Pictorial Imaging-Histopathology Correlation in a Rabbit with Hepatic VX2 Tumor Treated by Transarterial Vascular Disrupting Agent Administration.

Cancer vasculature is immature, disorganized and hyperpermeable and can serve as a target for anti-cancer therapies. Vascular disrupting agents (VDAs) are tubulin protein binding and depolymerizing agents that induce rapid tumoral vascular shutdown and subsequent cancer necrosis. However, two clinical problems exist with all VDAs, i.e. 1) incomplete anticancer effect and 2) dose-dependent toxicity. To tackle these problems, in our ongoing research, a novel VDA C118P is applied by transarterial administration of half the intravenous dose in rabbits with implanted VX2 liver tumor to assess its therapeutic efficacy. Nearly complete tumor necrosis was achieved by only a single arterial dose of C118P at 5 mg/kg, which was documented in a representative case by in vivo digital subtraction arteriogram (DSA) and magnetic resonance imaging (MRI), and further confirmed by ex vivo microangiogram and histopathology. This convincing and promising preliminary outcome would warrant further comprehensive studies to explore the potentials of VDAs by transarterial administration either in mono-drug or in combination for management of solid cancers.

Role of MR-DWI and MR-PWI in the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbits.

OBJECTIVE: To detect the activity of tumor cells and tumor blood flow before and after the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbit models by using magnetic resonance diffusion-weighted imaging (MR-DWI) and magnetic resonance perfusion weighted imaging (MR-PWI), and to evaluate the effectiveness and safety of the radiotherapy based on the changes in the MR-DWI and MR-PWI parameters at different treatment stages. METHODS: A total of 56 rabbit models with implanted pulmonary VX-2 carcinoma were established, and then equally divided into treatment group and control group. MR-DWI and MR-PWI were separately performed using a Philips Acheiva 1.5T MRI machine (Philips, Netherland). MRI image processing was performed using special perfusion software and the WORKSPACE advanced workstation for MRI. MR-DWI was applied for the observation of tumor signals and the measurement of apparent diffusion coefficient (ADC) values; whereas MR-PWI was used for the measurement of wash in rate (WIR), wash out rate (WOR), and maximum enhancement rate (MER). The radiation treatment was performed using Siemens PRIMUS linear accelerator. In the treatment group, the radiotherapy was performed 21 days later on a once weekly dosage of 1,000 cGy to yield a total dosage of 5,000 cGy. RESULTS: THE ADC PARAMETERS IN THE REGION OF INTEREST ON DWI WERE AS FOLLOWS: on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values at the center and the edge of the lesions were 1.352 and 1.461 in the treatment group and control group (P>0.05). During weeks 0-1 after treatment, the t values at the center and the edge of the lesions were 1.336 and 1.137 (P>0.05). During weeks 1-2, the t values were 1.731 and 1.736 (P<0.05). During weeks 2-3, the t values were 1.742 and 1.749 (P<0.05). During weeks 3-4, the t values were 2.050 and 2.127 (P<0.05). During weeks 4-5, the t values were 2.764 and 2.985 (P<0.05). The ADC values in the treatment group were significantly higher than in the control group. After the radiotherapy (5,000 cGy), the tumors remarkably shrank, along with low signal on DWI, decreased signal on ADC map, and remarkably increased ADC values. As shown on PWI, on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values of the WIR, WOR, and MER at the center of the lesions were 1.05, 1.31, and 1.33 in the treatment group and control group (P>0.05); in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.35, 1.07, and 1.51 (P>0.05). During weeks 0-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 1.821, 1.856, and 1.931 (P<0.05); in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.799, 2.016, and 2.137 (P<0.05). During weeks 1-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.574, 2.156, and 2.059 (P<0.05) and the t values of the WIR, WOR, and MER at the edge of the lesions were 1.869, 2.058, and 2.057 (P<0.05). During weeks 2-3 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.461, 2.098, and 2.739 (P<0.05) and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.951, 2.625, and 2.154 (P<0.05). During weeks 3-4 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.584, 2.107, and 2.869 (P<0.05) and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.057, 2.637, and 2.951 (P<0.05). During weeks 4-5 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.894, 2.827, and 3.285 (P<0.05) and the t values of the WIR, WOR, and MER at the edge of the lesions were 3.45, 3.246, and 3.614 (P<0.05). After the radiotherapy (500 cGy), the tumors shrank on the T1WI, WIR, WOR, and MER; meanwhile, the PWI parameter gradually decreased and reached its minimum value. CONCLUSIONS: MR-DWI and MR-PWI can accurately and directly reflect the inactivation of tumor cells and the tumor hemodynamics in rabbit models with implanted pulmonary VX-2 carcinoma, and thus provide theoretical evidences for judging the clinical effectiveness of radiotherapy for the squamous cell carcinoma of the lung.

RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Evaluation of the safety and efficacy of transarterial sevelamer embolization in a rabbit liver cancer model: A challenge on the size rule for vascular occlusion.

As the most efficient method to treat hepatocellular carcinoma in the immediate or advanced stage, transarterial chemoembolization (TACE) is coming into the era of microsphere (MP). Drug-eluting beads have shown their huge potential as an embolic agent and drug carrier for chemoembolization, but their sizes are strictly limited to be above 40 µm, which was considered to occlude vessels in a safe mode. microsphere smaller than 40 µm is easy to be washed out and transported to the normal liver lobe or other organs, causing severe adverse events and failed embolization. To determine whether sevelamer ultrafine particle (0.2-0.5 µm) is qualified as a safe and efficient embolic agent, we investigated the safety and therapeutic efficiency of transarterial sevelamer embolization (TASE) in the VX2 rabbit liver cancer model, aiming to challenge the "40 µm" rule on the selection criteria of the MP. In a four-arm study, blank bead (Callisphere, 100-300 µm), luminescent polystyrene microsphere (10, 100 µm), and sevelamer particle were transarterially administered to evaluate the threshold size of the MP size for intrahepatic or extrahepatic permeability. Another four-arm study was designed to clarify the safety and efficiency of preclinical transarterial sevelamer embolizationTASE tests over other techniques. Sham (saline), TASE, C-TACE, and D-TACE (n = 6) were compared in terms of serum chemistry, histopathology, and tumor necrosis ratio. In the first trials, the "40 µm" rule was detectable on the VX2 cancer model, but the regulation has no application to the new embolic agent as sevelamer ultrafine particles have not been found to leak out from the VX2 lesions, only found in the embolized vessels. Pathology proves that less viable tumor residue was found 2 weeks after the procedure, evidencing a better therapeutic outcome. No adverse events were found except for a short stress response. These results indicate that sevelamer is a safe and efficient embolic as an alternative to the current MP-based embolization therapy techniques.

In Vivo Models for Studying Interstitial Photodynamic Therapy of Locally Advanced Cancer.

Interstitial photodynamic therapy (I-PDT) is a promising therapy considered for patients with locally advanced cancer. In I-PDT, laser fibers are inserted into the tumor for effective illumination and activation of the photosensitizer in a large tumor. The intratumoral light irradiance and fluence are critical parameters that affect the response to I-PDT. In vivo animal models are required to conduct light dose studies, to define optimal irradiance and fluence for I-PDT. Here we describe two animal models with locally advanced tumors that can be used to evaluate the response to I-PDT. One model is the C3H mouse bearing large subcutaneous SCCVII carcinoma (400-600 mm3). Using this murine model, multiple light regimens with one or two optical fibers with cylindrical diffuser ends (cylindrical diffuser fiber, CDF) can be used to study tumor response to I-PDT. However, tissue heating may occur when 630 nm therapeutic light is delivered through CDF at an intensity ≥60 mW/cm and energy ≥100 J/cm. These thermal effects can impact tumor response while treating locally advanced mice tumors. Magnetic resonance imaging and thermometry can be used to study these thermal effects. A larger animal model, New Zealand White rabbit with VX2 carcinoma (~5000 mm3) implanted in either the sternomastoid (neck implantation model) or the biceps femoris muscle (thigh implantation model), can be used to study I-PDT with image-based pretreatment planning using computed tomography. In the VX2 model, the light delivery can include the use of multiple laser fibers to test light dosimetry and delivery that are relevant for clinical use of I-PDT.

