Rabbit VX2 Liver Cancer Model Research Articles

Sustained release hypoxia-activated prodrug-loaded BSA nanoparticles enhance transarterial chemoembolization against hepatocellular carcinoma

Transarterial chemoembolization (TACE) is the standard of care for patients with advanced hepatocellular carcinoma (HCC), but facing the problem of low therapeutic effect. Conventional TACE formulations contain Lipiodol (LP) and chemotherapeutic agents characterized by burst release due to the unstable emulsion. Herein, we developed a novel TACE system by inducing bovine serum albumin (BSA) loaded hypoxia-activated prodrug (tirapazamine, TPZ) nanoparticle (BSATPZ) for sustained drug release. In the rabbit VX2 liver cancer model, TACE treatment induced a long-term hypoxic tumor microenvironment as demonstrated by increased expression of HIF-1α in the tumor. BSATPZ nanoparticles combined with LP greatly enhanced the anti-tumor effects of the TACE treatment. Compared to conventional TACE treatment, BSATPZ nanoparticle-based TACE therapy more significantly delayed tumor progression and inhibited the metastases in the lungs. The effects could be partially mediated by the rebuilt immune responses, as BSATPZ nanoparticle can served as an immunogenic cell death (ICD) inducer. Collectively, our results suggest that BSATPZ nanoparticle-based TACE therapy could be a promising strategy to improve clinical outcomes for patients with HCC and provide a preclinical rationale for evaluating TPZ therapy in clinical studies.

Dynamic contrast-enhanced magnetic resonance imaging assessment of residual tumor angiogenesis after insufficient microwave ablation and donafenib adjuvant therapy

To analyze the correlation between dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) permeability parameters and serum vascular endothelial growth factor (VEGF) levels in a rabbit VX2 liver cancer model with insufficient microwave ablation (MWA), to observe the dynamic changes in residual tumor angiogenesis in the short term after MWA, and to assess the effectiveness of donafenib as adjuvant therapy. Forty rabbits with VX2 liver tumors were randomly divided into three groups: an insufficient MWA group (n = 15), a combined treatment group (n = 15) and a control group (n = 10). The dynamic changes in VEGF expression after MWA and the effectiveness of donafenib as adjuvant therapy were evaluated by DCE-MRI and serum VEGF levels before surgery and 1, 3, 7, and 14 days after surgery. The correlation between the volume translate constant (Ktrans) of DCE-MRI parameters and serum VEGF levels fluctuated after ablation, but the coefficient was always positive (all p < 0.001). Repeated-measures ANOVA revealed significant changes in the serum VEGF concentration (F = 40.905, p < 0.001; partial η2 = 0.689), Ktrans (F = 13.388, p < 0.001; partial η2 = 0.420), and tumor diameter in each group (F = 34.065, p < 0.001; partial η2 = 0.648) at all five time points. Pairwise comparisons showed that the serum VEGF level, Ktrans value and tumor diameter in the insufficient MWA group and combined treatment group were significantly lower at 1 d than in the control group, but these values gradually increased over time (all p < 0.05). Ktrans and tumor diameter were significantly greater in the insufficient MWA group than in the control group at 14 days (all p < 0.05). The serum VEGF concentration, Ktrans, and tumor diameter were significantly lower in the combined treatment group than in the other two groups at 3, 7, and 14 days (all p < 0.05). Ktrans is positively correlated with the serum VEGF concentration. Ktrans and the serum VEGF concentration changed significantly after treatment with insufficient ablation or in combination with donafenib, and Ktrans may change faster. Insufficient MWA promotes the progression of residual tumors. Adjuvant treatment with donafenib is effective.

