|Year : 2018 | Volume
| Issue : 8 | Page : 152-158
The effect of temperature-control microwave on HELA and MG-63 cells
Zhenwei Ji1, Yunlei Ma1, Haien Zhao2, Wei Li1, Xiaoxiang Li1, Zhe Yun1, Guangyi Zhao1, Baoan Ma1, Qingyu Fan1
1 Department of Orthopedic, Orthopedic Oncology Institute of Chinese PLA, Tangdu Hospital, The Fourth Military Medical University, 710038 Xi'an, Shaanxi, China
2 Xi'an Research Institute of Hi-Tech, 710025 Xi'an, Shaanxi, China
|Date of Web Publication||26-Mar-2018|
Orthopedic Oncology Institute of Chinese PLA, Tangdu Hospital, The Fourth Military Medical University, 710038, Xinsi Road, Xi'an, Shaanxi
Source of Support: None, Conflict of Interest: None
Context: Hyperthermia has now been used to treat many kinds of solid malignancies. However, the applied thermal parameters about heat temperature and time varied all over the world, and no consensus about the optimal formula had been reached. Microwave ablation, as one of thermal ablation methods, is usually applied based on the fixed parameters of power and duration. As a result, too high temperature or overheating might not be avoided and excessive heating might cause some additional side effects to normal tissues.
Aims: To explore the optimal parameters of power and duration for the HELA and MG-63 cells in vitro.
Settings and Design: With a temperature-controlled microwave workstation, a microwave thermal ablation experiment was performed in vitro.
Subjects and Methods: The HELA and MG-63 cells were heated with 40°C, 45°C, 50°C, 55°C, and 60°C lasting for 5–30 min, respectively. Then, the cell viability was detected using four methods: Flow cytometer assay, nicotinamide adenine dinucleotide-diaphorase staining, Calcein-acetoxymethyl ester staining immediately after treatment, and CCK-8 assay 24 h later.
Results: The temperature-controlled microwave has an excellent ablation effect on both cell lines. Furthermore, when the thermal stimulation reached 55°C 25 min and 55°C 20 min for the HELA and MG-63 cells, respectively, or 60°C 5 min for both, all the viability indexes indicated immediately devitalization.
Conclusion: It presented a preliminary minimum lethal dose of heat was validated on the cellular level in vitro, which should be verified and corrected further in vivo.
Keywords: Hyperthermia, osteosarcoma, tumor treatment
|How to cite this article:|
Ji Z, Ma Y, Zhao H, Li W, Li X, Yun Z, Zhao G, Ma B, Fan Q. The effect of temperature-control microwave on HELA and MG-63 cells. J Can Res Ther 2018;14, Suppl S1:152-8
| > Introduction|| |
It is generally known that thermal ablation, as an alternative to conventional surgical management, could obtain a targeted and complete killing of focal tumors while having little destruction to the surrounding healthy tissues. Presently, some kinds of thermal ablation techniques have been applied clinically, such as radiofrequency, laser, high-intensity focused ultrasound and microwave., Microwave ablation, as one of the ablation methods, has been accepted and used to treat many kinds of solid tumors involving liver, lung, kidney, and bone tumor.,,,,
Compared with other ablation techniques, the microwave ablation has several advantages including faster ablation times, induction of larger ablation volumes, simultaneously multiple applicators, higher ablation temperatures, optimal heating of cystic masses, and absence of impedance problem., Different from other thermal ablation techniques, the microwave can produce two kinds of biological effects, thermal effects and nonthermal effects., Although the nonthermal effects might be helpful for tumor treatment, presently, the thermal effects of microwave on tumor cells, mainly the cell-killing were still the most important treatment goal for thermal ablation. Furthermore, although both kinds of cell death, apoptosis and necrosis, could be induced by microwave,, clinically the immediately coagulation necrosis was the main treatment objective for the thermal ablation.
As for the treatment effects of thermal ablation, it was well-known that the thermal dose, which concluded the applied heating temperature and lasting time should play a decisive role. For thermal ablation, the recommended temperature and heating time should be as precise as possible. However, presently the applied thermal parameters about heat temperature and time varied greatly all over the world, and no consensus about the optimal formula had been reached.