Modification of the method to establish a hepatic VX2 carcinoma model in rabbits.

The hepatic VX2 carcinoma model in rabbits is widely used for the preclinical study of hepatocellular carcinoma. In the present study, a modification was made to the conventional method to establish the animal model, as the conventional method gives rise to frequent tumor seeding due to the drop-out of tumor fragments. In order to evaluate each distinct method of establishing the model, the rabbits were divided into two groups: Group A (the conventional method; n=20) and group B (the modified method; n=20). All surgical details were recorded for reference. At 14 days post-surgery, contrast-enhanced computed tomography (CECT) and autopsy were conducted. Microscopic morphology of tumor cells was observed using hematoxylin and eosin (H&E) and transmission electron microscopy (TEM). Vascular endothelial growth factor (VEGF) and cluster of differentiation (CD)31 were detected via immunochemistry and reverse transcription-polymerase chain reaction. In total, 19 rabbits in each group succeeded in model establishment. Throughout the surgery, group A experienced a longer surgery time compared with group B (group A vs. group B, 22.57±1.34 vs. 20.17±1.50 min; P<0.001), an increased tumor fragment drop-out frequency (group A vs. group B, 1.84±0.96 vs. 1.16±0.38; P=0.008) and an increased peritoneal nodule incidence (group A vs. group B, 35 vs. 5%, P=0.042). As for CECT, H&E and TEM, hepatic VX2 allografts in the two groups demonstrated similar imaging presentations and tumor cell morphology. In addition, VEGF and CD31 levels did not differ between the two groups. In conclusion, the modified method for the establishment of hepatic VX2 carcinoma model in rabbits may decrease tumor fragment drop-out frequency during surgery and incidence of tumor seeding without affecting the properties of VX2 carcinoma.

Porphyrin-containing Gadolinium Complex as a Tumor-targeting Magnetic Resonance Imaging (MRI) Contrast Agent.

OBJECTIVE: Gadolinium diethylenetriaminepentaacetic di[5-(4'-amidophenyl)-10,15,20- tris(4'-sulfonatophenyl) porphyrin trisodium salt] (Gd-DTPA-2APTSPP) was synthesized by the reaction of diethylenetriaminepentaacetic dianhydride with 5-(4'-aminophenyl)-10,15,20-tris(4'-sulfonatophenyl) porphyrin and subsequently chelation with gadolium chloride. METHODS: This gadolinium complex was characterized and its properties in vitro and in vivo were also evaluated. Compared with Gd-DTPA, Gd-DTPA-2APTSPP possessed high relaxivity r1, low cytotoxicity to HeLa cells and high enhanced signal intensities of the VX2 carcinoma in rabbits for a prolonged time. CONCLUSION: Moreover, Gd-DTPA-2APTSPP can distinguish the VX2 carcinoma from the reactive hyperplasia incited by inflammation and normal tissues in rabbits. Therefore, Gd-DTPA-2APTSPP can be taken up selectively by tumors and show the potential as a tumor-targeting MRI contrast agent.

Indirect computed tomography lymphography identifies lymph node metastasis in rabbit pyriform sinus VX2 carcinoma.

Indirect computed tomography lymphography (CT-LG) could be used to determine the regional spread of cancer and assess lymphatic function by the interstitial delivery of diagnostic agents. Few studies have been reported on its use in pyriform sinus carcinoma. The aim of the present study was to establish the rabbit VX2 tumor as a model for pyriform sinus carcinoma and to observe its neck lymph node metastasis by indirect CT-LG. VX2 tumor tissue suspension was transplanted into the pyriform sinus submucosa of 15 rabbits under direct laryngoscope. Rabbits were randomly placed into one of three groups, each comprised of five rabbits. Observation of the tumor growth and neck lymph node metastases were taken on days 14 (group 1), 21 (group 2) and 28 (group 3) following transplantation using the method of indirect CT-LG. VX2 tumors were transplanted successfully in all rabbits. Deep cervical lymph nodes were enhanced clearly in indirect CT-LG. The contrast agent filling defected appeared on the metastasis nodes while the lymph node without metastasis was smooth. The metastasis rates of deep cervical lymph nodes were 100% in all three groups on CT-LG. The CT attenuation value of CT-LG reached peak values of 400 and 600 Hu at 1 and 3 min after the injection, which then decreased gradually. In this study, CT-LG could demonstrate the internal architecture of lymph nodes and their lymphatic vessels, and therefore may have the advantages of radiological methods such as B ultrasound, CT, magnetic resonance imaging and positron emission tomography.