Intra-arterial delivery of doxorubicin-loaded hollow gold nanospheres—photothermal ablation and chemoembolisation therapy in a rabbit VX2 liver cancer model

Hepatocellular carcinoma (HCC) is associated with a high mortality rate. In this study, we aimed to investigate the therapeutic effect and safety of hepatic arterial administration of doxorubicin (Dox)-loaded hollow gold nanospheres (Dox@HAuNSs) combined with photothermal ablation (PTA) in a rabbit VX2 liver cancer model established by implanting VX2 tumour cells into rabbit livers. Rabbits were randomly divided into four treatment groups: physiological saline solution (control); lipiodol and doxorubicin (Dox + Lp); lipiodol and Dox@HAuNS (Dox@PEG-HAuNS + Lp); and lipiodol, Dox@PEG-HAuNS, and photothermal ablation (Dox@PEG-HAuNS + Lp + PTA). Dox release from Dox@PEG-HAuNS in the tumour was detected using fluorescence microscopy. Tumour size and liver-kidney function were assessed in each rabbit preoperatively and on postoperative days 3, 7, and 14. Necrosis, proliferation, and micro-vessel density of VX2 tumours were estimated in peripheral tumour tissues. Dox release from Dox@PEG-HAuNS was increased with a subsequent increase in tumour necrosis in liver tumours after PTA, whereas the tumour volume and proliferation decreased. However, the aspartate aminotransferase and alanine aminotransferase levels indicated transient liver damage. Thus, intra-arterial delivery of Dox@PEG-HAuNS combined with PTA can suppress VX2 liver cancer growth. Dox@PEG-HAuNS + Lp + PTA is also associated with transient liver damage in rabbits. The combination of Dox@PEG-HAuNS chemoembolisation and PTA could be a potential therapeutic approach for HCC and it has broad application prospects.

Pi-induced in-situ aggregation of sevelamer nanoparticles for vascular embolization

Decades have witnessed rapid progress of polymeric materials for vascular embolic or chemoembolic applications. Commercially available polymeric embolics range from gelatin foam to synthetic polymers such as poly(vinyl alcohol). Current systems under investigation include tunable, bioresorbable microspheres composed of chitosan or poly(ethylene glycol) derivatives, in situ gelling liquid embolics with improved safety profiles, and radiopaque embolics that are trackable in vivo. In this paper, we proposed a concept of ‘responsive embolization’. Sevelamer, clinically proved as an inorganic phosphate binder, was ground into nanoparticles. Sevelamer nanoparticle is highly mobile and capable of swelling and aggregating in the presence of endogenous inorganic phosphate, thereby effectively occluding blood flow in the vessel as it was administered as an embolic agent for interventional therapy. Moreover, citrated sevelamer nanoparticles delayed the aggregation, preferable to penetrate deeply into the capillary system. On the rabbit VX2 liver cancer model, both sevelamer particles aggregates occlude the tumor feeding artery, but backflow was found for the pristine one, thereby citrate passivation of sevelamer nanoparticles endows it have potential from ‘bench to bedside’ as a new type of vascular embolic.

Tirapazamine-loaded CalliSpheres microspheres enhance synergy between tirapazamine and embolization against liver cancer in an animal model

Tirapazamine (TPZ) is a promising hypoxia-selective cytotoxic agent that may exert synergistic tumor-killing activity with transcatheter arterial embolization (TAE) for liver cancer. To investigated whether TPZ-loaded microspheres enhance the synergy between TPZ and TAE in liver cancer, we prepared TPZ-loaded CalliSpheres microspheres (CSMTPZs) and characterized their properties as a chemoembolization agent in vitro. Tumor hypoxia after TAE was detected in the rabbit VX2 model of liver cancer using a modified Clark-type microelectrode research system. CSMTPZ therapy was performed in the animal model. The plasma and tumor concentrations of TPZ and its metabolites were measured, and the efficacy and safety of CSMTPZ therapy were evaluated and compared with those of the conventional combination of intraarterial TPZ injection and embolization. The results showed that CSMTPZs displayed favorable in vitro properties including drug loading and release and microsphere size, shape, and surface profiles. TAE induced acute tumor hypoxia, but residual tumor cells responded to hypoxia through hypoxia-inducible factor 1α. CSMTPZ therapy improved TPZ delivery into tumor tissue with minimal systemic exposure. Accordingly, CSMTPZ therapy exhibited advantages in terms of hypoxia-selected cytotoxicity, tumor apoptosis and necrosis, animal survival, and safety over the conventional combination of TPZ and TAE. We revealed the improved synergistic anti-tumor effects of CSMTPZ therapy in the rabbit VX2 liver cancer model. Our data support the clinical evaluation of CSMTPZs in the treatment of hepatocellular carcinoma, and CSMTPZ administration might serve as a successful therapeutic strategy for this malignancy.

