercellular space widening, the presence of high electro＇n-density apoptotic bodies and vacuoles in cytoplasm. CONCLUSION： The non-thermal effects of PHIFU combined with UCA can be used to ablate rabbit liver VX2 tumors.

Assessment of hepatic VX_2 tumors with combined percutaneous transhepatic lymphosonography and contrast-enhanced ultrasonographic imaging

AIM： To evaluate the feasibility and efficacy of percutaneous transhepatic lymphosonography （PTL） as a novel method for the detection of tumor lymphangiogenesis in hepatic VX2 of rabbits and to evaluate combined PTL and routine contrast-enhanced ultrasonographic imaging for the diagnosis of liver cancer. METHODS： Ten rabbits with VX2 tumor were included in this study. SonoVue （0.1 mL/kg） was injected into each rabbit via an ear vein for contrast-enhanced ultrasonographic imaging, and 0.5 mL SonoVue was injected into the normal liver parenchyma near the VX2 tumor for PTL. Images and/or movie clips were stored for further analysis. RESULTS： UItrasonographic imaging showed VX2 tumors ranging 5-19 mm in the liver of rabbits. The VX2 tumor was hyperechoic and hypoechoic to liver parenchyma at the early and later phase, respectively. The hepatic lymph vessels were visualized immediately after injection of contrast medium and continuously vi- sualized with SonoVue during PTL. The boundaries of VX2 tumors were hyperechoic to liver parenchyma and the tumors. There was a significant difference in the values for the boundaries of VX2 tumors after injection compared with the liver normal parenchyma and the tumor parenchyma during PTL.CONCLUSION： PTL is a novel method for the detection of tumor lymphangiogenesis in hepatic VX2 of rabbits. Combined PTL and contrast-enhanced ultrasonographic imaging can improve the diagnosis of liver cancer.

Expression of MMP-2 in residual VX2 liver tumor after transcatheter arterial embolization combined with portal venous embolization in an animal model

Objective:This study aimed to analyze the effects of transcatheter arterial embolization(TAE)combined with portal venous embolization(PVE)on the expression of MMP-2 in residual VX2 liver tumor tissues,liver function and non-embolic lobe regeneration.Methods:A total of 72 rabbits were randomly divided into Sham,TAE,PVE and TAE+PVE groups(n=18/group).The tissue samples from each group were taken at 6 h,3 days and 7 days after interventional operation,respectively.MMP-2 expression was detected by immunohistochemistry,Real-time PCR,and Western-blotting.The main indicators(such as AST,ATL,and TBIL)of liver function and the volume of non-embolized hepatic lobes were measured in each group after operation.One-way ANOVA and Kruskal-wallis method were used for statistical analysis.Results:The expression of MMP-2 mRNA and protein remained the highest in the Sham group,and the expression of MMP-2 mRNA and protein in TAE,PVE and TAE+PVE groups were successively increased,and the expression of MMP-2 in TAE+PVE group was always significantly higher than TAE group.The AST and ALT levels in each group on day 7 after operation showed a significant declination,and all groups have recovered to the preoperative baseline level and TBIL has a slight fluctuation in each group after operation with no statistical difference.On day 7 after operation,the increasing volume of non-embolized liver lobes in TAE+PVE group showed a more significant effect than those in PVE group,but there was no statistical significance(37.62±1.54 ml VS 36.18±1.15 ml,P=0.881),and its volume was significantly higher than those in the sham group(27.03±1.11 ml).Conclusion:TAE+PVE is considered to be an efficient and safe approach for treating rabbit VX2 liver transplantation tumor,but the expression of MMP-2 increased fastest after TAE+PVE,which might promote tumor cell invasion and metastasis.

Establishing models of portal vein occlusion and evaluating value of multi-slice CT in hepatic VX2 tumor in rabbits

