The human prostate cancer PC-3 cell line (American Type Culture Collection, Manassas, VA, USA) was used. The PC-3 cell line was initiated from a bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old male Caucasian. The PC-3 cell line was cultured in RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with a 5% CO₂ atmosphere.

Each chamber of a Lab-Tek^® 4-chamber cell culture slide^™ (Thermo Fisher Scientific, Inc.) was seeded with 2×10⁴ PC-3 cells. The slide was placed in a water bath at 37°C. A 0.032 inch heating guidewire was attached under the bottom of chamber 4 of the four-chamber cell culture slide and was then connected to a radiofrequency (RF) generator (MPG-4; OPTHOS Instruments, Inc., Rockville, MD, USA) When the RF generator was operated at 2–3 W through the heating guidewire, the temperature in chamber 4 increased from 37–42°C, creating a heat gradient along the four chambers (Fig. 1). The temperature of each chamber was recorded using a micro-thermometer (Photon Control Inc., Richmond, Canada).

To compare the therapeutic effects of different RFH temperatures, the same amount of docetaxel (52 nM) (Hopira, Lake forest, IL, USA) was placed into each of the four chambers as the bottom of chamber 4 was heated to 42°C for 20 min, and the temperature of chamber 1 was maintained at 37°C. To compare the therapeutic effects of different treatments, the prostate cancer cells were divided into four groups: No treatment (control), RFH only (42°C for 20 min), chemotherapy only (chemo-only, 52 nM docetaxel) and combination therapy (chemotherapy plus RFH). A docetaxel concentration of 52 nM was selected according to previous studies (15,16). Following this, the cells were then cultured at 37°C for 72 h, and subjected to different laboratory evaluations, including cell proliferation and apoptosis assays, to examine and compare the effects of the different treatments on prostate cancer cells.

The treatments were initiated when the cell confluency reached 80%. RFH-enhanced chemotherapy was performed by adding docetaxel into the medium and heating the cells to ~42°C with the RF power set to 12–14 W for 20 min. Docetaxel was maintained in the chamber for 24 h following RFH. The cells in the chemotherapy-only group were treated with docetaxel for the same duration (24 h).

Cell proliferation was evaluated by the MTS assay (Promega Corp., Madison, WI, USA). Briefly, 120 µl MTS reagent was added to 0.6 ml of medium in each chamber, and the cells were incubated at 37°C for 3 h. Then, 120 µl medium was transferred to a 96-well plate, and the absorbance was detected at 490 nm with a Microplate Reader (VersaMax; Molecular Devices, LLC, Sunnyvale, CA, USA). The relative cell proliferation rates of the different cell groups were evaluated using the following equation: A_treated/A_control, where A is the absorbance. All of the experiments for all cell groups were repeated six times.

The animal protocol was approved by Sir Run Run Shaw Hospital Institutional Animal Care and Use Committee (Hangzhou, China). A total of 24 male mice (athymic nu/nu; 4–6 weeks; weight, ~20 g; Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were used to generate a tumor model. The mice were maintained on Alpha-Dri bedding in temperature (22±2°C) and humidity (30–50%) in controlled rooms with a 12/12 h light/dark cycle. The feeding method for mice was ad libitum. A suspension of 1×10⁷ PC-3 cells in 100 µl PBS was injected subcutaneously and unilaterally into the back of each mouse to seed a prostate cancer mass. Within three weeks, the tumor masses had grown to 5–10 mm in diameter (Fig. 2).

A total of 24 mice bearing human prostate cancer xenografts were randomly stratified into four study groups (6 mice per group) for receipt of different intratumoral treatments: i) PBS (control); ii) RFH-only (42°C for 20 min via the heating guidewire); iii) chemo-only (intratumoral injection of 75 mg/m² docetaxel); and iv) combination therapy (chemo plus RFH). RFH was conducted by inserting the 0.022 inch heating-guidewire into the tumor such that its hot spot was at the center of the tumor. A 400 µm micro-optical temperature fiber (Photon Control Inc.) was placed subcutaneously parallel to the heating guidewire, such that the real temperature in the tumor mass was measured continuously (Fig. 2A). By adjusting the RF output power to ~10 W, the temperature of the tumor mass was maintained at ~42°C. Throughout the entire procedure, the animals were anesthetized with 1–3% isoflurane in 100% oxygen.

The animals were placed in the supine position in a 3.0 Tesla MR scanner with a 100 mm micro-imaging coil (GE Healthcare, Chicago, IL, USA). MRI scans were acquired prior, and 7 and 14 days subsequent to the treatment. Axial and sagittal T2-weighted imaging was performed using a Fast Spin Echo sequence with the following parameters: TR/TE=3,100 ms/80 ms, field of view=8 cm, matrix=256×256, section thickness=1.5 mm, intersection gap=0.5 mm, and Number of Excitation (NEX)=3. Axial T1-weighted imaging was then achieved using a spin echo (SE) sequence with the following parameters: TR/TE=550 ms/15 ms, field of view=8 cm, matrix=256×256, section thickness=1.5 mm, intersection gap=0.5 mm, and NEX=2.