Large animal model for retroperitoneal lymphatic and lung metastasis.

Retroperitoneal lymph node and lung metastasis are important prognostic factors for gynecologic cancer. The present study aimed to develop a new animal model for retroperitoneal lymph node and lung metastasis. VX2 squamous cell carcinoma tumor tissues were injected into the left gastrocnemius muscle of 38 healthy female New Zealand white rabbits. Animals were randomized into three groups according to day of sacrifice: 1, day 19; 2, day 22; and 3, day 25. Implanted primary tumor (IPTu), left and right retroperitoneal lymph node volumes and lung wet weights were measured on the day of sacrifice. The IPTu and left and right retroperitoneal lymph node volumes increased in a time­dependent manner. In addition, the proportion of animals with metastasis to the left peritoneal lymph nodes and the number of nodes involved increased over time. For days 19, 22 and 25, the proportion of animals with nodal metastasis was 58.3, 84.6 and 100%, respectively, and the number of affected nodes (range) was 3 (2­3), 3 (3­5) and 4 (4­5), respectively. No metastasis was detected in the right peritoneal lymph nodes. Metastasis to the lungs also increased with time, but was not statistically significant at days 19, 22 and 25 with metastasis present in 33.3, 38.5 and 76.9% of animals, respectively. Rates of metastases to the left retroperitoneal lymph nodes and lungs were found to positively correlate with the volumes (r=0.416 and 0.449, respectively). The current study assessed the characterization of a rabbit VX2 carcinoma model. This animal model is likely to be useful for evaluating retroperitoneal lymph node and lung metastasis.

The antitumor effect and hepatotoxicity of a hexokinase II inhibitor 3-bromopyruvate: in vivo investigation of intraarterial administration in a rabbit VX2 hepatoma model.

OBJECTIVE: The purpose of this study was to compare the antitumor effect and hepatotoxicity of an intraarterial delivery of low-dose and high-dose 3-bromopyruvate (3-BrPA) and those of a conventional Lipiodol-doxorubicin emulsion in a rabbit VX2 hepatoma model. MATERIALS AND METHODS: This experiment was approved by the animal care committee at our institution. VX2 carcinoma was implanted in the livers of 36 rabbits. Transcatheter intraarterial administration was performed using low dose 3-BrPA (25 mL in a 1 mM concentration, n = 10), high dose 3-BrPA (25 mL in a 5 mM concentration, n = 10) and Lipiodol-doxorubicin emulsion (1.6 mg doxorubicin/ 0.4 mL Lipiodol, n = 10), and six rabbits were treated with normal saline alone as a control group. One week later, the proportion of tumor necrosis was calculated based on histopathologic examination. The hepatotoxicity was evaluated by biochemical analysis. The differences between these groups were statistically assessed with using Mann-Whitney U tests and Kruskal-Wallis tests. RESULTS: The tumor necrosis rate was significantly higher in the high dose group (93% +/- 7.6 [mean +/- SD]) than that in the control group (48% +/- 21.7) (p = 0.0002), but the tumor necrosis rate was not significantly higher in the low dose group (62% +/- 20.0) (p = 0.2780). However, the tumor necrosis rate of the high dose group was significantly lower than that of the Lipiodol-doxorubicin treatment group (99% +/- 2.7) (p = 0.0015). The hepatotoxicity observed in the 3-BrPA groups was comparable to that of the Lipiodol-doxorubicin group. CONCLUSION: Even though intraarterial delivery of 3-BrPA shows a dose-related antitumor effect, single session treatment seems to have limited efficacy when compared with the conventional method.