AIM： To establish models of portal vein occlusion of hepatic VX2 tumor in rabbits and to evaluate the value of multi-slice CT. METHODS： Forty New Zealand rabbits were divided into 4 groups according to digital table： Immediate group （group A; transplantation of tumor immediately after the portal vein occlusion）, 3-wk group （group B; transplantation of tumor at 3 wk after the portal vein occlusion）, negative control group （group C） and positive control group （group D）, 10 rabbits in each group. Hepatic VX2 tumor was transplanted with abdominalembedding innoculation immediately after the portal vein occlusion and at 3 wk after the portal vein occlusion. Meanwhile, they were divided into negative control group （Left external branch of portal vein was occluded by sham-operation, and left exite was embedded and inoculated pseudoly） and positive control group （Transplanted tumor did not suffer from the portal vein occlusion）. All rabbits were scanned with multi-slice CT. RESULTS： All 40 animals were employed in the final analysis without death. Tumor did not grow in both immediate group and 3-wk group. In 3-wk group, left endite was atrophied and growth of tumor was inhibited. The maximal diameter of tumor was significantly smaller than that in positive control group （2.55±0.46 vs 3.59±0.37 cm, t = 5.57, P 〈 0.001）. Incidences of metastasis in the liver and lung were lower in 3-wk group than those in positive control group （10% vs 400, and 90% vs 100%, respectively）. The expression intensities of the vascular endothelium growth factor （VEGF） in groups A, B, C and D were 0.10±0.06, 0.66±0.21, 0.28±0.09 and 1.48±0.32, respectively. VEGF expression level in the test group A was significantly lower than that in the negative control group C （t = 5.07; P 〈 0.001）.In addition, VEGF expression in the test group B was significantly lower than that in the positive control group D （t = 6.38; P 〈 0.001）. Scanning with multi-slice CT showed that displaying rate of hepatic artery branches was obviously lower in grade Ⅲ（40%） than that in grade Ⅰ（70%） and Ⅱ（100%） （P 〈 0.05）; but there was no significant difference in displaying rate of the portal vein at various grades. Values of blood flow （BF） of the liver, blood volume （BV）, mean transit time （MTT） and permeability of vascular surface （PS） were lower in the immediate group and 3-wk group than those in control groups, but values of hepatic arterial fraction （HAF） were increased. Significant positive correlations were existed between BF and BV （r = 0.905, P 〈 0.01）, and between BF and PS （r = 0.967, P 〈 0.01）, between BV and PS （r = 0.889, P 〈 0.01）. A significant negative correlation existed between PV and HAF （r = -0.768, P 〈 0.01）, between PS and HAF （r = -0.557, P 〈 0.01）. The values of BF, BV and PS had a positive correlation with VEGF （rBF = 0.842, rBV = 0.579, rPS = 0.811, P 〈 0.01） . However, there was no significant correlation between the values of MTT and HAF and the VEGF expression （rMTt = 0.066, rHAF = -0.027）. CONCLUSION： Ligating the left external branch of portal vein is an ideal way to establish models of portal vein occlusion in rabbits with hepatic VX2 tumor. Multi slice CT plays a key role in evaluating effect of portal vein occlusion.

Inhibitory Effect of Extract of Fungi of Huaier on Hepatocellular Carcinoma Cells

This study investigated the inhibitory effect of the extract of fungi of Huaier （EFH） on the growth of hepatocellular carcinoma （HCC） cells. Hep-G2 cells, a human HCC cell line, were cultured in DMEM containing 10% fetal bovine serum and treated with EFH of different concentrations （1, 2, 4, 8 mg/mL） for 24, 48 and 72 h respectively. The apoptosis rate of the cells was flow cytomet-rically measured. Thirty-six tumor-bearing New Zealand rabbits were randomly divided into 3 groups group A （control group）, in which the rabbits were infused with 0.2 mL/kg normal saline via the hepatic artery; group B （transhepatic artery cbemoembolization [TACE] group）, in which the rabbits were given lipiodol at 0.2 mL/kg plus MMC at 0.5 mg/kg via the hepatic artery; group C （TACE ＋ EFH group ）, in which EFH （500 mg/kg） were orally administered after TACE. Two weeks after TACE, the rabbits were sacrificed and the implanted tumors were sampled. The tumor volume and the necrosis rate were determined. The tumor tissues were immunohistochemically detected for the expressions of factor Ⅷ, VEGF, P53, Bax and Bcl-2. The microvessel density （MVD） was calculated by counting the factor Ⅷ-positive endothelial cells. Our results showed that after treatment with EFH the apoptosis rate of Hep-G2 cells was enhanced in a concentration- and time-dependent manner. Two weeks after the treatment, the average tumor volume, the necrosis rate and the growth rate of the implanted tumor in group C were significantly different from those in groups A and B （P〈0.05）. MVD and VEGF expressions were significantly decreased in the group C when compared with those in groups B （P〈0.05 for all）. The Bax expression was weakest in group A and strongest in group C. The expressions of P53 and Bcl-2 were minimal in group C and maximal in group A. There were significant differences in the expressions of P53, Bax and Bcl-2 among the 3 groups （P〈0.05 for all） and there was significant difference between group B and group C （P〈0.05）. It was concluded that EFH could suppress not only the growth of HCC cells but also tumor angiogenesis and it can induce the apoptosis of HCC cells. EFH serves as an alternative for the treatment of HCC.