Low Inorganic Phosphate Stress Inhibits Liver Cancer Progression: from In Vivo to In Vitro

AbstractMetabolic stress (for example, low pO2, low pH) stimulates cancer progression in a complex and largely unresolved manner. Excessive inorganic phosphate burden is being considered as another stimulator, until now there is no well‐designed study to examine the potential benefits of reducing phosphate burden on cancer progression. Sevelamer microspheres, a polymeric phosphate binder, are introduced as embolic material for interventional treatment of rabbit VX2 liver cancer model. This technique is named as “transarterial sevelamer embolization (TASE).” The microspheres prove to be highly biocompatible, and TASE is found to be a safe local‐regional technique. Compared with conventional transarterial chemoembolization (TACE), TASE is found to not only occlude the tumor‐feeding vessel, but simultaneously deplete intratumoral inorganic phosphate (Pi), thereby inducing severe necrosis as well reducing metastasis and recurrence. Reduced Pi stress inhibits tumor vascularity, invasion, and metastasis by downregulating the angiogenic factors and oncoprotein expression. Energy metabolomics indicate that Pi stress also suppresses tumor anaerobic glycolysis and glutaminolysis. This systemic anti‐cancer effect indicates a promising new application for sevelamer and TASE as a potential alternative to conventional TACE for liver cancer treatment.

Development and evaluation of an ultrasound-triggered microbubble combined transarterial chemoembolization (TACE) formulation on rabbit VX2 liver cancer model.

Transarterial chemoembolization (TACE) is an image-guided locoregional therapy used for the treatment of patients with primary or secondary liver cancer. However, conventional TACE formulations are rapidly dissociated due to the instability of the emulsion, resulting in insufficient local drug concentrations in the target tumor.Methods: To overcome these limitations, a doxorubicin-loaded albumin nanoparticle-conjugated microbubble complex in an iodized oil emulsion (DOX-NPs-MB complex in Lipiodol) has been developed as a new ultrasound-triggered TACE formulation.Results: (1) Microbubbles enhanced therapeutic efficacy by effectively delivering doxorubicin- loaded nanoparticles into liver tumors via sonoporation under ultrasound irradiation (US+). (2) Microbubbles constituting the complex retained their function as an ultrasound contrast agent in Lipiodol. In a rabbit VX2 liver cancer model, the in vivo study of DOX-NPs-MB complex in Lipiodol (US+) decreased the viability of tumor more than the conventional TACE formulation, and in particular, effectively killed cancer cells in the tumor periphery.Conclusion: Incorporation of doxorubicin-loaded microbubble in the TACE formulation facilitated drug delivery to the tumor with real-time monitoring and enhanced the therapeutic efficacy of TACE. Thus, the enhanced TACE formulation may represent a new treatment strategy against liver cancer.

Early Identification of Residual Tumors following Microwave Ablation Using Contrast-Enhanced Ultrasonography in a Rabbit VX2 Liver Cancer Model.

Objective It is difficult to evaluate the ablation effect immediately after thermal ablation of liver cancer by clinical imaging methods, due to the immediate formation of an annular inflammatory reaction band (IRB). This study is aimed at exploring the early identification indicators of the IRB and residual tumor postmicrowave ablation (MVA) using contrast-enhanced ultrasonography (CEUS). Methods MVA was used to inactivate part of the tumor nodules in rabbit VX2 liver cancer models, leading to the coexistence of the IRB with residual tumors. Quantitative analysis of the perfusion parameters of the tumor and ablation zone was performed using CEUS, followed by liver biopsy and VEGFR-2 immunohistochemical staining. Results All rabbits successfully tolerated VX2 tumor inoculation and MVA operation. No statistically significant difference existed between the IRB vs. residual tumors, the IRB vs. junctional areas, and residual tumors postablation vs. VX2 tumors before ablation in regional blood volume, blood velocity, and blood flow estimated by parameters A, k, and A∗k of CEUS quantitative analysis. There was a statistically significant difference between the IRB and normal liver parenchyma in regional blood velocity and blood flow (p = 0.005 and p = 0.023, respectively). Normal liver parenchyma showed nonspecific VEGFR-2 staining, while VX2 tumor before ablation and residual tumor after ablation both showed positive VEGFR-2 staining; the necrosis zone showed negative staining by VEGFR-2 immunohistochemical staining. Conclusion MVA had no significant effect on the residual tumor hemodynamics. The blood flow in the IRB increased significantly as compared to normal liver parenchyma, resembling tumor hemodynamic patterns. CEUS can detect residual tumors immediately postablation only when they protrude from the annular-shaped IRB. In addition, VEGFR-2 targeted CEUS may have a great potential for detecting residual tumor after thermal ablation of hepatocellular carcinoma.

