The effects of percutaneous ethanol injection followed by 20‑kHz ultrasound and microbubbles on rabbit hepatic tumors
Abstract
Objective Low-frequency ultrasound (US) in combination with microbubbles (MBs) is able to inhibit the growth of VX2 rabbit liver tumors. In this study, we investigated the feasibility of using percutaneous ethanol injection (PEI) followed by low-frequency ultrasound and microbubbles (USMB) to inhibit VX2 tumor growth.Methods Eighteen New Zealand rabbits with hepatic VX2 tumors were divided into three groups: PEI, low-frequency ultrasound and MBs followed by PEI (USMB + PEI), and PEI followed by USMB (PEI + USMB). PEI was performed by ultrasound-guided injection of 95 % anhydrous alcohol into internal liver tumors in rabbits twice a week for 2 weeks. The US parameters were 20 kHz, 2 W/cm2, 40 % duty cycle, 5 min, and once every other day for 2 weeks. Magnetic resonance imaging (MRI) was used to observe tumors before and after treatment, to examine changes in the tumors, and to measure the diameters of the tumors. All animals were followed up for 180 days after tumor implantation. Autopsy was performed at the end of the scheduled follow-up or immediately after death. Anatomically observed metastatic sites included the liver, lung, abdomen, and pelvic cavity. The survival time of all rabbits was recorded.Results After 4 weeks of treatment, on MRI, the tumor diameters in the PEI, USMB + PEI, and PEI + USMB groups were 8.33 ± 1.83, 19 ± 2.61, and 4.5 ± 1.22 mm, respectively. There was a significant difference in tumor size indicated by MRI in the three groups. Tumor size was smaller in the PEI + USMB group than in the PEI and USMB + PEI groups, with t = 4.54, p = 0.0062, and t = 16.38, p < .0001, respectively. The PEI + USMB group showed the fewest metastasis sites (χ2 = 11.7333, p = 0.0194) and the longest survival period (χ2 = 7.448, p = 0.0241).Conclusion Percutaneous ethanol injection followed by low-frequency ultrasound and microbubbles can be effective in inhibiting rabbit liver tumors and prolonging survival time.
Keywords
Percutaneous ethanol injection · Ultrasound · Low frequency · Microbubble · Cavitation · Hepatic · VX2 tumor · Rabbit
Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Asia. According to some reports, the annual incidence is 400,000, accounting for a global incidence of 60 % (Asia-Pacific Working Party on Prevention of Hepatocellular Carcinoma 2010). The majority of HCC patients』 tumors are found to be inoperable (Shin et al. 2014); and even if the patients undergo hepatic tumor resection or orthotopic liver transplantation, the recurrence rate is very high. Therefore, interventional therapy or local ablation is an important adjuvant treatment (Zheng et al. 2014; Crissien and Frenette 2014), even the only choice for some advanced HCC patients (Molla et al. 2014). Local ablation methods mainly include percutaneous ethanol injection (PEI) (Silva et al. 2014), radiofrequency ablation, microwave ablation (Crissien and Frenette 2014), and transcatheter arterial chemoembolization (TACE). Ultrasound-guided PEI, which was developed in the early 1980s, appears to be widely applicable for hepatic tumors (Giorgio et al. 2000). Compared to radiofrequency, microwave, and TACE (Watanabe et al. 2014), PEI is the simplest, cheapest, and most convenient operation. For tumors adjacent to a big, important blood vessel (Cha et al. 2013), such as the hepatic vein, portal vein, or bile ducts, which is a relative contraindication to local ablation (Zhang et al. 2014), PEI is an alternative operation. Another benefit is that PEI is considered to be effective for treating relatively small HCC (Xu et al. 2014), especially for tumors <2 cm in diameter, as there was no difference between radiofrequency ablation and PEI in terms of efficacy (Salhab and Canelo 2011). However, the limitations of PEI include the need for many sessions, and recurrent injections, and the fact that the relative survival rate is slightly lower than that of radiofrequency ablation (Xu et al. 2014). So, it is necessary, important, and urgent to find another synergistic therapy with PEI to make up for its shortages. Low-frequency ultrasound (US) is a novel tumor irradiation therapy that has recently been developed (Shen et al. 2013); its mechanism of action involves the use of ultrasonic cavitation (Shen et al. 2014; Xenariou et al. 2010). Compared to high-frequency ultrasound (MHz US), low-frequency US (20–100 kHz) produces weaker thermal effects, leading to reduced skin burns, less epidermal detachment, and less normal tissue necrosis (Ahmadi et al. 2012), at the same time, the technique generates a stronger cavitation effect, in which microbubble (MB) oscillation, growth, and collapse in microvessels occur, eventually leading to vessel wall rupture (Shen et al. 2014), decreased tumor blood supply. In our previous studies, we found that low-frequency US combined with MBs (USMB) could inhibit the growth of rabbit hepatic tumors (Shen et al. 2014). We further incorporated the effects of PEI; thus, the purpose of this early-stage in vivo study was to examine the efficacy of the combined PEI-USMB technique for possible future clinical application.
