The Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine approved this retrospective study (reference number: AF-0406). Written informed consent was obtained from each patient. From October 2016 to August 2019, 61 patients in our center underwent IRE treatment (including 26 liver tumors, 17 pancreatic tumors, 4 renal tumors, 5 adrenal gland tumors, 6 retroperitoneal tumors; one patient with both liver and pancreatic tumors, and two patients with both liver and retroperitoneal tumors). Due to the immunity of these types of patients were relative to the types of malignancies that were treated were highly variable, we selected a single tumor type--- hepatocellular carcinoma (HCC) for immunological analysis, 3 cholangiocarcinoma and 12 hepatic metastases were excluded. Data were collected on a total of 11 consecutive patients with HCC (8 men, 3 women, mean age, 60.8 ± 9.3 years) in the study. All patients had a history of chronic HBV infection and cirrhosis. No chemotherapy or interferon therapy was received previously. After the diagnosis of HCC, all patients were treated with oral nucleoside antiviral drugs. During the follow up, all patients received no adjunctive therapies.
IRE procedure in patients and sample collection
Two experienced interventional radiologists performed all the procedures. All patients were administered with muscle relaxants and general anesthesia. IRE was performed using a NanoKnife system (AngioDynamics, Latham, NY, USA) with an electrocardiogram (ECG) synchronization device under the guidance of CT. Nineteen gauge monopolar needles were placed in parallel around the tumors percutaneously at the intervals of 1.2–2.2 cm. Tip exposure of the needles was 1.0–2.0 cm. The number of needles was decided according to the tumor size. The parameters of IRE ablation were set as follows: average electric field intensity, 1500 V/cm; pulse length, 70–90 μs; 90 pulses. The ablation range covered the whole tumor with an ablation margin of at least 5 mm. Peripheral blood samples were collected 1 day before IRE therapy and used as baseline values. Additional blood samples were collected 1 day, 3 days, 7 days, 2 weeks, and 4 weeks after IRE treatment. Blood tests included blood cells analysis and immune cells analysis. A routine clinical flow cytometry test protocol was followed for analyzing immune cells in the peripheral blood. During the IRE treatment and the follow up period, no collateral events related to IRE were observed.
Cell culture and animal models
All animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals with approval by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine prior to initiation of experiments. The mouse hepatic carcinoma cell lines, H22 were purchased from China Center for Type Culture Collection (Wuhan, China). H22 cells were cultured in RPMI containing 10% FBS and 1% penicillin-streptomycin, in an incubator with a humidified atmosphere of 5% CO2 at a temperature of 37 °C. H22 cells (5 × 106) were suspended in 200 μL phosphate buffered saline and injected subcutaneously into the right flank of 5- to 6-week-old male BALB/c mice (commercially obtained from LINGCHANG BIOTECH, Shanghai, China). Two weeks after the injection, the diameter of the tumors reached nearly 1 cm.
IRE procedure in mice and sample collection
After 2 weeks of modeling, a total of 30 mice were randomly divided into two groups: the control group (n = 15) and the IRE group (n = 15). For the IRE group, the mice were anesthetized by injecting sodium pentobarbital (10 mg/mL, 50 mg/kg body weight) intraperitoneally. Then, each mouse was fixed on an insulating plate, and the IRE procedure was performed using an ECM 830 Square Wave Electroporation system (BTX Harvard Apparatus, Holliston, MA, USA) with a pair of genetrodes (BTX item #45–0161, BTX Harvard Apparatus, Holliston, MA, USA). The genetrodes with a 10 mm gap were inserted into the tumors to deliver electric pulses with the following parameters: voltage, 1200 V; pulse length, 90 μs; 90 pulses. This protocol was selected to produce a complete ablation for the tumors. The mice in the control group received sham procedures with the genetrodes inserted into the tumors but no electric pulses were given. The blood samples and tumor samples were collected at 3, 7, and 14 days post-IRE procedure from five mice separately. Samples from the control group were collected at the same time. At the end of the experiments, the mice were killed by standard CO2 asphyxiation.
