Cell lines and mice
CT26, a murine colon carcinoma cell line that is derived from a BALB/c mouse, was purchased from the Culture Collection and Research Center (Hsinchu,Taiwan), where fresh batches are thawed every year. CT26 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) was supplemented with 10 % fetal bovine serum (FBS), 100 ng/ml of streptomycin, and 100 U/ml of penicillin (Invitrogen). Female BALB/c mice were obtained from the National Science Council Animal Center, Taipei, Taiwan, and were used at between 6 and 8 weeks of age. This study was approved by the Institutional Animal Care and Use Committee of the Shin Kong Wu Ho-Su Memorial Hospital (Approval No. 0990827008).
Modulated electro-hyperthermia treatment (mEHT)
Electromagnetic heating was conducted using capacitive-coupling with an amplitude-modulated 13.56-MHz radiofrequency (LabEHY, Oncotherm, Germany). The mEHT technical details of the method can be found elsewhere . An in vitro heating model was established in an electrode chamber (LabEHY in vitro applicator), which was heated to 42 °C for 30 min at a mean power of 8 ~ 9 W. The cells were placed in a chamber with a culture medium at 42 °C for 30 min. Tumor implants in the right femoral area of BALB/c mice were placed in the parallel electric condenser of the heating circuit, as described elsewhere . The treatment groups were givena single shot of mEHT for 30 min at a mean power of 1.5 W under 100 mg/kg Ketamine and 10 mg/kg Xylazine anesthesia. Intratumoral temperature was maintained at ~ 42 °C on the treated side of each mice, as measured using optical sensors (Luxtron FOT Lab Kit, LumaSense Technologies, Inc., California, USA). The subcutaneous temperature underneath the electrode was maintained at 38 ~ 40 °C.
Water bath-treated and mEHT-treated CT26 cells were cultured for 24 h, then trypsinized, and washed twice with PBS. Apoptosis was verified using an Annexin V Apoptosis Kit (BD Pharmingen), following the manufacturer’s instructions. Briefly, tumor cells were washed three times with PBS; then, some cells were analyzed immediately for apoptosis using Annexin V/PI staining. Washed cells were supplemented with 1 % BSA and then stained directly with 10 μL of PI and 2.5 μL Annexin V-FITC, following the addition of 222.5 μL of binding buffer. Immediately after 10 min of incubation in the dark on ice, the cells were analyzed by flow cytometry. The percentage of positive cells was determined using a FACSCalibur cytometer and Cell Quest Pro software (Becton Dickinson, Mountain View, CA).
Western blot analysis
For protein analysis, the water bath-treated control and mEHT-treated CT26 cells were lysed for 5 min at room temperature in a buffer of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1 % (v/v) Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride, 20 μg/mL aprotinin, and 25 μg/mL leupeptin (Sigma). The total protein concentration was measured using the Bio-Rad protein assay reagent. Cell lysates (100 μg) were electrophoresed on a 12 % polyacrylamide gel, transferred onto an Immobilon-P PVDF membrane (Millipore, Bedford, MA), and blocked in PBS-Tween 20 and 10 % nonfat milk for 2 h at room temperature. The filter was incubated with specific antibodies to anti-Hsp70 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-HMGB-1 (Abcam, Cambridge, MA, USA) for 2 h at room temperature in PBS-0.05 % Tween 20 that contained 5 % nonfat milk, followed by 1 h incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) in the same buffer. Blots were developed using a chemiluminescent detection system (ECL; GE Life Science, Buckinghamshire, UK).
Hsp70 release assay
mEHT treated CT26 cells were cultured for 24 h. The culture supernatants were harvested and Hsp70 was measured by using an enzyme-linked immunosorbent assay (ELISA) (Enzo Life Sciences, Farmingdale, USA). A Multiskan Plus (Thermo Scientific, Hudson, NH, USA) was utilized to measure absorbance at 450 nm.
Generation of bone marrow-derived dendritic cells
Bone marrow-derived DCs (BM-DCs) were produced as described elsewhere . Briefly, BM-DCs were isolated from BALB/c mice by culturing red blood cell-depleted BM cells in a complete medium (RPMI 1640 that was supplemented with 10 % FBS, L-glutamine, and 5 mM 2-mercaptoethanol) that contained 20 ng/ml of recombinant mouse GM-CSF (Peprotech, Rocky Hill, NJ, USA) at 37 °C in a humidified atmosphere with 5 % CO2 and fed every third day with a medium that contained fresh GM-CSF. On day nine of the culture, the DCs were mixed with 10 μg/ml AH1 (SPSYVYHQF) that had been manufactured at 95 % purity by AnaSpec (Fremont, CA) and 50 μg/ml Hsp70 which was prepared in our laboratory as described elsewhere  for 24 h. On day ten of the culture, non-adherent cells were harvested, washed once in a complete medium, and examined to evaluate the expression of the DC surface markers (MHC class II molecule I-Ad/I-Ed, CD80 (B7-1), CD86 (B7-2), CD11c, and DEC205). The BM-DCs (5–10 × 105) were stained with 50 μL of FITC-conjugated antibodies in phosphate-buffered saline (PBS) that contained 1 % bovine serum albumin (BSA) and 0.1 % azide, which was also used as the washing buffer, before being subjected to fluorescence-activated cell sorting (FACS) analysis using a FASCalibur flow cytometer (BD Bioscience, San Diego, California, USA). Cells were stained with the corresponding isotype-matched control IgG (BD Pharmingen, San Diego, CA, USA). Endocytic activity was quantifiedby incubating cells for 2 h with FITC-dextran (100 μg/ml) (Sigma) at 4 °C or 37 °C. Cells were washed extensively with PBS, before being subjected to FACS analysis. Non-specific binding of FITC-dextran to the cell surface was measured by incubating the cells at 4 °C . The percentage of positive cells was obtained using a FACSCalibur cytometer and Cell Quest Pro software (Becton Dickinson, Mountain View, CA).