Methods
Eighteen New Zealand rabbits were divided into three groups, with six rabbits in each group: group A, PEI group; group B, USMB followed by PEI (USMB + PEI) group; and group C, PEI followed by USMB (PEI + USMB) group. A control (no treatment) and USMB group were absent in this study. As in our previous research (Shen et al. 2014), the effect of USMB was better than that of the control. Presently we focused on the effects of comparison among PEI, PEI + USMB and USMB + PEI groups, so the control and USMB were absent to prevent duplication of previous research.
Animal protocol and tumor inoculation
The experiments were performed with New Zealand white rabbits weighing 2.0–2.5 kg (average 2.2 kg). The experiments were approved by the Animal Care Committee of Nantong University Medical School, Jiangsu, China, and were performed in accordance with the corresponding institutional guidelines. The animals were anesthetized with an intravenous injection of 30 mg/kg pentobarbital. VX2 carcinoma was used to create the hepatic tumors. VX2 tumors were first grown for 2 weeks on the hind legs of the carrier rabbits and then were harvested after they reached a size of 1.5 cm. The harvested tumors were placed in saline solution and cut into cubes with a volume of 1 mm3. The experimental method employed has been previously described elsewhere (Shen et al. 2014). An iU22 ultrasound system (Philips, Bothell, Washington, USA) with a 12–15 MHz broadband linear probe was used to monitor tumor growth daily after tumor inoculation. VX2 carcinoma nodules reaching 0.8–0.9 cm in diameter were considered appropriate for low-frequency US treatment. The time required for tumors to reach a size of 0.8 cm ranged from 12 to 13 days.
Experimental protocol
PEI protocolAfter successful anesthesia, the hepatic tumors were injected with ethanol twice per week for 2 weeks. PEI was performed with a fine needle (21G × 180 mm, puncture needle, Ba guang company, Japan) guided using an ultrasound machine (iU22 ultrasound system, Philips, Bothell, Washington, USA) with a 12–15-MHz broadband linear probe. The concentration of ethanol was standardized to 95 %.The general guideline for the required injection volume was calculated according to the following numerical expression V = (4/3) π(r + 0.5)3, where V (in mL) is the volume of ethanol and r (in cm) is the radius of the tumor; the constant 0.5 was added to provide a safety margin, which is based on the concept that some surrounding liver parenchyma around the tumor must be ablated along with the tumor itself (Shiina et al. 2012).USMB protocolThe hepatic tumors were sonicated using a focused low-frequency US transducer manufactured by Taizhou Research Institute of Ultrasound Technology in Jiangsu Province, China. The probe was placed on the shaved abdominal skin of the rabbits, with an US transmission gel (A lotus medical ultrasonic coupling agent, Yi Jie Guangzhou Pharmaceutical Technology Co., Ltd, China) being interposed to ensure US propagation. The low-frequency US parameters were set to 20 kHz, 2 W/cm2, duty cycle 40 % (on 2 s, off 3 s), and a duration of 5 min once every other day for 2 weeks. The MBs used were an ultrasound contrast agent (SonoVue: Bracco SpA, Milan, Italy). The MB contrast agent was administered simultaneously with US wave irradiation via the auricular vein by continuous infusion (the flow rate was 0.2 mL/min). The method has been previously described elsewhere (Shen et al. 2014).