The overall objectives of thermal ablation are to eradicate all viable malignant cells with a safe margin and minimize the damage to surrounding normal tissues. Clinically, microwave ablation is usually applied with fixed parameters of power and duration. As a result, the temperatures under the targeted tumor tissues were always found to be so high which might cause some adverse effects to the surrounding normal tissues. Therefore, to achieve the optimum therapeutic effect, an accurate combination of temperature and heating time is of first importance, which is also our research purpose.
In this study, with a temperature-controlled microwave workstation, an in vitro microwave thermal ablation experiment was performed. A series of gradually enhanced microwave-induced heating models, as the different combinations of temperature and duration, were applied on two kinds of cell lines, HELA and MG-63 cells. Then, the cell viability was detected with four methods: Flow cytometer assay, nicotinamide adenine dinucleotide (NADH)-diaphorase staining, Calcein-acetoxymethyl ester (Calcein-AM) staining immediately after microwave treatment, and CCK-8 assay 24 h later.
| > Subjects and Methods|| |
Cell lines and cell culture
Two human cell lines, HELA and MG-63, derived originally from cervical cancer tissue and osteosarcoma tissue respectively, were adopted in this study. HELA cells were routinely maintained in RPMI-1640 medium, and MG-63 cells in Eagle's minimal essential medium supplemented with 10% fetal bovine serum (FBS), 5% glutamine and without penicillin and streptomycin, in 5% CO2 humidified incubator at 37°C.
For the microwave treatment, a microwave workstation (MAS-II, Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China), which has a microwave source producing continuous waves at 2.45 GHz with powers between 25 W and 1 kW, was used. When a temperature was set, the workstation could maintain this temperature by self-regulation, whose error was <1°C.
Cells were washed twice with phosphate buffered saline (PBS). Then cells were trypsinized with 0.25% Trypsin solution and centrifuged at 1000 rpm for 5 min in room temperature. After two washings in respective medium without serum, the cells were re-suspended in serum-free medium to a final concentration of 1 × 106 cells/ml. For exposure to microwaves treatments, 1 ml aliquots of the cell suspension were placed in sterile glass ampoules. Moreover, each ampoule was immersed in a beaker filled with 30 ml respective medium without serum for the subsequent processes.
Cells at appropriate concentration were seed in 24-well plates, and coverslips were laid in each well in advance. After 24 h in the incubator, the coverslips were washed twice with PBS and twice with respective medium without serum, and then they were laid, respectively, on a glass brace in a beaker filled with 30 ml respective medium without serum for the subsequent processes.
The specimens were maintained at 25°C. The beakers with the specimens were placed individually in the center of the workstation cavity and exposed to microwaves at powers regulated automatically (300 W upper limit was set). We chose five temperatures, 40°C, 45°C, 50°C, 55°C, and 60°C to evaluate the microwave effects, and the heating time ranged from 5 min to 30 min with an interval of 5 min. The times needed to reach the preset temperatures above mentioned were 17 s, 22 s, 26 s, 32 s, and 36 s, respectively. The temperature observed was generally consistent when the microwave radiation was repeated. All materials used inside the workstation cavity were highly transparent to microwaves. After microwave radiation, the specimens for flow cytometer assay were stored at 4°C and others at room temperature for the following experiments.
Cell viability assay
Following microwave treatment, cell viability was determined by four methods: Flow cytometer assay, NADH-diaphorase staining, Calcein-AM staining immediately after treatment, and CCK-8 assay 24 h later.
Cells were removed from the ampoules. Hundred microliters of the suspension were mixed with 900 μl respective medium supplemented with 10% FBS. Each 100 μl of the new suspension was plated in 96-well plates for 24 h in 37°C humidified incubator, 5% CO2. After 24 h, the plates were performed with CCK-8 as the following protocol. Ten microliters of CCK-8 (CCK-8 kit, Dojindo, Japan) reagent were added to each well. Plates were incubated for 4 h in 37°C protected from light. Optical density was measured using a spectrophotometer at wavelength of 450 nm (reference absorbance was determined at a wavelength of 630 nm – nonspecific readings). To correct for background absorbance, prepare three control wells without cells for each group, and subtract the average absorbance of the control wells from that of the other wells. Cell viability was calculated as a percentage of the control group that was not subjected to MV treatment. This assay was based on the ability of dehydrogenases in metabolic active cells to reduce tetrazolium salt (WST-8) to yellow colored product (formazan), which was soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells was directly proportional to the number of metabolically active cells.
