Repeated inductive heating using a sintered MgFe₂O₄ needle for minimally invasive local control in breast cancer therapy

Abstract

Purpose: This study investigated the efficacy of repeated thermotherapy for breast cancer utilising a novel sintered MgFe₂O₄ needle and alternating current (AC) magnetic field in xenograft animal models mimicking human breast cancer.

Materials and methods: A sintered MgFe₂O₄ needle and an apparatus to apply an AC magnetic field were prepared for this study. Animals bearing BT-474 tumours (mean (±standard deviation) volume, 471 ± 153 mm³) were divided into four groups. A sintered MgFe₂O₄ needle (length, 5 mm) was placed in the centre of each tumour. An AC magnetic field (amplitude, 4 kA/m; 2 kW; 540 kHz) was applied for 10 min once, twice or three times for the first, second and third groups, respectively, and was not applied for the control group. Temperature during treatment and tumour volume 8 weeks after first treatment were assessed.

Results: Maximum tumour temperature tended to increase in repeated-application groups: group 1, 59.2 ± 4°C; group 2, 58.9 ± 3.3°C and 61.2 ± 8.9°C for the first and second applications; and group 3, 60.4 ± 4.6°C, 62.1 ± 7.8°C and 71.1 ± 6.1°C for the first, second and third applications. Tumour volumes in control, groups 1, 2 and 3 at 8 weeks after treatment were 3633 ± 2478 mm³, 3240 ± 1031 mm³, 1252 ± 1289 mm³ and 0 mm³, respectively. Tumours were significantly smaller in group 3 than in the control and group 1 at 8 weeks.

Conclusions: The efficacy of repeated inductive heating utilising a sintered MgFe₂O₄ needle was demonstrated. Thermotherapy using the present method may offer an effective non-surgical treatment for human breast cancer.

• Inductive heating
• thermotherapy
• hyperthermia
• breast cancer

Introduction

In the treatment of breast cancer, improvements in chemotherapy, radiotherapy and other treatments have enabled increasingly less invasive surgical treatment. In recent years, use of radiofrequency ablation (RFA) for local control of breast cancer has been proposed [1–7]. Side effects of this thermotherapy are limited to local complications such as minor burns and slight bruising [1], [2]. Attractive results have been achieved in these studies and the feasibility of thermotherapy against breast cancer has been identified.

We have previously reported another method of thermotherapy using inductive heating [8–10]. We investigated inductive heating with various kinds of metals and identified MgFe₂O₄ as offering the most effective heating. This material was then formed into a needle shape for easy and long-term placement within the target. We have also developed a new apparatus for use with the sintered MgFe₂O₄ needle in this ablation therapy. Conventional heating methods such as RFA involve dielectric heating, utilising thermal heating from friction produced by a high-frequency alternating current (AC) delivered into tissue, causing excitation and motion of intracellular ions. The tissue surrounding the electrode, rather than the electrode itself, is the primary source of heat [11] (Figure 1A). Inductive heating utilises the heat produced by the hysteresis loss effect within the magnetic material itself when an alternating magnetic field is applied externally. Rather than the tissue surrounding the magnetic material, the material itself forms the source of heat (Figure 1B). This represents the essential difference between dielectric and inductive heating.

Figure 1. (A) Dielectric heating. Rather than the applicator, tissues around the applicator produce heat by friction of ions agitated by a high-frequency electric current. (B) Inductive heating. The magnetic material in the target itself produces heat by the hysteresis loss effect.

With inductive heating, once the magnetic material is placed in the target, the target has only to move into an alternating magnetic field to receive treatment. This means the patient can receive treatments repeatedly simply by moving into an alternating magnetic field, with no need for further puncture or contact as required in conventional thermotherapies. This characteristic of inductive heating may provide patients with less invasive tumour control.

The objective of this study was to evaluate the efficacy of repeated inductive heating using a sintered MgFe₂O₄ needle in xenograft animal models mimicking human breast cancer.

Methods

Animals

Female immunodeficient mice (4–6 weeks old) were obtained from Charles River Japan (Yokohama). All animals were treated in accordance with the guidelines for animal research of Ehime University School of Medicine. Mice were anaesthetised using intraperitoneal administration of 50 µg/g pentobarbital (Nembutal; Dainippon Sumitomo Pharma, Osaka).

Human breast cancer cells from the BT-474 line (JCRB Cell Bank, Osaka) were used. A suspension of 1 × 10⁶ BT-474 cells in 0.1 mL of phosphate-buffered saline was injected subcutaneously into the flank of a mouse using a 27-G needle. After 12 weeks, formation of a subcutaneous tumour 2 cm in diameter was confirmed. The tumour was resected and cut into 2-mm blocks. Blocks were inoculated into the right flank of 30 mice through small skin incisions. The study was started after 6–8 weeks, once the tumour had exceeded 10 mm in diameter (mean diameter, 11.8 ± 1.6 mm). Mean weight of mice at this stage was 23.8 ± 2 g.

