Percutaneous microwave coagulation for eradication of VX2 tumors subcutaneously in rabbits

Animal experiments were approved by the Animal Care and Use Subcommittee at our University. Adult female New Zealand White rabbits weighing between 2.0 and 3.0 kg were used in this study. The VX2 cell line, used for tumor implantation, was obtained from the Surgery Department of our Hospital. The rabbits were anesthetized using an intravenous injection of pentobarbital (30 mg/mL, 0.8 to 1.2 mL/kg, pentobarbital sodium; Sigma, St, Louis, MO, USA) before the VX2 tumor inoculation and other procedures. The required tumor tissue, taken from a tumor carrier rabbit, was cut into small strips (1.5 × 1.5 × 6 mm). Each strip of tumor tissue was inserted subcutaneously underneath the right second nipple by a 16-gauge needle. The animals were followed up every week by physical examination. The tumor growth was 100% in the implantation sites. When the animals refused solid and fluid intake for more than four days with concomitant apathy and weight loss of more than 20%, the animals were sacrificed for ethical reasons [29].

The larger and shorter diameters of the tumor were measured using a caliper. After the larger diameter of the tumor reached about 15 mm (10 to 21 days), 15 rabbits were included in this study with the following treatments: PMC group (n = 9), nine rabbits with nine VX2 tumors were treated with PMC and control group (n = 6), six rabbits with six VX2 tumors served as untreated controls.

After the same anesthesia as in tumor implantation, the rabbits were positioned supine. The cooled-shaft antenna (2 mm in diameter) was inserted into the tumor along the long axis without any navigation. This microwave delivery system consists of a microwave generator, a flexible coaxial cable and an internally-water-cooled-shaft antenna. The microwave irradiation frequency is 2,450 MHz. An output power of 40 W was chosen in this study. After testing the cold water (4°C) cycling system, the PMC procedure was started for one to two minutes to eradication of the entire tumor according to the tumor size [8, 11‐13] and our own experience. Since the larger diameter of all the tumors was less than 2.5 cm, two minutes may be enough. Therefore, when the shorter diameter of the tumor was shorter than 1.5 cm, one minute was chosen. When the shorter diameter of the tumor was shorter than 2.0 cm, one and a half minutes was used. When the shorter diameter of the tumor was shorter than 2.5 cm, two minutes was chosen in our study.

After PMC treatment, the antenna was removed and the incision closed. All the rabbits were monitored during the PMC treatment and for one month after surgery for any complications that might be induced by the intervention. The degree and extension of the burns to the skin were evaluated. The behavior of the rabbits after the treatment was observed. Furthermore, the weight and food intake of the rabbits were monitored.

The efficacy of PMC therapy was evaluated with survival rate, MRI, physical examination of the coagulated tumors and histopathology. MRI was performed to monitor the long-term results of the coagulated tumors at day 0, week 1, week 3 and week 7 after PMC therapy. Core needle biopsy was randomly performed on two rabbits for histological examination at week 1 and week 3 after PMC therapy. Since there was little tissue under the skin at week 7, core needle biopsy was not performed at this time-point. Incomplete ablation was determined by core needle biopsy or MRI follow-up without treatment control. When the tumors were not reabsorbed with local growth or the needle biopsies were positive, the tumors were recognized as incomplete ablation. Survival rate was used to assess the therapeutic effect between the two groups.

Prior to MRI scanning, the animals were anesthetized by the procedure described as in tumor implantation to decrease respiratory rates for clear images. The animals were strapped but not intubated to reduce motion artifacts. Respiratory-triggered sequence with prospective acquisition correction was applied to MRI examination for respiration control. Rabbits in the PMC group were subject to three Tesla (T) MRI examinations (Trio, Siemens Healthcare) at pre-microwave therapy, one week, three weeks and seven weeks post-PMC therapy. T2-weighted 2D fast spin-echo (FSE) sequences were acquired in the axial plane using a phased-array coil. The 2D FSE sequence was performed with the following parameters: TR, 3,948 to 4,960 milliseconds; TE, 80 to 89 milliseconds; matrix size (168 to 256) × 320; field of view, (135 to 150) × (150 to 180) mm; slice thickness, 4 mm; bandwidth, 205 to 260 Hz; and acquisition time, 104 to 150 seconds. The techniques of MRI were the same during the procedure.