Improved ADM Penetration Distance and Therapeutic Efficiency in a Rabbit VX2 Liver Cancer Model by Relaxin Infusion Combined with Transcatheter Chemoembolization Through Hepatic Artery.

PurposeTo evaluate the adriamycin (ADM) pervasion distance within tumor stroma after relaxin (RLX) infusion through tumor feeding artery and further investigate the therapeutic effects of RLX infusion combined with transcatheter chemoembolization (TACE) on the rabbit VX2 liver cancer, since the chemotherapy impaired due to limited drug distribution hindered by stiffened tumor stroma.Materials and MethodsIn the first part, rabbits received normal saline (NS), RLX or combined with TACE, and the penetration distance of ADM was measured by immunofluorescence and the matrix metalloproteinases (MMPs) were evaluated by gelatin substrate zymography in each group. In the second part, the rabbits received NS, TACE and RLX combined with TACE, respectively. The tumor growth rates, necrosis rates and intrahepatic metastasis were measured, and hematoxylin-eosin (HE), transferase-mediated dUTP-biotin nick end labelling (TUNEL) and Ki67 staining were conducted in each group.ResultsIn the first part, the expression of MMP-9 was increased in groups treated by RLX compared with NS group, especially three days after RLX infusion (p=0.001). The ADM penetration distance was significantly increased in groups treated by RLX compared with NS group (p<0.05), and it was farthest three days after RLX infusion. In the second part, compared with the NS and TACE groups, the tumor growth rates, the positive staining rates of Ki67 and the tumor growth rates were significantly decreased in RLX+TACE group (p<0.05). However, the positive staining rates of TUNEL and the tumor necrosis rates were significantly increased (p<0.05), and HE staining also revealed higher necrosis rates. The intrahepatic metastasis indicates no difference between the three groups (p=0.273).ConclusionAn increased penetration distance was obtained by RLX infusion through tumor feeding artery, and better therapeutic effects were achieved by RLX combined with TACE.

Efficacy of transcatheter arterial chemoembolization combined with sorafenib in inhibiting tumor angiogenesis in a rabbit VX2 liver cancer model

BackgroundThe aim of this study was to investigate the effects of transcatheter arterial chemoembolization (TACE) combined with sorafenib on tumor angiogenesis. Materials and methodsThirty New Zealand rabbit VX2 liver cancer model animals were divided into five groups, which received either normal saline (A), TACE (B), sorafenib (C), sorafenib followed by TACE (D), or TACE followed by sorafenib (E). Serum vascular endothelial growth factor (VEGF) levels were measured before and after TACE via ELISA. Immunohistochemistry for CD34 was performed to evaluate microvessel density (MVD), and ultrasonography was used to access tumor volume. ResultsVEGF levels declined in group C but increased significantly on the 3rd post-operative day in groups B, D, and E. Levels decreased after the 7th post-operative day. Peak levels were significantly lower in group D than in groups B and E. On the 14th post-operative day, VEGF levels were the lowest in group C, followed by those in groups D and B. MVD was the lowest in group C followed by that in group D and E, and was the highest in group B. Group D had the smallest tumor volume. HE staining of tumor tissues from group C showed apoptosis in a scattered patchy pattern, whereas in groups B, D, and E, large areas of tumor cell necrosis were visible. ConclusionTACE can up-regulate serum VEGF levels, which in turn accelerates the formation of new blood vessels. Thus, TACE combined with sorafenib inhibits VEGF and angiogenesis, and pre-operative administration of sorafenib has a more superior anti-angiogenic effect than post-operative administration.