MRI protocol
The liver tumor size was measured on day 0 of tumor treatment and 28 days after tumor treatment using a horizontal open MRI system (1.5 T coil, Siemens Espree, Germany). To obtain these images, we used a spin-echo pulse sequence T1-weighted image (time to repeat 117 ms, time to echo 4.76 ms, field of view 150 mm, matrix size 224 × 192, slice thickness 6 mm, interslice gap 1 mm). Pre-contrast axial acquisitions included a T2-weighted fast spin echo with fat suppression, breath-free diffusionweighted echo planar images (matrix 128 × 128; slice thickness 6 mm; interslice gap 1 mm; B values 0 and 500; repetition time 3800 ms; echo time 108 ms; and receiver bandwidth 64 kHz), and three-dimensional T1-weighted spoiled gradient echo with and without fat suppression. All animals received a 0.025-mmol dose of 0.25-mol/L GdEOB-DTPA per kilogram of body weight (Schering AG, Berlin, Germany) intravenously at a speed of 2 mL/s. The line was flushed with 5 mL of 0.9 % saline. Dynamic imaging was performed in the arterial (2–5 s after injection) and portal venous phases (20 s after injection) using a dynamic three-dimensional T1-weighted GRE sequence without fat suppression. In addition, delayed T1-weighted GREs with fat suppression were performed 10 min after contrast administration. When measuring tumor size on MR images, the two radiologists were blinded to the groups and exposure to treatments.
Survival time follow‑up
Survival time was calculated based on the date of tumor incubation and date of death. All animals were followed for 180 days. Animals that survived to the defined end point of 180 days after implantation were killed. During the follow-up period, autopsies were performed 8 h after the animal keeper’s report. Finally, an anatomic evaluation was performed for all rabbits. The ablation area in the rabbit hepatic tumors was observed, and metastasis of other organs, including the liver, peritoneum, mesentery, pelvis, and lungs, was identified. The number of rabbits with metastases was calculated for each group.
Statistics
Statistical analysis was performed using SPSS version 11.0 (SPSS Inc.). Student’s t test for tumor size was used to make a statistical comparison between the groups. For the metastasis sites in each group, a Chi-square test was performed using Cochran–Mantel–Haenszel statistical analysis. Survival curves were calculated using the Kaplan–Meier method and were analyzed using the log-rank test. All testing was performed using Prism 3.0 (GraphPad, San Diego, CA, USA). Error bars were displayed as the standard error above the mean. Statistical significance was determined using p < 0.05.
Results
PEI procedure results
PEI of the hepatic tumor was performed by US-guided 21G needle puncture. The appropriate dose of ethanol in the 10-mL syringe was injected into the rabbit hepatic tumors slowly over a period of 30–60 s. When the ethanol was injected inside the tumor, a high-echodensity liquid bubble was visible by US originating on the needle tip and spreading around the tumor. When the high-echodensity bubble completely covered the tumor and expanded to the tumor margin by at least 0.5 cm, the injection was stopped. After injection, the needle remained inserted for a few seconds and then was pulled to within 1–1.5 cm of the tumor’s edge. No liquid reflux was observed on the monitor, and then the needle was withdrawn completely. Gauze was used to cover the puncture site, an abdominal bandage was placed, and the rabbit was returned to the animal room.Reaction of animals: Ethanol injection in the PEI and PEI + USMB groups occurred without any problems. Because a larger amount of alcohol was used in the USMB + PEI group, generally up to 7.4 mL, even with good initial anesthesia, the rabbits became uncomfortable during alcohol injection. In one case, the rabbit appeared intolerant to the procedure, even after increasing the anesthetic drugs; the procedure was finally completed successfully.Amount of alcohol injected: The average amount of ethanol injected into each liver neoplasm in the PEI group was 4 ± 0.3 mL; the average amount in the PEI + USMB group was 4.2 ± 0.4 mL. The tumor in the USMB + PEI group grew slowly during the first 2 weeks after USMB
Fig. 1 Representative MRI images of tumors (arrows) before and after treatment of rabbits in the PEI, USMB + PEI, and PEI + USMB groups
was performed, the average tumor volume reached 2 cm3, the diameter reached 1.3 cm, and the subsequent average alcohol dose was 5.8 ± 1.6 mL.
