A total of sixty mice with H22 tumors were used in this phase and were randomized into the three groups: (a) control, (b) direct RFA, (c) indirect RFA. The mice were sacrificed at different time points after RFA, including 6 h, 24 h,72 h and 7 days for pathological analysis (n = 5 for each subgroup). Tissue was fixed in 10% formalin overnight at 4 °C, embedded in paraffin, and sliced at a thickness of 5 μm. The tissue was stained with hematoxylin eosin for gross pathologic examination. The specific immunohistochemistry (IHC) or immunofluorescence (IF) staining was used to detect the changes of the tumor microenvironment, containing the expression of the following markers, HSP70 (ab181606, Abcam, Cambridge, MA), SMA (ab32575, Abcam, Cambridge, MA), Ki-67 (12202S, Cell Signaling, Danvers, MA), VEGF (AF469, R&D Systems), CD31 (77,699 T, Cell Signaling, Danvers, MA), HIF-α (MAB1536-SP, R&D Systems), IL-6 (AF-406-SP, R&D Systems) and c-Met (AF527, R&D Systems).
The positive cells of different markers were analyzed according to IHC/IF staining of tumor specimen at 6 h, 24 h, 72 h and 7d after RFA. The expression and distribution of the markers in each specimen were observed under the microscope, and were analyzed blindly to the treatment to eliminate bias. The positive rate of each marker was calculated for five random high-power fields in each slide. The expression of each marker in different time points was semi quantitatively presented by bar graph. We analyzed the quantitative data of pathological results and evaluated the possible target markers that may have the most significant changes after RFA.
Evaluation of the effect of specific inhibitor
In the third phase, we evaluated the role of inhibiting one of the key pathways underlying post ablation tumor response. The effect of an adjuvant small-molecule c-Met receptor inhibitor (PHA-665752, subsequently referred to as PHA; Sigma-Aldrich, St Louis, Mo) on tumor microenvironment after RFA ablation was studied. Sixteen mice with H22 tumors were used for tumor growth study. In this phase, the range of 8-10 mm diameter was selected as the appropriate tumor size when we performed RFA treatment. The mice were randomized into two groups (n = 8 in each group): (a) RFA alone, (b) PHA + RFA. PHA (0.83 mg/kg, 200 μl each) was injected intraperitoneally every 2 days for four times. RFA was performed 2 h after first injection of PHA. To mimic the residual tumor during ablation of large tumors in clinical practice, about half of the tumor was completely ablated. The diameter of the tumor and the body weight of each mouse was measured every 2 days. Next, 10 mice from the above two groups were sacrificed at 7 days after RFA (24 h after the last injection of PHA) (n = 5 in each group) for pathological analysis. IHC staining was used to compare the changes of the tumor microenvironment after administration of PHA, including Ki-67, CD31, HIF-α and c-Met.
For all experiments and procedures, anesthesia was induced by means of intraperitoneal injection of pentobarbital sodium (45 mg/kg, chemical reagent factory of Foshan, China). Animals were sacrificed in a CO2 chamber. RFA was performed in an established H22 liver adenocarcinoma model in BALB/C mice (female, weighing 18–20 g, aged 6–8 weeks). 0.2 ml of H22 cells (at a density of 1 × 107/ml) suspended in serum-free RPMI-1640 and matrigel (1:1) were injected subcutaneously into the abdominal wall with an 18-gauge needle for each tumor to establish the liver adenocarcinoma model. Animals were monitored every 2 or 3 days to measure tumor growth. Ultrasonography was used to determine solid nonnecrotic tumors before treatment in this study. Mean starting tumor size was similar for all comparative treatment groups at initial treatment. The longitudinal and transverse directions of the tumor were measured with mechanical calipers and the body weight of the mouse was weighed on the electronic balance every 2 days after RFA. The measurement was performed by A-NJ and KZ, with 4 and 3 years of experience in animal experiments, respectively and verified by WY, with 12 years of experience in animal experiments, who was blinded to the treatment group. Tumor volume was calculated as D*d2*0.5, where D and d were the two diameters of the tumor measured above.
In the animal experiments, the 17-gauge monopolar electrode (ACT1507 electrode; Valleylab, Tyco Healthcare) and the 480-kHz RFA generator (Model CC-1-220; Valleylab, Tyco Healthcare, USA) were used during RFA. The animal was shaved off on the back and applied electrolytic contact gel and then placed on the conventional metallic grounding pad (Cosman Medical, Inc. USA) to complete the RFA circuit. About 0.7 cm of the electrode tip was placed at the center of the target tumor part first and the RFA generator was set to the tip temperature at 65 ± 2 °C and applied for 5 min. Here, we chose temperature 65 °C as the ablation parameter because the temperature point was the highest level for mice can tolerate during RFA treatment.
The paraffin tissue sections were firstly soaked in xylene to deplete paraffin, rinsed in ethanol to deplete xylene and soaked in hydrogen peroxide solution to reduce endogenous peroxidase activity. Then, antigen retrieval was performed by placing slides in a container and heated (97 °C) to enhance the accessibility of antibody to antigen for 10 minutes. After the slides were rinsed with PBS, they were blocked in goat serum to reduce nonspecific binding of the antibody. The blocked tissue slides were incubated with primary antibodies overnight. After washed with PBS, they were incubated with species matched secondary antibodies for 20 minutes and washed. For IHC reactions, the slides were then incubated with DAB (Beyotime Institute of Biotechnology) until desired stain intensity was observed, counterstained with hematoxylin and rinsed. Then, the slides were dehydrated in ethanol and emerged in xylene. After that, the slides were covered with cover glass and dried on a flat surface. For IF reactions, the slides were then incubated with DAPI (Beyotime Institute of Biotechnology) to visualize nucleic acid in each cell. After washed with PBS, anti-fluorescence quenching agent (Beyotime Institute of Biotechnology) was added. Then the slides were covered with cover glass, dried and kept in the dark to prevent bleaching fluorescent signal. Pathological slides were imaged and analyzed by using a microscope (Micromaster I; Westover Scientific, Mill Creek, Wash). For quantitative analysis, the percent of positively stained cells was counted on at least five high power (× 400) fields per slide and was assigned scores in a blinded fashion to remove observer bias .
In this study, SPSS 27.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. P < 0.05 was statistically significant. All continuous data were provided as means ± SD. Kruskal Wallis test was used to evaluate the significance of different treatments. When the total P was less than 0.05, Nemenyi test was used for multiple comparison. The two-way analysis of variance was used to determine the significance of treatment in tumor growth or body weight.