Flow cytometry analysis
Tumors isolated from the mice were digested mechanically to obtain single-cell suspensions. The cell suspensions were surface stained with PE-Cy7-labeled anti-CD3 (eBioscience, San Diego, CA, USA), FITC-labeled anti-CD4 (eBioscience, San Diego, CA, USA) and PE-Cy5-labeled anti-CD8 (eBioscience, San Diego, CA, USA) monoclonal antibodies, and then treated with Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ, USA). Then, the cells were stained intracellularly with PE-labeled anti-FoxP3 (eBioscience, San Diego, CA, USA) and Pacific Blue-labeled anti-Granzyme B monoclonal antibodies (Biolegend, San Diego, CA, USA). The stained cells were analyzed with CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using CytExpert software (Beckman Coulter, Brea, CA, USA).
Lactate dehydrogenase (LDH) cytotoxicity assay
Single-cell suspensions from mice tumors were prepared. T cells were isolated from the single-cell suspensions by negative selection using the Pan T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated T cells were stimulated with recombinant murine IL-2 (PeproTech, Rocky Hill, NJ, USA) for 3 days. Then, the T cells were added to H22 cells with an effector to target cell ratio of 20:1, and the cells were co-cultured in 96-well plates for 24 h. Evaluation of T cell cytotoxicity activity was performed using an LDH-Cytox™ Assay kit (BioLegend, San Diego, CA, USA), according to the manufacturer’s protocol. The cytotoxicity percentage was calculated as follows: (LDH experimental − LDH spontaneous) / (LDH maximum − LDH spontaneous) × 100%. LDH experimental represents the LDH release activity from the T cells and tumor cells co-culture. Spontaneous LDH release activity was obtained from tumor cells cultured separately. The maximal LDH release activity was obtained following lysis of the tumor cells.
The tumor tissue removed from each mouse was fixed in 4% paraformaldehyde and embedded in paraffin for 5 μm-thick sections. After being deparaffinized and rehydrated, the sections were treated with sodium citrate buffer (pH = 6), and the microwave was used for antigen retrieval. The activity of endogenous peroxidase was blocked with 3% H2O2 in methanol. The sections were then incubated with anti-CD3 monoclonal antibody (Abcam, Cambridge, UK), anti-CD4 monoclonal antibody (Abcam, Cambridge, UK), and anti-CD8 monoclonal antibody (Abcam, Cambridge, UK) at 4 °C overnight, respectively. Afterwards, the sections were stained with HPR-conjugated secondary antibody, and the positive reactions were visualized with diaminobenzidine (DAB). Finally, the sections were counterstained with Mayer’s hematoxylin. Digital images of the stained sections were obtained in five randomly selected fields both at the interior regions and the margin of tumors using a fluorescence microscope. The positive cell numbers were counted and the results from the five areas were averaged and used in the statistical analysis.
Cytometric bead array (CBA) analysis
Blood serum was separated from the blood sample obtained from the tumor-bearing mouse by centrifugation at 3000 g for 20 min and then stored at − 80 °C, until later analysis. Serum cytokine analysis was performed using the CBA Flex Set (BD Biosciences, Franklin Lakes, NJ, USA), containing mouse IFN-γ, IL-1β, IL-2, IL-10, and TNF-α. Mouse Soluble Protein Flex Set Standards and samples were prepared according to the manufacturer’s instruction. The samples were acquired on the flow cytometer (BD LSRFortessaTM X-20, Franklin Lakes, NJ, USA). The data were analyzed using FCAP Array software (BD Biosciences, Franklin Lakes, NJ, USA).
RNA sequencing analysis
Tumors from IRE and control groups 7 days postoperative were separated for RNA sequencing analysis. Total RNA was extracted using Trizol reagent (Roche, Basel, Switzerland). Total RNA quality was evaluated on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Library preparation was performed from the pooled RNA using an Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) and sequenced on the Illumina HiSeq 4000 platform (Illumina, San Diego, CA, USA). The sequenced reads were aligned to the mouse genome mm10 by HISAT2 . FeatureCounts was used to quantitate the transcriptome, using the GTF annotation files . Differential analyses were performed to the count files using DESeq2 packages, following standard normalization procedures . The differentially expressed genes (DEGs) were identified with p values < 0.05 and absolute log2 fold change > 1. Gene Ontology (GO) enrichment analysis was performed using Metascape (metascape.org).
Statistical analysis was performed using SPSS statistical software, version 23 (IBM, Armonk, NY, USA). The immunohistochemistry results were analyzed with the Mann-Whitney test. For other analysis, Student’s t-test was used. All data were expressed as mean ± SEM (standard error of the mean) of n independent measurements. GraphPad Prism 7 software (GraphPad, San Diego, CA, USA) was used to plot graphs. P < 0.05 was considered statistically significant.