Study of the Dextran-DTPA-Gd at rabbit popliteal fossa lymph node metastasis from thigh transplanted tumor with interstitial magnetic resonance lymphography

Objective： The aim of the study was to investigate the developing situation of the interstitial magnetic resonance （MR） lymphoid contrast agent Dextran-DTPA-Gd through the rabbit popliteal fossa lymph node metastasis from thigh VX2 transplanted tumor injection to show targeting enhanced metastatic lymph nodes and lymphatics. Methods： VX2 tumor was transplanted to the right hind limb quadriceps of 12 healthy New Zealand rabbits and the left side as a contrast. Eight rabbits had homonymy popliteal lymph node metastasis after 1 month through 3.0 GE MRI and they were later injected with lym phatic targeting contrast agent Dextran-DTPA-Gd 0.4 mL （3.96 x 10^-3 tool/L） through bilateral hindlimb toe web respectively. Enhanced MR images were obtained with interval 10 min, 15 min, 20 rain, 25 min, 30 rain, 35 min, 40 rain, 45 rain, 50 min, 55 min, 60 min, 2 h, 4 h, and 24 h. The signal intensities before and after enhancing were measured to calculate the enhanc- ing rates （E%） of popliteal lymph node and the popliteal lymph node signal intensity-time curves were drawn to observe the development of cancer metastasis lymph nodes and lymphatics and to compare the differences of interval sides. Results： Ten minutes after injected into the rabbit＇s bilateral hindlimb toe web, we could see hind lymphatic and popliteal lymph nodes were strengthened significantly and evenly without blood vessels developing. The signal reached a peak after 35 rain with E% to 315%, which decreased to 205% after 4 h and would be undifferentiated with the surrounding tissues after 24 h. Sta- tistical analysis was made to popliteal lymph node enhancement rate. It was considered statistically significant as long as P 〈 0.05. The tumor-side popliteal lymph node manifested as coarse and irregular shape, lymphatic vessels tortuous dilated and lymphatic chain incomplete as a result of tumor infection. Conclusion： Dextran-DTPA-Gd is specific to lymphoid tissue development. It can targeting display regional lymphatic drainage concretion and the morphology of normal and cancer cells metastasis lymph nodes rapidly.

Impact of microbubble enhanced, pulsed, focused ultrasound on tumor circulation of subcutaneous VX2 cancer

Background Intravascular microbubble-enhanced acoustic cavitation is capable of disrupting the vascular walls of capillaries and small vessels. This study was designed to investigate the impact of microbubble-enhanced, pulsed and focused ultrasound （MEUS） on the blood perfusion of subcutaneous VX2 tumors in rabbits. Methods Subcutaneous VX2 cancers in twenty New Zealand rabbits were treated by combining high-pressure amplitude, pulsed and focused therapeutic ultrasound （TUS） and intravenous microbubble injections. The TUS transducer was operated with a peak negative pressure of 4.6 MPa and a duty cycle of 0.41%. Controls were subcutaneous VX2 cancers treated with TUS or microbubbles only. Contrast-enhanced ultrasound （CEUS） and intravenous Evans Blue （EB） perfusion were performed to assess the tumor circulation. The tumor microvascular disruption was assessed by histological examination. Results CEUS showed that the tumor circulation almost vanished after MEUS treatment. The average peak grayscale value （GSV） of tumor CEUS dropped significantly from 84.1±22.4 to 15.8±10.8 in the MEUS-treated tumors but no significant GSV changes were found in tumors in the two control groups. The mean tumor EB content of the MEUS-treated tumors was significantly lower than that of the controls. Histological examination found scattered tumor microvascular disruption with intercellular edema after MEUS treatment. Conclusion The tumor circulation of VX2 cancers can be arrested or significantly reduced by MEUS due to microvascular disruption. Chin M~.cl ,I 2014：127 （14）： 2605-2611

Tumor growth and metastasis can be inhibited by maintaining genomic stability in cancer cells

The existence of cancer stem cells, stem-like cancer cells （SLCCs）, or tumor-initiating cells is considered as the cause of tumor formation and recurrence, indicating the importance of studying novel therapy that targets SLCCs. The origin of SLCCs is controversial because of two competing hypotheses： SLCCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Our previous research demonstrates that SLCCs are inducible by increasing genomic instability in cancer cells. In this study, to block the emergence of SLCCs, aminoethyl isothiourea （AET）, a compound that clears free radicals and is used to protect patients from radioactive exposure, was used as an agent that maintains genomic stability in combination with mitomycin C （MMC）, a commonly used chemotherapeutic drug that damages DNA. Using a rabbit tumor model with VX2 hepatic carcinoma, we found that MMC alone increased lung metastases and disadvantaged survival outcome, but the combination of MMC and AET reversed this effect and even prolonged overall survival. Moreover, in a VX2 xenograft model by immunocompromised mice, MMC alone enriched tumor-initiating cells, but the administration of MMC in combination with AET eliminated tumor cells effectively. Furthermore, MMC alone enhanced genomic instability, but MMC combined with AET attenuated the extent of genomic instability in primary VX2 tumor tissue. Taken together, our data suggest that the genomic protector AET can inhibit the induction of SLCCs, and this combination treatment by AET and cytotoxic agents should be considered as a promising strategy for future clinical evaluation.