Detection of ALDH1 activity in rabbit hepatic VX2 tumors and isolation of ALDH1 positive cancer stem cells.

BackgroundAldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells.Materials and methodsWe used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein.ResultsWe used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver.ConclusionThe overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0785-0) contains supplementary material, which is available to authorized users.

Abstract 4913: Development of Fe3O4@TiO2 core-shell nanocomposites as radiosensitizers

Abstract Purpose: To test in vivo, in a rabbit VX2 liver cancer model, a nanocomposite made with Fe3O4 core and TiO2 shell as a possible radiosensitization agent. Methods: This study was approved by the institutional ACUC. Rabbits were implanted in liver with pairs of VX2 tumors distantly enough to allow irradiation of only one of the two tumors. The tumors were allowed to grow until clearly detectable by magnetic resonance imaging, followed by Computed Tomography (CT) simulation to identify and contour the tumors. Each rabbit was anesthetized, and immobilized in supine position. Isocenter was placed during the CT simulation and the rabbit was tattooed. After the CT, each rabbit was injected with nanoparticles and put under observation until recovery. The core-shell nanoparticles were surface covered with glucose (glut-NPs) and delivered intravenously to 6 rabbits. A radiation oncologist contoured the target volumes. The gross target volume included a hepatic tumor in one lobe; 1 - 3 mm expansion was done to create the clinical target volume and another 1-2 mm expansion to create the planning target volume (PTV). The control hepatic tumor was contoured and the treatment plan objective was to deliver more than 95% of the prescribed dose to the PTV while sparing the control tumor. A certified radiation physicist created the treatment plan. Dose Volume Histograms were created and the radiation oncologist approved the plan. Irradiation usually utilized 2 - 3 beams, delivering 2 Gy as a single fraction using Linear Accelerator. All 6 rabbits treated with nanoparticles the day before (14-18h previously) and 3 radiation-only (not glut-NPs treated) animals were irradiated so that only one of the tumors received radiation. The animals were terminated 3 days after irradiation and the tumors were harvested. Several view-fields with at 3-10 x 103 cells were automatically scored using the NanoZoomer and percentage of Ki67 positive cells, as a marker of proliferation was determined in each tumor. TUNEL assay was done as well. Results: In each rabbit the larger tumor of the pair was irradiated. In no-NP treated animals that led to the fact that irradiated tumors at 3 days after irradiation generally still had greater number of proliferating cells than the non-irradiated tumor sections. Conversely, in glut-NPs treated rabbits, irradiation led to a decrease of Ki-67 positive cells compared to the paired non-irradiated glut-NPs treated tumors. Three-day time-point was better suited for evaluation of tumor proliferation changes. TUNEL analysis showed higher apoptotic cell numbers in glut-NPs treated tumors (p=0.049). Combination of radiation with glut-NP treatment showed only a mild trend for increased apoptosis compared to non-irradiated glut-NPs treated tumors, this trend was not statistically significant (p=0.53). Conclusion: These data suggest that the glut-NPs show a considerable promise for radiosensitization. Note: This abstract was not presented at the meeting. Citation Format: Tamer Refaat, Derek West, kathleen R. Harris, Vamsi Parini, William Liu, Beau Wanzer, Lydia Finney, Andrew C. Larson, Jonathan Bautista, Vythialinga Sathiaseelan, Bharat Mittal, Tatjana Paunesku, Gayle Woloschak. Development of Fe3O4@TiO2 core-shell nanocomposites as radiosensitizers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2014-4913

The correlation of contrast-enhanced ultrasound and MRI perfusion quantitative analysis in rabbit VX2 liver cancer.

