Cell lines and culture
The VX2 cell line, used for tumor implantation, was obtained from the Surgery Department of the First Affiliated Hospital of Nanjing Medical University. The cells were cultured in RPMI 1640 (Hyclone, USA) supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin (Shanghai Sangon, China) at 37 °C with 5% CO2.
Animal and tumor model
Treatment procedures of animals
The larger and shorter diameters of the tumor were measured by using a caliper. After the larger diameter of the tumor reached about 15 mm (12 to 20 days), altogether the 65 rabbits involved in this study was given the following treatments: (1) control group (n=16), tumor-bearing without any treatments (one for NADH-diaphorase staining), (2) low-power MWA group (n=17), an output power of 20W was used along the tumor’s long axis for 2 mins (two for NADH-diaphorase staining), (3) high-power MWA group (n=17), an output power of 40W was used along the tumor’s long axis for 1 min (two for NADH-diaphorase staining), (4) operation group (n=15), in which tumors were removed. For low-power MWA, the output power of 20 W was applied for 2 mins by the use of a microwave generator (ECO-100E, Yigao Microwave Electric Institute, Nanjing, China) with the microwave irradiation frequency of 2,450 MHz. For high-power MWA, the output power of 40 W was applied for 1 mins with the same microwave irradiation frequency.
Pathologic Evaluation
After MWA, the tumor specimen was sliced sequentially into 5-mm sections. The sections were snap-frozen, and cryosections of 8-μm thick were made for cell viability staining with slices, and stained with NADH-diaphorase (Sigma, USA). A section of tumor from control group was used as a negative control. The frozen unfixed sections were mounted on glass slides and covered with 150μl of incubation media for 20 minutes at room temperature. This incubation medium was prepared as described previously [14]. After incubation, viable cells showed an intense blue cytoplasmic pigment, which were absent in nonviable cells.
Lung metastasis analysis
The animals were autopsied within six hours after death. The lung specimens were fixed in 10% formalin solution, embedded in paraffin, sectioned into 4 μm slices, and stained with hematoxylin and eosin (H&E). The histological slides were evaluated by one experienced pathologist.
Immunohistochemical analysis
The tumor tissues were fixed in 4% formalin solution and paraffin embedded. Paraffin sections were stained with Rabbit-anti-CD8 (RM-9116-SO, Thermo Fisher, USA) and
Mouse-anti-CD4 (ab194998,ABCAM,British) followed by horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz, USA) and 3,3’-diaminobenzidine (DAB kit, Beyotime, Nanjing, China). The numbers of positive cells were counted in five randomly selected fields at 400-fold magnification. Results from the five areas were averaged and used in the statistical analysis.
Flow cytometric analysis
The rabbits were anesthetized and blood was drawn from the ear vein in EDTA tubes 14 days after operation. The blood were lysed with RBC lysis buffer (BD Biosciences, San Jose, USA) to remove red cells and obtained single cell suspension. Subsequently, all single cell suspensions were resuspended in Flow cytometry staining buffer. Then single cell suspensions were stained with surface markers at 4 °C for 30 min, after Fc blocking, to characterize the immune cell subsets: CD4(clone KEN-4, BIO-RAD), PE(clone A85-1, BD), FITC-anti-CD5 (clone KEN-5, BIO-RAD) and APC-anti-CD8A (clone 12.C7, Novusbio). Flow cytometric analysis was performed by using a FACS flow cytometer (Beckman Coulter, Miami, USA), and data analyzed by FlowJo software.
ELISA assay
Blood was collected from ear vein into EDTA tubes. Plasma was obtained by centrifugation at 1000 ×g for 10 mins and stored at -80°C until analysis. Concentrations of INF-γ, IL-4, IL-10, IL-6 and IL-12 in plasma were measured by enzyme-linked immune sorbent assay (ELISA) using High Sensitivity ELISA Kit (m1027824, mm030302, mm030702, mm030202, m1027340, MLBIO, CHINA) according to manufacturer’s instruction respectively. TNF-α and IL-2 were quantifified using High Sensitivity ELISA Kit (DY5670, DY6994, R&D, USA).
Statistical analysis
Software (SPSS 16.0, Chicago, IL) was used for statistical analysis in the current study. The differences between the four groups were compared by repeated measurement ANOVA. Pairwise comparisons were made using the bonferroni corrected t-test. The Kaplan-Meier method and the log-rank test were used for the end-point survival analysis. Results with P values less than 0.05 were considered significant. *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.