Flow cytometer assay
Cell suspensions were removed from the ampoule and washed cells twice with PBS. After incubated at room temperature for 15 min in the dark with 100 μl incubation buffer including 2 μl Annexin-V-fluorescein and 2 μl proidium iodid (Cat. No: 11988549001, Roche), the cells were analyzed by flow cytometer. According to the status of the two kinds of staining, the cells' conditions can be divided into Annexin-V(−)/propidium iodide (PI)(−), Annexin-V(+)/PI(−), Annexin-V(+)/PI(+), and Annexin-V(−)/PI(+). Among the four conditions, the Annexin-V(−)/PI(−) was usually considered as normal cells. Annexin-V was a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). This protein could be used as a sensitive probe for PS exposure upon the outer leaflet of the cell membrane and was therefore used to detect apoptotic cells. PI was a fluorescent, DNA-binding dye, which could easily penetrate into nonviable cells with damaged, permeable plasma membranes and could be excluded by live cells with intact plasma membranes. It had been widely used to assess the cell viability and label the nucleic acids of necrosis cells.
Nicotinamide adenine dinucleotide-diaphorase staining
Washing the cell-attached coverslips twice with PBS. For NADH-diaphorase staining, the coverslips were placed in incubation medium for 15 min under aerobic conditions at room temperature. The incubation medium consisted of 6.8 ml of reduced α-NADH-diaphorase (Sigma-Aldrich, USA) at a concentration of 1.5 mg/ml, 12.0 ml of nitroblue tetrazolium chloride (Sigma-Aldrich, USA) at a concentration of 2.0 mg/ml, 4.8 ml of PBS, and 3.8 ml of Ringer's solution. The pH of the medium was adjusted to 7.2 before incubation of the sections. After incubation, each coverslip was washed in distilled water for 2 min. After mounting with a glass slide, slides were evaluated for characterization of staining within 24 h of processing. Cell viability was determined on the basis of the reduction of nitroblue tetrazolium chloride, a redox indicator, by NADH-diaphorase. Viable cells express NADH-diaphorase, which causes them to have an intense blue cytoplasmic pigment. Intense blue cytoplasmic staining in the cells was considered to indicate viable cells, whereas the absence of blue cytoplasmic staining was taken as an indicator of nonviable cells. It had been mentioned that cell death could cause an immediate cessation of NADH-diaphorase activity, the blue cytoplasmic stain is absent in nonviable cells.
Calcein-acetoxymethyl ester staining
To wash the cell-attached coverslips twice with PBS. Dilute the Calcein-AM (Sigma-Aldrich, USA) stock solution in respective medium to make a 2 μM working solution immediately prior to use. Incubation the cover clips in the working solution for 15 min at 37°C, 5% CO2. After washing twice with PBS, the cover clips were detected under fluorescence microscopy using 490 nm excitation filters and a 515 nm emission filter. Calcein-AM was a nonfluorescent, hydrophobous compound that easily permeates intact, live cells. The hydrolysis of Calcein-AM by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that was well-retained in the cell cytoplasm. Green fluorescence staining in the cells was considered to indicate viable cells, whereas the absence of it was taken as an indicator of nonviable cells.
The data were reported as mean value ± standard deviation. Statistical differences between groups were analyzed by ANOVA test. Differences between groups were regarded as statistically significant if P was <0.05. All statistical analysis was performed with statistical analysis software (SPSS, version 17.0).
| > Results|| |
CCK-8 and flow cytometer assay
[Figure 1]a and [Figure 1]b summarized the CCK-8 results of HELA and MG-63, respectively, assayed 24 h after microwave treatment. The cell viability percentage was defined as the ratio of cell lines after microwave treatment and 24 h incubation to controls. The data were expressed as mean value ± standard deviation. When the temperature was set at 40°C, there was no statistically difference between the heated and control cells in viability, even if the duration of microwave treatment was extended to 30 min. At 45°C, the cell viability began to decline with the extension of heating time, and compared with MG-63, the cell viability of HELA had a relatively gentle decline. At 50°C, the cell viability of HELA declined drastically, and when the heating time extended to 25 min, it reached nearly 0% and did not fluctuate evidently even if along with longer heating time or higher heating temperatures. While the MG-63 was treated at 50°C, the cell viability decreased drastically 5 min later, and when the heating time extended to 20 min, the cell viability reached nearly 0% and did not fluctuate evidently. [Figure 1]c and [Figure 1]d summarized the flow cytometer results of HELA and MG-63, respectively. The Annexin-V(−)/PI(−) cells percentage were defined when compared with the total number of cells in each specimen. The data were reported as mean value ± standard deviation. For HELA at 40°C and 45°C and MG-63 at 40°C, the percentages of Annexin-V(−)/PI(−) cells were not statistically different from the control. While for HELA at 50°C and MG-63 at 45°C and 50°C, the percentages decreased significantly with the extended heating time. When the thermal dose parameter combination reached 55°C for 15 min for HELA cells and 55°C for 5 min for MG-63 cells, the Annexin-V(−)/PI(−) cells percentages did not change evidently even if treated with longer heating times or higher heating temperatures.