MRI results
MRI features: The rabbit liver tumors observed by T1-weighted MRI showed a low signal, enhanced tumor peripheral ring signal intensity, and low central signal. After treatment, the tumor showed a low signal on T1-weighted imaging. The tumor size changed. Among the various groups, the PEI-only group showed tumor shrinkage. Tumors in the USMB + PEI group increased in size compared to tumors examined within the first week of treatment, whereas tumors in the PEI + USMB group shrank significantly. After 4 weeks of treatment, the MRI results indicated that the tumor diameters in the PEI, USMB + PEI, and PEI + USMB groups were 8.33 ± 1.83, 19 ± 2.61, and 4.5 ± 1.22 mm, respectively. There were significant differences in tumor size between the three groups. Tumor size was smaller in the PEI + USMB group than in the PEI and USMB + PEI groups (t = 4.54, p = 0.0062 and t = 16.38, p < .0001, respectively) (Figs. 1, 2).
Metastatic results
Tumor metastatic sites and the number of rabbits are shown in Table 1. The PEI + USMB group showed the fewest metastases, compared to the other two groups (χ2 = 11.7333, p = 0.0194).
Fig. 2 Tumor size in each group by MRI. The tumor size significantly differed between the PEI + USMB group and the other two groups (triangle p < 0.05 vs. PEI; star p < 0.05 vs. USMB + PEI)
Survival results
Two rabbits in the PEI group lived up to 180 days; the survival times for the remaining four cases were 88, 98, 128, and 143 days, respectively. The survival times for the USMB + PEI rabbits were 76 88, 103, 104, 128, and 132 days, respectively. The PEI + USMB group had three cases that survived for 180 days; the remaining survival times were 124, 148, and 154 days. There were significant differences in survival time in the three groups, and the PEI + USMB group showed the longest survival time of the three groups, with χ2 = 7.448, p = 0.0241 (Fig. 3).
Discussion
This study found that anhydrous alcohol or ethanol injection combined with low-frequency ultrasound could inhibit rabbit liver tumors and significantly prolong survival.The mechanisms of synergistic role of combination therapy of PEI + USMB are as follows. Anhydrous alcohol leads to liver cancer tissue coagulative necrosis, and followed USMB damages the internal blood vessels of residual tumor. Ultrasound guidance accurately delivered anhydrous alcohol into the tumor, and the drug concentration reached a high level inside the tumor within a short time, resulting in rapid tumor protein dehydration, solidification, degeneration, and necrosis of the tumor cells and vascular endothelial cells (Lin and Lin 2006). Inert cavitation effect of the USMB plays an important role in the vascular destruction. Firstly, the cavitation threshold reduced as the MBs were infused to aerial vein of rabbits (Maxwell et al. 2013). Sonicated by US, the MBs excited seriously and inert cavitation produced (Shen et al. 2015), which could lead to blood vessel rupture, thrombosis, endothelial cell injury, and apoptosis of tumor cells (Shen et al. 2014).Secondly, 20-kHz ultrasound combined with microbubbles could download the expression of VEGF, COX-2 (Shen et al. 2013, 2014; Wood and Sehgal 2015), and CD31 (Huang et al. 2013) in the tumor tissues. Thirdly, anti-vascular ultrasound therapy could even induce the systemic post-therapy immune response against the cancer cells. For example, it was reported that after USMB treatment, one kind of lymphocyte-mediated inflammation was incited by the presence of T lymphocytes within the tumors (Hunt et al. 2015). Finally, as the velocity of blood flow in the tumor neovasculature was slower than that in the vessels of the normal contiguous tissues, the bio-effects within a neoplasm resulting from the interaction between US and MBs were so much greater than those within the adjacent normal tissues (Wood and Sehgal 2015). Thus, the therapy technique accurately targets the neovasculature of the tumor while sparing the normal blood vessels in the adjacent healthy tissues. All these mechanisms may be used to explain the synergistic role of PEI + USMB leading to vessel damages and tumor inhibition.MRI showed that, in the USMB + PEI group, tumor inhibition was not as good as that in the PEI + USMB