Our objective is to explore the value of liver cancer contrast-enhanced ultrasound (CEUS) and MRI perfusion quantitative analysis in liver cancer and the correlation between these two analysis methods. Rabbit VX2 liver cancer model was established in this study. CEUS was applied. Sono Vue was applied in rabbits by ear vein to dynamically observe and record the blood perfusion and changes in the process of VX2 liver cancer and surrounding tissue. MRI perfusion quantitative analysis was used to analyze the mean enhancement time and change law of maximal slope increasing, which were further compared with the pathological examination results. Quantitative indicators of liver cancer CEUS and MRI perfusion quantitative analysis were compared, and the correlation between them was analyzed by correlation analysis. Rabbit VX2 liver cancer model was successfully established. CEUS showed that time-intensity curve of rabbit VX2 liver cancer showed "fast in, fast out" model while MRI perfusion quantitative analysis showed that quantitative parameter MTE of tumor tissue increased and MSI decreased: the difference was statistically significant (P < 0.01). The diagnostic results of CEUS and MRI perfusion quantitative analysis were not significantly different (P > 0.05). However, the quantitative parameter of them were significantly positively correlated (P < 0.05). CEUS and MRI perfusion quantitative analysis can both dynamically monitor the liver cancer lesion and surrounding liver parenchyma, and the quantitative parameters of them are correlated. The combined application of both is of importance in early diagnosis of liver cancer.

In vivo experimental study on the effects of fluid in increasing the efficiency of radiofrequency ablation.

Radiofrequency ablation (RFA) is the most widely used and studied method internationally for the local treatment of liver tumors. However, the extension of coagulation necrosis in one RFA procedure is limited and incomplete coverage of the damaged area can lead to a high local recurrence rate. In this study, we compared the effects of different solutions in enhancing hepatic radiofrequency by establishing a rabbit VX2 liver cancer model. We also determined the optimal solution to maximise effects on the extent of RFA-induced coagulation necrosis. Thirty VX2 tumor rabbits were randomly assigned to five groups: group A, RFA alone; group B, RFA with anhydrous ethanol injection; group C, RFA with 5% hypertonic saline injection; group D, RFA with lidocaine injection; and group E, RFA with a mixed solution. Routine ultrasound examinations and contrast-enhanced ultrasound (CEUS) of the ablation areas were performed after RFA. Then, we measured the major axis and transverse diameter and compared the areas of coagulation necrosis induced by RFA. The mean ablation area range increased in groups B, C and especially E, and the scopes were greater compared with group A. Preoperative application of anhydrous ethanol, hypertonic saline, lidocaine and the mixed solution (groups B, C, D and E, respectively) resulted in larger coagulation necrosis areas than in group A (p<0.05). Among the groups, the coagulation necrosis areas in group E was largest, and the difference was statistically significant compared with other groups (p<0.05). Pathological findings were consistent with imaging results. A mixture of dehydrated alcohol, hypertonic saline and lidocaine injected with RFA increases the extent of coagulation necrosis in the liver with a single application, and the mixed solution is more effective than any other injection alone.

Intra-arterial interleukin-12 gene delivery combined with chemoembolization: anti-tumor effect in a rabbit hepatocellular carcinoma (HCC) model

Interleukin-12 (IL-12), a cytokine naturally secreted by activated dendritic cells and monocytes/macrophages, is known as a key anti-tumor agent in many tumor models, including hepatocellular carcinoma (HCC) models. To evaluate the anti-tumor effect of intra-arterial IL-12 gene delivery alone and in combination with transcatheter arterial chemoembolization (TACE) in rabbit VX2 liver cancer model. Rabbits with VX2 liver tumors were randomized into four groups, eight in each group. After laparotomy and insertion of a 30-gauge needle into the proper hepatic artery, the following interventional procedure protocols were applied: 0.9% saline solution (group A, control), TACE (group B, TACE alone, lipiodol + mitomycin), intra-arterial interleukin-12 gene infusion (group C, IL-12 alone), and intra-arterial interleukin-12 gene infusion in combination with TACE (group D, IL-12 plus TACE). Growth ratio was estimated by computed tomography. To analyze apoptotic index, tumor tissues were explanted for terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining, 14 days after therapy. Significant differences of the relative tumor growth ratio were observed in TACE alone group and IL-12 plus TACE group in comparison with control (P < 0.05, ANOVA, Tukey's HSD correction) but not between IL-12 alone and control, or IL-12 plus TACE group and TACE alone group (P > 0.05). Significant changes of the apoptotic index were observed in group D in comparison with remaining three groups (P < 0.05). The difference between group C and group A was not significant statistically (P > 0.05). Intra-arterial interleukin-12 gene therapy combined with TACE has a potent anti-tumor effect in rabbit VX2 liver cancer in comparison with TACE alone.