For the tumor size measurements, the tumor volume was expressed in cubic millimeters (mm³) and was calculated based on the axial T2-weighted images. The tumor border was delineated manually, and the area was automatically calculated using ImageJ 1.46r software (Wayne Rasband, National Institutes of Health, USA). Given the irregular contours of the tumors, the tumor volume was calculated according to the following equation: V= s·(a₁ + a₂ + a₃ + … + a_n), where s is the section interval and a₁- a_n are the areas of various sections.

The relative tumor volume (RTV) at different time points was then calculated for each tumor using the following equation: RTV=TV_Dn/TV_D0, where TV is the tumor volume, Dn is day 1, 7, or 14 following treatment and D0 is the day prior to treatment (8).

As the present study primarily focused on proving the principle of this novel concept, a longitudinal follow-up of the animals to time points at which the treated tumors had completely disappeared was not performed.

Following satisfactory MRI, the mice were sacrificed with 5% CO_2, and the tumor masses were harvested. The tumor tissues were embedded in Tissue Freezing Medium (O.C.T compound; Thermo Fisher Scientific, Inc.), frozen in liquid nitrogen, maintained in a freezer at −80°C and then sectioned into 5 um thick slices. The tissue slices were stained with the In Situ Cell Death Detection kit and fluorescein (Roche Diagnostics, Basel, Switzerland), and were then exposed to freshly prepared proteinase K working solution for 15–30 min at 37°C (10–20 µg/ml in 10 mM Tris/HCl; pH 7.4–8). Subsequent to washing with PBS, the slices were incubated in 50 ml TUNEL reaction mixture for 60 min in a dark and humidified environment. The slides were washed twice with PBS, stained overnight with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) and sealed with Prolong Gold Antifade reagent (Thermo Fisher Scientific, Inc.). All of the slides were examined under a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany), and a total of 200 cells were counted in 7 fields of view to calculate the mean percentage of apoptotic cells as the apoptotic index. Apoptotic cells were counted at ×400 magnification using Image-Pro Plus 6 software (Media Cybernetics, Inc., Rockville, MD, USA).

The tissue slices (5 µm in thickness) were stained with Hematoxylin and Eosin Staining kit (Nanjing SenBeiJia Biological Technology, Nanjing, China). The slides were immersed for 30 sec and agitated by hand in H₂O. Slide were submerged into a Coplin jar containing Mayer's hematoxylin (Nanjing SenBeiJia Biological Technology, Nanjing, China) and agitated for 30 sec prior to being washed in H₂O for 1 min. Slides were stained with 1% eosin Y solution (Nanjing SenBeiJia Biological Technology, Nanjing, China) for 30 sec with agitation. Sections were then dehydrated with two changes of 95% alcohol and two changes of 100% alcohol for 30 sec each. The alcohol was then extracted with two changes of xylene. All of the slides were examined at ×400 magnification under a light microscope (Leica, Wetzlar, Germany).

Data are presented as the means ± standard deviation. The statistical analyses were performed using SPSS 13.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance was performed to compare the mean cell proliferation rate, cell apoptotic index, tumor volume and RTV, and Tukey's post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

In the in vitro experiments, the combination therapy significantly reduced the proliferation of prostate cancer cells compared with the control, RFH-only and chemo-only groups (Fig. 3; 20.50±4.99 vs. 96.18±1.82%, P=0.005; 95.06±2.95%, P=0.003; 48.98±11.74%, P=0.007, respectively).

All of the animals survived throughout the in vivo experiments. A MRI analysis demonstrated significant reductions in tumor size in the mice treated with combination therapy (chemo plus RFH) compared with those that received the control, RFH-only and chemo-only treatments (0.28±0.16 vs. 1.42±0.27; 0.96±0.23; 0.75±0.18 cm³, respectively; Fig. 4; P<0.001). These results were confirmed by subsequent pathological examination. The average RTV in the combination treatment group was significantly lower compared with the control, RFH-only and chemo-only groups (0.57±0.17 vs. 3.95±0.26; 2.68±0.43; 2.08±0.30, respectively; P<0.001; Fig. 5). The number of apoptotic cells and the average apoptotic index of the combination therapy group were significantly higher compared with the control, RFH-only and chemo-only groups (Fig. 6; 43.22±10.26 vs. 8.00±2.87%, P=0.003; 10.91±4.31%, P=0.032; 23.20±6.00%, P=0.025, respectively).