MgFe₂O₄ needle and electromagnetic heating device

MgFe₂O₄ particles (mean particle size, approximately 1 µm) were purchased from Wako Pure Chemicals (Osaka). Particles were forced into a thin quartz tube before sintering at 1100°C for 27 h. The completed MgFe₂O₄ needle is 1 mm in diameter and contains 2.21 mg of MgFe₂O₄/mm (Figure 2A). The entire manufacturing process was performed by the Department of Materials Science in the Faculty of Engineering at Ehime University.

Figure 2. (A) The MgFe₂O₄ needle (diameter, 1 mm; length, 5 mm; containing 2.21 mg of MgFe₂O₄/mm). (B) The device for applying alternating current (AC) magnetic fields comprises loops of copper pipe connected to a high-frequency power supply through an adjuster. Loops are cooled from within by flowing water in a cooling unit, so that the coil can consistently produce reproducible magnetic fields.

The device for applying an AC magnetic field comprised loops of copper pipe connected to a T162-5712B high-frequency power supply (Thamway, Shizuoka, Japan) through an adjuster. Loops were cooled from within by flowing water in a CLU33 cooling unit (Iwaki, Tokyo), so that the coil could produce consistently reproducible magnetic fields (Figure 2B).

Elevation of temperature at the centre of the tumour was monitored using an AMOTH FL 2000 sensor FS100-M fluorescence optic fibre thermometer (Anritsu Meter, Tokyo).

Inductive heating

A total of 30 tumour-bearing mice were divided into four groups. MgFe₂O₄ needles were placed in the centre of the tumour. All animals were anaesthetised and placed in the centre of the loops for 15 min 3 times at 24-h intervals. An AC magnetic field of amplitude 4 kA/m (2 kW, 540 kHz) was applied for 10 min once for group 1 (n = 7), twice for group 2 (n = 8) and 3 times for group 3 (n = 7), and was not applied for the control group (n = 8). One animal that died from anaesthesia in group 2 was excluded from the study.

Tumour volume

Tumour volume before and 8 weeks after first treatment was calculated as an elliptocyte according to the following formula:where a and b represent the long and short diameters, respectively, and c is the diameter of the vertical axis.

Microscopic observations

Animals were sacrificed 8 weeks after the first treatment. Tumours or residual tissue were fixed in 10% formalin and embedded in paraffin for 24 h. Paraffin-embedded sections were cut at a thickness of 4 µm, dewaxed, dehydrated and stained using haematoxylin and eosin (HE). Sections were then examined for microscopic evidence of residual viable cancer cells.

Statistical analysis

All values are expressed as mean ± standard deviation. Comparison of maximum tumour temperatures in groups 2 and 3 were made using a two-sided Wilcoxon's signed-rank test and Kruskal-Wallis test with Dunn's multiple comparison test. Tumour sizes at 8 weeks were compared using the Kruskal-Wallis test with Dunn's multiple comparison test. Differences of P < 0.05 were considered statistically significant in all tests.

Results

Elevation of temperature

Baseline tumour temperature was approximately 32°C before treatment. After starting AC application, temperature plateaued within a few minutes and fell immediately to baseline level after the end of application. Maximum temperature in group 1 was 59.2° ± 4.0°C. In group 2, maximum temperatures were 58.9° ± 3.3°C and 61.2° ± 8.9°C for the first and second applications. In group 3, maximum temperatures were 60.4° ± 4.6°C, 62.1° ± 7.8°C and 71°.1 ± 6.1°C for the first, second and third applications. No significant differences were seen between first and second applications in group 2 or first, second and third applications in group 3 (Figure 3).

Figure 3. Maximum temperature was 59.2° ± 4°C in group 1, 58.9° ± 3.3°C and 61.2° ± 8.9°C for the first and second applications in group 2, and 60.4° ± 4.6°C, 62.1° ± 7.8°C and 71.1° ± 6.1°C for the first, second and third applications in group 3. Although no significant differences were seen between groups 2 and 3, maximum temperature tended to increase with increased application time.

Tumour volume

One animal in the control group died from tumour 5 weeks after first application. Tumour volumes in control, and groups 1, 2 and 3 were 438 ± 121 mm³, 461 ± 158 mm³, 515 ± 198 mm³ and 474 ± 86.0 mm³ before application, and 3633 ± 2478 mm³, 3240 ± 1031 mm³, 1252 ± 1289 mm³ and 0 mm³ at 8 weeks. Tumour volume was significantly smaller in group 3 compared with the control and group 1 (P < 0.01 each) (Figure 4).

Figure 4. Tumor volumes at 8 weeks after treatment in control and groups 1, 2 and 3 were 3633 ± 2478 mm³, 3240 ± 1031 mm³, 1252 ± 1289 mm³ and 0 mm³. Volume was significantly smaller in group 3 than in the control and group 1 (p < 0.01, Kruskal-Wallis test with Dunn's multiple comparison test).

Microscopic observation

No viable tumour tissue was found in four animals from group 2 and all animals from group 3 (Figure 5), apparently indicating complete tumour regression.