Tumor Uptake of Hollow Gold Nanospheres After Intravenous and Intra-arterial Injection: PET/CT Study in a Rabbit VX2 Liver Cancer Model

This study was designed to investigate the intratumoral uptake of hollow gold nanospheres (HAuNS) after hepatic intra-arterial (IA) and intravenous (IV) injection in a liver tumor model. Fifteen VX2 tumor-bearing rabbits were randomized into five groups (n = 3 in each group) that received either IV (64)Cu-labeled PEG-HAuNS (IV-PEG-HAuNS), IA (64)Cu-labeled PEG-HAuNS (IA-PEG-HAuNS), IV cyclic peptide (RGD)-conjugated (64)Cu-labeled PEG-HAuNS (IV-RGD-PEG-HAuNS), IA RGD-conjugated (64)Cu-labeled PEG-HAuNS (IA-RGD-PEG-HAuNS), or IA (64)Cu-labeled PEG-HAuNS with lipiodol (IA-PEG-HAuNS-lipiodol). The animals underwent PET/CT 1h after injection, and uptake expressed as percentage of injected dose per gram of tissue (%ID/g) was measured in tumor and major organs. The animals were euthanized 24h after injection, and tissues were evaluated for radioactivity. At 1h after injection, animals in the IA-PEG-HAuNS-lipiodol group showed significantly higher tumor uptake (P < 0.001) and higher ratios of tumor-to-normal liver uptake (P < 0.001) than those in all other groups. The biodistribution of radioactivity 24h after injection showed that IA delivery of PEG-HAuNS with lipiodol resulted in the highest tumor uptake (0.33%ID/g; P < 0.001) and tumor-to-normal liver ratio (P < 0.001) among all delivery methods. At 24h, the IA-RGD-PEG-HAuNS group showed higher tumor uptake than the IA-PEG-HAuNS group (0.20 vs. 0.099 %ID/g; P < 0.001). Adding iodized oil to IA-PEG-HAuNS maximizes nanoparticle delivery to hepatic tumors and therefore may be useful in targeted chemotherapy and photoablative therapy. PET/CT can be used to noninvasively monitor the biodistribution of radiolabeled HAuNS after IV or IA injection.

Effects of transcatheter arterial chemoembolization with pingyangmycin-lipiodol emulsion on VX2 liver tumors in rabbits

To evaluate the changes induced in tumor tissue, the feeding artery, and neovascularization upon pingyangmycin-lipiodol emulsion treatment via transcatheter arterial chemoembolization (TACE) using the rabbit VX2 liver cancer model. The VX2 liver tumor model was established in 28 rabbits, and baseline tumor volume (V1, in mm3) was measured by spiral scan computed tomography (CT). Then, the rabbits were randomly divided into four groups (n = 7 each) and administered intraarterial therapies of: ultrafluid lipoidol embolization (group A); pingyangmycin (group B); pingyangmycin-lipiodol emulsion (group C); or saline (group D). All rabbits were sacrificed seven days later, and the response to therapy was determined by measuring the tumor volume (V2, in mm3), calculating the tumor growth rate, detecting expression of the vascular endothelial growth factor (VEGF) tumor biomarker, and performing histological analysis of the microvessel density (MVD) in the liver. Prior to therapy, the average V1 of the groups was statistically similar (A: 389.8+/-167.3, B: 404.1+/-184.9, C: 355.1+/-158.3, D: 378.1+/-189.0; (F = 0.257, P more than 0.05). In contrast, after therapy the average V2 of the groups was significantly different (A: 922.6+/-32.9, B: 665.9+/-99.9, C: 349.5+/-177.8, D: 1403.5+/-411.2; F = 26.23, P less than 0.05), as was the tumor growth ratio (A: 1.4, B: 0.6, C: -0.02, D: 2.7) and the mean positive ratio of VEGF (A: 57.1%, B: 42.9%, C: 28.6%, D: 100%; F = 8.407, P less than 0.05). MVD was highest in group D and lowest in group C (all, P less than 0.05). Bivariate correlation analysis revealed a positive correlation between VEGF expression and MVD (r = 0.743, P less than 0.01). Pingyangmycin exerts anti-tumor effects in the rabbit VX2 liver cancer model, but is more effective when administered as the combination therapy of pingyangmycin-lipiodol emulsion with TACE.