On day zero, the right femoral areas of BALB/c mice were injected subcutaneously with 5 × 105 CT26 tumor cells. On day 14 following injection, the mice received local mEHT treatment (as described above), and then, on the following day, 5 × 105 syngeneic DCs or PBS in 25 μL were injected into the right femoral tumor area. Each group comprised ten mice. Sampling was carried out 48 h following treatment, using three mice in each group. Each excised tumor was fixed in 10 % formalin, dehydrated, and embedded in paraffin wax (FFPE). The sizes of the tumors in the other seven mice in each group were measured at least three times weekly: length (L) and width (W) were recorded and the tumor volumes were calculated as L × W2/2. To evaluate whether specific immunologic memory responses were generated in mice that bore CT26 tumor cells, the mice were re-challenged with 1 × 105 tumor cells in the other flank 30 days following the first tumor inoculation . The mice were examined three times weekly to evaluate tumor development for 30 days after tumor cell transplantation or the first inoculated tumor growth until the tumor was more than 2 cm in diameter.
Cytotoxicity T lymphocyte (CTL) assay
On day 30 following tumor injection, the mice were killed and their spleens harvested. Erythrocyte-depleted splenocytes (1 × 106 cells/ml) were cultured for five days in vitro using mitomycin C-treated CT26 tumor cells (1 × 106 cells/ml) in 24-well plates, during which time 50 IU/ml of recombinant human IL-2 (Proleukin; Novartis Pharmaceuticals, East Hanover, NJ) was added daily. On day five, the cells were collected; dead cells were removed on a density gradient, and the viable cells were tested to evaluate specific cytotoxicity using LDH-release assay (Promega, Madison, WI, USA). The percentage-specific cytotoxicity was calculated as 100 x [(experimental release – spontaneous release)/(maximal release – spontaneous release)].
Enzyme-linked immunosorbent spot (ELISPOT) assay
The ELISPOT assay was conducted using a Mouse IFN-γ Development Module kit (R&D System), following the manufacturer’s instructions. Splenocytes were prepared as described for use in the CTL reactions. The harvested splenocytes (1 × 105 in 100 μL) were then mixed with 100 μL of CT26 tumor lysate (50 μg of protein/ml) in each well of a 96-well filtration plate (MultiscreenTM HTS) that had been previously coated with capture antibodies (1:60 dilution). The negative controls were the medium alone and the splenocytes alone and the positive control was splenocytes plus 20 μg/ml of Con A. After incubation overnight at 37 °C, color was developed using the streptavidin-alkaline peroxidase and BCIP/NBT that was provided in the ELISPOT kits. The spots were counted visually under a dissection microscope; the numbers of spots in the test samples (splenocytes + tumor lysate), spots obtained using splenocytes alone, and spots obtained using medium alone were calculated.
Immunohistochemistry and Luna stain
To conduct immunohistochemical studies, the tumor was resected and fixed in 10 % formalin for 24 h. To stain the sections immunohistochemically, paraffin sections were deparaffinized in xylene and rehydrated in a graded alcohol series, treated with 3 % H2O2 for 10 min, and boiled in a citrate buffer (pH 6) for 30 min (anti-F4/80 antibody, Bioss bs-7058R, anti-CD45 antibody, Bioss bs-0522R), before immunoblock (Bio TnA, TAHC03) was applied to prevent non-specific binding for 60 min at room temperature. The sections were incubated with rabbit anti-F4/80 antibody (diluted 1:100) and anti-CD45 antibody (diluted 1:100) for one hour at 37 °C, and analyzed by Mouse/Rabbit Probe HRP labeling (BioTnA, TAHC03) for 30 min at room temperature. Peroxidase activity was developed in a diaminobenzidine- H2O2 solution (Bio TnA, TAHC03) for 10 min at room temperature. The sections were then counterstained with hematoxylin. All stained slides were examined by two pathologists who were blind to the treatment group data. The percentage of positively stained cell membranes or cytoplasm was obtained by microscopically examining the entire tissue at high magnification (×400). The numbers of positive cells was calculated in ten fields. The Luna protocol was performed as described elsewhere with slight modifications . The sections were immersed in working Hematoxylin-Biebrich (Sigma, Cat # H-3136 and Acros, CI 26905, respectively) scarlet solution (for five minutes), and then dipped (∼8x) in 1 % acid alcohol and rinsed in tap water. The sections were then dipped (∼5x) in lithium carbonate solution until they turned blue and washed in running tap water (for two minutes). The numbers of eosinophil on the stained slide were calculated in ten fields (x400).
All results were compared using an unpaired t test (two-tailed) or one-way ANOVA. Differences were considered statistically significant at a P value of less than 0.05