Figure 5. HE sections from group 3 showing no residual tumour 8 weeks after treatment.

Discussion

The method we used in this study involves inductive heating. The heating principle in this method differs from the prevailing method of dielectric heating employed in RFA and microwave coagulation therapy (MCT). In dielectric heating, heat is produced by friction from the excitation of ions or water molecules surrounding the needle-shaped applicator positioned within the target. In comparison, the magnetic material itself achieves heating effects when entering a magnetic field in inductive heating. Hysteresis loss is thought to be greatly involved in the heating effect, although the precise mechanisms remain unclear. Using this method, repeated episodes of thermotherapy can be provided without subsequent contact with the body.

The first trial of inductive heating using iron oxide and an AC magnetic field was reported by Gilchrist in 1957 [13]. Subsequent experimental studies have reported using inductive heating as a method of local control for cancer. Almost all of these studies used FeFe₂O₄ as the metallic magnetic material, reaching temperatures of 42°–48°C by applying stronger AC magnetic fields with longer application times than those used in the present study [14–25]. Our previous study determined the heating ability (W/g) of various ferrite powders by placing 1 g of each powder into 10 mL of water under an alternating magnetic field [10]. This alternating magnetic field (370 kHz, 4.0 kA/m) was produced using a copper pipe 6 mm in diameter connected to a power supply through an impedance tuner. Heating abilities of MFe₂O₄ where M = Mg, Mn, Fe, Co, Ni, Cu and Sr were 1.44, 0.31, 0.16, 0.20, 0.41 and 0.45 W/g, respectively. That study identified MgFe₂O₄ as the most effective material for inductive heating, raising therapeutic temperature and reducing the time required in the AC magnetic field. Using this material with high potential for temperature elevation, inductive heating with a weaker AC magnetic field and shorter duration of application can be achieved. Another advantage of using MgFe₂O₄ is that this material has a lower possibility of further oxidisation than FeFe₂O₄. Ion oxidation and long-term insertion in the body may cause adverse events such as surrounding tissue injury, elution of materials into the circulation and finally reduced heating efficacy. For this reason, MgFe₂O₄ may provide equal or greater efficacy of heating after long-term placement, representing a potential advantage with repeated inductive heating.

The present study showed the efficacy of repeated treatment, with three applications at 24-h intervals appearing sufficient to achieve complete local control. Some studies have shown the efficacy of multiple thermal cycles compared to a single continuous session in vivo [20],[25]. The interval in those studies was set at 24 h, but the ideal interval remains unclear. We applied the alternative magnetic field with a 24-h interval on the basis of convenience for outpatients in clinical use. A preliminary study comparing double thermal sessions with 24-, 72- and 120-h intervals (data not shown) suggested the 24-h interval as the most effective for BT-474 tumour models. When we consider clinical use in outpatients, triple sessions with intervals of 6- or 12-h may prove impractical for use on an outpatient basis.

Maximum temperature during the treatment tended to rise with increasing treatment time. Friedman et al. suggested that the decrease in tumour blood flow makes following treatment more effective in combination therapy using radiotherapy and RFA [26]. The tendency for temperature increases seen in our study may be attributable to the same mechanisms. This may indicate that when a single session is insufficient, another session may complete total ablation of the tumour. Although conventional thermotherapies (RFA or MCT) require additional puncture for each session, our method has no such requirement once the needle has been placed.

Complete removal of the needle may be required in clinical use. Ohno et al. [20] formed a stick-type FeFe₂O₄ by mixing with carboxymethylcellulose for inductive heating. After thermotherapy, they showed diffused distribution of FeFe₂O₄ particles throughout the tissue. This diffusing characteristic of the stick may be advantageous in achieving diffused heat effects, but removing the material may be impossible. Our MgFe₂O₄ needle does not dissolve within the target, was easily removed and did not move until removal was performed. Although our HE sections showed no marked presence of residual particles in surrounding tissues, coating the needle in another material may facilitate complete removal of the needle.

In the future, a study using larger animals and larger tumours may be required for clinical use. Using multiple needles or applying the AC magnetic field for longer or more frequently may allow control of larger tumours. In this study, almost all animals developed skin burns over the tumour (data not shown). These lesions healed within 2 weeks, but may represent an issue in human patients in terms of cosmetic results. Animals in this study were very small, about 24g in weight. This may be the reason for severe skin burns. Monitoring of skin temperature during treatment by infrared thermometer showed a selective temperature elevation (Figure 6). Controlling the strength of the alternative magnetic field under a strict monitoring of skin temperature or active skin cooling may therefore help to avoid this problem.

Figure 6. Monitoring of skin temperature during treatment by infrared thermometer shows a selective temperature elevation.

This treatment appears to offer an effective, less invasive thermotherapy for clinical use against breast cancer.

Conclusion

This study showed that repeated inductive heating using a sintered MgFe₂O₄ needle achieved effective local control of breast cancer in xenograft animal models mimicking human breast cancer. This heating method may well become a treatment of choice for local control of human breast cancer in future.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.