|Figure 1: Cell viability assayed by CCK-8 and flow cytometer after microwave heating. (a and b) HELA and MG-63, respectively, assayed by CCK-8 after 24 h incubation. (c and d) HELA and MG-63, respectively, assayed by flow cytometer immediately after heating. Error bars indicate the standard deviation, some of which are too low to detect|
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Nicotinamide adenine dinucleotide-diaphorase staining
[Figure 2] showed the results of NADH-diaphorase staining (blue color). [Figure 2]a, [Figure 2]b, [Figure 2]c presented the NADH-diaphorase staining of normal cells, 55°C for 5 min and 55°C for 25 min of HELA, respectively. While [Figure 2]d, [Figure 2]e, [Figure 2]f showed the NADH-diaphorase staining of MG-63 for three different state, normal, 55°C for 5 min and 55°C for 20 min, respectively. It could be observed that for both of the cell lines, the controls [Figure 2]a and [Figure 2]d showed intense blue cytoplasmic staining. However, when HELA and MG-63 were stimulated by 55°C for 25 min and 55°C for 20 min, respectively, the [Figure 2]c and [Figure 2]f showed no blue cytoplasmic staining, which indicated the inactivation of enzyme activity and cell death in consequence. For comparison, [Figure 2]c and [Figure 2]f were stained with eosin. The [Figure 2]b and [Figure 2]e showed the decrease of the staining intensity when the heating time was shorter as 5 min, which indicated that the cell viability might be partially impaired. Furthermore, the blue cytoplasmic staining did not disappear in a sudden, but decreased gradually with heating temperatures increasing and duration extending.
|Figure 2: Nicotinamide adenine dinucleotide-diaphorase staining immediately after microwave heating. (a-c) Three groups of HELA: The control, treated by 55°C 5 min and 55°C 25 min, respectively. (d-f) Three groups of MG-63: The control, treated by 55°C 5 min and 55°C 20 min, respectively|
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Calcein-acetoxymethyl ester staining
[Figure 3] reported the results of Calcein-AM staining. [Figure 3]A, [Figure 3]B, [Figure 3]C, [Figure 3]D showed the results of HELA, and [Figure 3]E, [Figure 3]F, [Figure 3]G, [Figure 3]H showed the results of MG-63. The [Figure 3]A and [Figure 3]E presented the normal cells of HELA and MG-63, respectively, stained by Calcein-AM. The [Figure 3]B, [Figure 3]C, [Figure 3]D and [Figure 3]F, [Figure 3]G, [Figure 3]H presented the changes of HELA and MG-63, respectively, when treated by 50°C for 15 min, 55°C for 5 min, and 60°C for 5 min. The [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f, [Figure 3]g, [Figure 3]h below each Calcein-AM staining image showed the nuclear staining by Hoechst in the same sections. It could be observed that the normal cells showed an entire well-distributed green staining. The [Figure 3]b and [Figure 3]f showed cells with bubbles induced by 50°C for 15 min heating. Actually, when HELA and MG-63 were stimulated with 50°C 5 min, the cells began to produce bubbles, and with the time extending, more bubbles appeared. The [Figure 3]C and [Figure 3]G showed that after stimulated with 55°C 5 min, the cells presented a weak fluorescence. When the stimulation reached 60°C for 5 min, the enzyme activity was totally absent.
|Figure 3: Calcein-acetoxymethyl ester staining immediately after microwave heating. (A-D) The control, treated by 50°C 15 min, 55°C 5 min, and 60°C 5 min for HELA. (E-H) The control, treated by 50°C 15 min, 55°C 5 min, and 60°C 5 min for MG-63. (a-h) Below each Calcein-acetoxymethyl ester image showed Hoechst staining|
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| > Discussion|| |
Microwave belongs to the electromagnetic waves with frequency ranging from one GHz to 300 GHz, and the clinically applied frequency band was between 915 MHz and 2.4 GHz. The alternating electric field generated by the microwave could excite the water molecules in tumor tissues, and the resultant friction heat could be an effective tumor killer. However, the microwave does not directly affect macromolecules.