The animals were autopsied within six hours after death. The tissue specimens were fixed in 10% formalin solution, embedded in paraffin, sectioned into 4 μm slices, and stained with H & E. The histological slides were evaluated by one experienced pathologist.

All rabbits in the PMC and control groups tolerated the experimental procedures. All the animals recovered well after the experiment. Although output power was only 40 W, epidermal burns, measuring 0.5 to approximately 1 cm in the greatest dimension, were found in seven rabbits in the PMC group. However, no necrosis of the skin was observed. Due to a high rate (7/9, 77.8%) of epidermal burns, it seems a very likely occurrence. No other immediate adverse effects occurred in all rabbits. Only about ten minutes was needed for the total PMC procedure.

The detailed information is shown in Table 1. The rabbits in the control group without PMC therapy died of end-stage malignancies (mean 47.7 ± 14.2 days). Five rabbits in the control group showed pulmonary metastases (Figure 1). In the PMC group, the mean greatest diameter of the tumor was 1.72 ± 0.36 cm (range, 1.5 to 2.5 cm) before PMC therapy. The mean coagulation time was 1.06 ± 0.17 minutes (range, 1.0 to 1.5 minutes). Tumor eradication was achieved in six rabbits (66.7%) without any evidence of tumor recurrence and metastasis. The mean greatest tumor diameter of these six rabbits was 1.83 ± 0.41 cm (range, 1.5 to 2.5 cm), and the short diameter was 1.33 ± 0.41 cm (range, 1.0 to 2.0 cm) before PMC treatment. Of these six rabbits, two rabbits died on the 16th and 34th day after PMC therapy without evidence of tumor recurrence or metastasis. An infectious disease, which may be caused by the PMC therapy, induced the death on day 16 after PMC treatment. Diarrhea, which is one of the most common causes of death in immature rabbits, induced the death on day 34 after PMC therapy. The other four rabbits survived longer than three months free of disease, of which three rabbits survived longer than six months. In contrast, no rabbits in the control group survived longer than 70 days. Survival analysis showed that rabbits in the PMC group had a significantly higher survival than those in control group (P = 0.0097) (Figure 2).

Tumor palpability was evaluated by physical examination every week. One week after PMC, the size of the ablated area was larger than before. Except for three rabbits with local recurrence, the tumors treated with PMC in the other six rabbits were progressively absorbed. Of these six rabbits, four survived longer than three months. All the tumors of these four rabbits became nonpalpable in three months after PMC therapy. The epidermal burns in the rabbits were healed after approximately 2 to 3 weeks.

Progressive resorption was also detected by MRI. The details are shown in Figure 3. In accordance with physical examinations, the image at one week (Figure 3B) after PMC therapy showed obvious edema and hyperemia around the coagulated tumor. At week three after PMC therapy (Figure 3C), the edema and hyperemia of the lesion was partly absorbed and the size of the coagulated area was much smaller than that at week one. The coagulated VX2 tumor was absorbed completely at week seven after PMC therapy (Figure 3D).

Local recurrence was detected in three rabbits in the PMC group by MRI (Figure 4). At one week after PMC therapy, no obvious local recurrence was detected by physical examination and MRI (Figure 4B) due to obvious edema and hyperemia. However, local recurrence was detected by MRI at week three after PMC therapy, but it was not very clear (Figure 4C). At week seven after PMC therapy, local recurrence was very obvious (Figure 4D). Of these three rabbits with local recurrence, metastases were observed in two rabbits. The first metastasis was detected by MRI 35 days after PMC therapy.

Histologically, the coagulated tissues appeared almost unchanged after PMC therapy, especially at week one (Figure 5A). At the H & E staining, the coagulated tumors looked almost intact in tissue architecture and were stained a little lighter than viable tissues. The outline of the coagulated tumor cells was still clear, but the nuclei were fractured. Some apoptotic bodies were observed in the area, the coagulation of which was insufficient. The cell architecture in the viable area was clearer at week three than week one. The difference between the coagulated tumors and viable tissues was more obvious at week three than week one (Figure 5). A sharper demarcation between the coagulated tumors and viable tissues was observed at week three after PMC therapy than at week one.