Confirmation of drug delivery after liver chemoembolization: direct tissue doxorubicin measurement by UHPLC‐MS‐MS

Because liver cancer is rarely suitable for surgery, transcatheter arterial chemoembolization (TACE) is used for palliative therapy. In this procedure, an emulsion of doxorubicin in iodized oil is injected directly into liver tumors through a catheter positioned within the artery supplying blood flow to the tumor. At present, there is limited understanding of factors affecting the delivery and dispersion of doxorubicin within treated tumors during TACE. This study addresses the development and application of an ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) method for rapid confirmation of drug delivery after TACE in a rabbit VX2 liver cancer model. Doxorubicin levels in liver tumors were measured using UHPLC-MS-MS and compared with computed tomography measured levels of iodized oil, a metric used clinically to indicate drug delivery. We found that tissue drug levels determined using UHPLC-MS-MS did not correlate with the regional iodized oil concentration (vehicle) within tumors following TACE, suggesting that chemotherapeutic drugs like doxorubicin spread throughout tumors, and that lack of iodized oil staining in portions of a tumor does not necessarily indicate inadequate therapy during TACE.

Experimental studies of rAd-p53 injection by interventional approach for the treatment of rabbit VX2 liver cancer

To evaluate the efficacy of recombinant human adenovirus p53 gene therapy (rAd-p53) in the rabbit VX2 liver cancer model using different interventional therapy approach. Thirty New Zealand rabbits implanted with VX2 tumor in the liver were randomized into five groups with six of each. The tumor volumes (V1) were measured by MRI and CT scan 11 days after tumors implanted. The interventional therapy scheme performed as below: intraarterial 0.9% saline solution perfusion in group A, transcatheter arterial embolization with 0.5 ml ultrafluid lipiodol in group B, intraarterial rAd-p53 gene perfusion in group C (1 x 10(6)/VP); intraarterial rAd-p53 gene perfusion (1 x 10(6)/VP) in combination with transcatheter arterial embolization (ultrofluid lipiodol, 0.5 ml) in group D and intratumoral rAd-p53 gene (1 x 10(6)/VP) injection in group E. The tumor volumes (V2) were measured by MRI and CT scan, and the tumor growth ratios were calculated 14 days after interventional procedures. Then all animals were sacrificed. The tumor tissues were explanted for immunohistochemistry to observe the expressions of vascular endothelial cell growth factor (VEGF) and factor VIII. Microvessel density (MVD) of the tumor tissues was assessed by factor VIII immunohistochemical analysis. In addition, apoptotic index was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The tumor volumes before therapy were (79.4+/-8.2), (75.3+/-7.8), (74.6+/-6.6), (78.7+/-9.1), (75.8+/-8.4) mm(3) respectively, without differences found among them (F = 12.248, P = 0.0636). But the tumor volumes after therapy were (564.7+/-96.7), (176.5+/-83.2), (239.6+/-42.8), (159.8+/-58.6), (334.7+/-32.6) mm(3) respectively (F = 24.537, P = 0.0218). The tumor growth ratios were 6.9, 2.6, 3.1, 1.6 and 4.1 respectively. The mean apoptosis index were 12.0%+/-1.1%, 14.5%+/-2.1%, 17.6%+/-2.3%, 18.6%+/-2.3% and 19.6%+/-2.5% respectively. with significant differences in group E in comparison with the other four groups. Mean positive ratio of VEGF was 50.0%, 83.3%, 83.3%, 50.0% and 50.0% respectively, with significant differences observed in group B and group C compared with the other three groups (F = 7.84, P = 0.019). The differences of VIII factor positive expression ratio among each group were significant (F = 0.854, P = 0.018). Statistical analysis showed a positive correlation between the expression of VEGF and MVD (r = 2.400, P = 0.0233). The rAd-p53 has effective treatment outcomes in VX2 rabbit liver cancer, and intra-arterial rAd-p53 gene perfusion in combination with transcatheter arterial embolization is the best approach in comparison with intra-arterial rAd-p53 gene perfusion, transcatheter arterial embolization and intratumoral rAd-p53 gene injection alone.