It has been reported that the mechanism of cell death induced by hyperthermia was sophisticated, which depended on the cell-type and thermal dose. In fact, most cell lines die of apoptosis under hyperthermia., However, when suffering from the higher temperatures in clinical application, such as microwave ablation, the microscopic morphology and antigenicity of the cells could be preserved well, while the enzymatic activity would disappear, just as the so-called coagulative necrosis.,,,, Presently, an optimum temperature-time combination for microwave ablation had not been determined in practice.
In our study, the assessment of cell viability depended on the following properties: (a) The intactness of the cell membrane and (b) the physiological state of the cell. It had been proposed that a cell should be considered dead once the integrity of its plasma membrane was damaged, which could be defined by the incorporation of vital dyes into cell. Moreover, as an physiological index of cell, the enzyme activity assay was being used more widely in clinic and had been utilized as a criteria to evaluate the viability of the tissues and cells.,,,
After the thermal stimulation, the cell viability was assessed by CCK-8 assay 24 h later. The CCK-8 assay was based on the ability of dehydrogenases in metabolic active cells to reduce tetrazolium salt to yellow colored product. Therefore, it could be recognized as a physiological index of cells. In our study, when the thermal stimulation reached 50°C 25 min and 50°C 20 min for HELA and MG-63, respectively, the dehydrogenases activity was almost absent, which showed the cell death after 24 h incubation. It was generally known that adherence ability was a basic characteristic to adherent cells. In this study, we also found that the cells had already lost their adherence ability even when the stimulation was 45°C 30 min and 45°C 20 min for HELA and MG-63, respectively (data not shown). However, as there was still major enzyme activity remained as shown in CCK-8 assay; the above-mentioned cells could not be determined to be dead.
The immediate effect of thermal stimulation induced by microwave was detected by flow cytometer with Annxin-V and PI staining. The Annxin-V could bind with PS exposing upon the outer leaflet of cell membrane and was widely used to detect apoptotic cells. The PI was a DNA-binding dye, which was being used to evaluate the intactness of the cell membrane for it could only penetrate into nonviable cells with damaged and permeable plasma membranes. The dose-time combination of 55°C for 15 min and 55°C for 5 min for HELA and MG-63, respectively were enough to damage the cell membrane severely. Although the percentage of Annexin-V(−)/PI(−) cells in specimen might continue to decrease with stronger stimulations, the 0% could not be obtained easily. The reason was probably that the system errors of flow cytometer, which made it difficult to differentiate the contaminations and cell debris.
Meanwhile, the immediately outcomes of microwave heating were also evaluated with NADH-diaphorase staining. The NADH-diaphorase staining had already been widely used to assess the cells and tissue viability after thermal ablation in recent years.,,, It was based on the ability of NADH-diaphorase in viable cells to reduce nitroblue tetrazolium chloride to an intense blue cytoplasmic pigment. In addition to, the NADH-diaphorase staining, we had also performed Calcein-AM staining, which was based on the ability of intracellular esterases in viable cells to hydrolyze Calcein-AM into calcein, a strongly fluorescent compound. As so many kinds of enzymes with different inactivation temperatures were included in a cell, it thus might be possible to derive different devitalization temperature from different enzyme histochemistry methods. In this study, the NADH-diaphorase activity was absent when the thermal stimulation was stronger than 55°C 25 min and 55°C 20 min for HELA and MG-63, respectively. However, in another enzyme histochemistry, the Calcein-AM staining showed that the intracellular esterases activity was absent when the stimulation was 60°C for 5 min.
| > Conclusion|| |
The temperature-controlled microwave acted as an excellent ablation effect to the chosen cell lines in this study and when the thermal stimulation reaching 55°C 25 min and 55°C 20 min for the HELA and MG-63 cells, respectively or 60°C 5 min for both, all the viability indexes indicated immediately and complete devitalization. However, for other cell lines those effection remained to be verified. Furthermore, in clinical practice, the solid tumor was comprised tumor cells, supporting cells, extracellular substance, and vessels. Given its complexity, the conclusion derived from this study should be corrected further by animal experiment in the future.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]