Fig. 3 Rabbits treated with PEI + USMB showed significantly longer overall survival than those in the PEI and USMB + PEI groups (χ2 = 7.448, p = 0.0241)
group. Moreover, the survival time in the former group was relatively short. One reason for this finding is that low-frequency ultrasound combined with microbubbles mainly inhibits the formation of tumor blood vessels; however, cancer cells are not completely killed, and slow tumor growth still occurs. Additionally, at 2 weeks, the tumor volume was approximately 2 cm3. Compared to the PEI and PEI + USMB groups, the USMB + PEI group began treatment with a larger tumor size, leading to a higher ethanol concentration requirement. Due to increased pain, the rabbits tolerated the procedure poorly, with one rabbit even requiring more anesthesia.In contrast, in the PEI + USMB group, we first performed ethanol injection, leading to better tumor control compared to that achieved for the USMB + PEI group, and less ethanol was used. With subsequent USMB irradiation, tumor microvessel proliferation was then further blunted, inhibiting tumor growth. Therefore, the best control group was PEI + USMB. In the PEI + USMB group, the rabbits showed the longest survival period (Fig. 3), with three cases surviving to 180 days. In the absence of our defined end point based on institutional regulations, the rabbits would have continued to live. Moreover, after autopsy, the rabbits in the PEI + USMB group showed the fewest metastases to other areas, such as the abdomen, pelvic cavity, and pleura (Table 1). Therefore, we believe that PEI + USMB had a significant inhibitory effect on rabbit liver tumors.There are two limitations in this study. First, there were not enough rabbits included in our research and this probably led to selection bias. This was mainly due to the fact we had limited financial support, including complex therapeutic plans (PEI and USMB treatment), expensive examination (SonoVue and MRI application), and a long animal breeding (the longest time to 200 days). Second, in this study, we performed the PEI and USMB irradiation treatments for small tumors, due to the small livers and thin liver parenchyma found in rabbits. Therefore, in this study, only tumors measuring 1–2 cm could be studied. If the tumor were to grow larger, the tumor would develop internal necrosis and metastasis to other organs. In addition, an intolerable amount of alcohol would be needed. Therefore, if the larger tumors are to be ablated, larger experimental animals, such as dogs or pigs, may be needed.In conclusion, this study found that ethanol injection combined with ultrasound irradiation had a good effect on the inhibition of rabbit hepatic tumors. Anhydrous alcohol could cause rapid tumor protein necrosis and microvascular closure, and then auxiliary low-frequency ultrasound further led to tumor capillary destruction, reducing the tumors』 blood supply. PEI followed by USMB better inhibited tumor growth and prolonged survival of the rabbits.
Funding This study was partially supported by Nantong Municipal Science and Technology Project (Grant No. HS149072), Nantong Municipal Youth Fund Project (Grant No. WQ2015054), and the National Natural Science Foundation of China (Grant No. 81271597).
Compliance with ethical standards
Conflict of interest The authors have no conflicts of interest to declare.
Ethical approval All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
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