A total of 114 patients (age range, 35–91 years old) with HCC were enrolled in the present study at Qingdao Sixth People's Hospital (Qingdao, China). Date of the first diagnosis of HCC was from April 2013 to February 2020. Date of the last follow-up was April 2020. A total of 50 patients were treated by surgery, 10 by biopsies and the rest had neither surgery nor biopsy. The following inclusion criteria were used for the patients: i) First TACE treatment; ii) conformed to the indications of TACE treatment, with no history of radiotherapy or systemic chemotherapy before TACE treatment; iii) no autoimmune diseases present or no drugs affecting immune function were taken 3 months before treatment. The exclusion criteria used were as follows: i) Patients with metastatic liver cancer; ii) patients with serious heart, brain, lung, kidney and blood system diseases; iii) pregnant and lactating women; iv) patients had communication disorders, such as cases with hepatic encephalopathy or depression; and v) unwilling to participate.

The criteria for diagnosis included clinical manifestations, imageology, α-fetoprotein (AFP) levels and pathological examination, which met the diagnostic criteria of the Chinese Society of Clinical Oncology guidelines for the diagnosis and treatment of primary liver cancer (v1; 2018) (13). All patients provided signed informed consent for surgery and were treated with TACE. Clinical data were collected by the hospital system. Fresh peripheral blood was collected and stored in tubes with EDTA anticoagulation for assessment of immune function before intervention and 1 month after treatment. Written informed consent was signed by all patients and healthy donors before participation in the study. The present study was approved by the Ethics Committee of Qingdao Sixth People's Hospital (Qingdao, China).

Child-Pugh grading system (14) was used to evaluate patient liver function. A total of five indices (including the hepatic encephalopathy, ascites, total bilirubin, albumin and prothrombin time) of patients were divided into 3 levels, corresponding to 1,2,3, respectively. Finally, scores were added up and a high score indicated the severity of liver damage. See Table I for details.

All patients were treated with TACE using the Seldinger technique (15). Ultrasound-guided percutaneous insertion of a retrograde catheter was performed from the femoral artery through the celiac trunk and hepatic artery, and the final location of the catheter was determined by the tumor location. Chemotherapeutic drugs, including fluorouracil, mitomycin and carboplatin were mixed with iodized oil to form a pingyangmycin lipiodol emulsion. The mixture was injected into gelfoam to block blood supply to the tumor as embolic agents. The dose and type of chemotherapeutic drug depended on the patient's age, weight, and liver and kidney function. In addition, patients also underwent concomitant therapy, such as disodium cantharidinate (0.25 g; once daily; intravenous injection) for antitumor therapy, tiopronin (0.2 g; once daily; intravenous injection) for liver protection and supportive treatment, such as hemostasis and rehydration to prevent electrolyte disturbances and prophylactic antibiotics for preventing infection in patients after surgery. According to the Response Evaluation Criteria In Solid Tumors (16), the therapeutic effect was evaluated as follows: Complete remission (CR), where all target lesions had disappeared; partial response (PR), where the sum of the longest diameter of target lesions decreased by ≥30%; stable disease (SD), indicating neither PR nor progressive disease (PD); and PD, where the sum of the longest diameters of the target lesions increased by ≥20% or new lesions or metastases appeared. CR and PR were defined as a satisfactory response, while SD and PD were regarded as a poor response. Overall survival was defined as the time interval between patients undergoing TACE and death or the last follow-up.

The diagnostic criteria for cirrhosis included: i) Pseudolobules were diagnosed through percutaneous liver biopsy; and ii) liver stiffness measurement ≥17.5 kPa using FibroScan (transient elastography). Patients with either of the aforementioned criteria were diagnosed with liver cirrhosis.

Peripheral blood was collected from 114 patients with HCC who received TACE intervention and 20 healthy donors. The recruitment of healthy blood donors at the Sixth People's Hospital of Qingdao (Qingdao, China) began in February 2014 and ended in October 2017. The age range of the healthy controls was 21–67 years. There were 14 men and 4 women. The inclusion criteria used for the healthy contacts were as follows: i) Body mass index (BMI) between 19–26; >45 kg for women, >50 kg for men; iii) understood fully the purpose of the study and voluntarily signed the informed consent; and iv) had no history of chronic or serious diseases, such as cardiovascular disease, and were in good general health. The exclusion criteria were as follows: i) Had taken any medication within 3 months; ii) Had any medical condition; PBMCs were separated using the Ficoll-Hypaque density gradient deposition method (17). At room temperature, the blood diluted by PBS was centrifuged at 300 × g for 20 min. Cells of the mononuclear cell layer were collected and cleaned with PBS at room temperature at 300 × g for 10 min. The precipitated cells were suspended in the medium for later use. PBMCs were washed for 3 min using PBS at room temperature and further analyzed by flow cytometry or reverse transcription-quantitative PCR (RT-qPCR).

Flow cytometry was used to detect CD3⁺, CD4⁺ and CD8⁺ cells. PBMCs were collected, washed for 3 min using PBS at room temperature and centrifuged at 200 × g for 5 min at room temperature and the supernatant was discarded. FITC-anti-CD3 (1:200; cat. no. 561806; BD Biosciences), phycoerythrin-anti-CD4 (1:200; cat. no. 561843; BD Biosciences) and allophycocyanin-Cy7-anti-CD8 (1:200; cat. no. 557760; BD Biosciences) antibodies were added, followed by incubation at 4°C for 20 min. PBMCs were washed for 5 min using PBS at room temperature. Following centrifugation at 200 × g for 5 min at room temperature, the PBMCs were resuspended with PBS containing 1% paraformaldehyde for fixation at room temperature, detected (FACSCanto II; BD Biosciences) and analyzed by CellQuest software v.5.1 (BD Biosciences).

Total RNA was extracted from isolated cells using TRIzol^® reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. Extracted total RNA was then reverse transcribed into cDNA. The reaction tube containing total RNA was first held at 37°C for 15 min for reverse transcription, then heated at 85°C for 5 min for inactivation of reverse transcriptase, and cooled to 4°C for storage. cDNA was analyzed by qPCR according to the Takara two-step method (Takara Bio, Inc.). The thermocycling conditions were as follows: Firstly, 1 cycle at 95°C for 30 sec for initial denaturation; followed by 40 cycles at 95°C for 5 sec for denaturation and at last 60°C for 30–34 sec for annealing. The primer sequences of programmed cell death 1 ligand (PD-L1) and programmed cell death protein 1 (PD1) are shown in Table II.

The 2^−ΔΔCq method (18) was used to quantify gene expression. GAPDH was used as the internal reference gene.

All experiments were performed three times. Quantitative data are presented as the mean ± standard deviation, and categorical data is shown as the number of cases. Comparisons between before and after treatment were performed using a paired t-test. The association between the levels of PD1/PD-L1 and the efficacy of TACE was assessed using one-way ANOVA followed by Tukey's multiple comparisons test. The association between PD1/PD-L1 expression and survival time was analyzed by Kaplan-Meier analysis with a log-rank test. All data were analyzed using SPSS v22.0 (IBM Corp.) and GraphPad Prism 8.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

A total of 114 patients with HCC, including 84 male and 30 female patients, were included in the present study (Table III). The mean age was 66.5±12.6 years. There were 54 patients who were >65 years old and the remaining 60 patients were ≤65 years old. A total of 97 patients had hepatitis B, while 17 patients had hepatitis C. According to the Child-Pugh grading system (14), 89 cases were classified as grade A, 20 cases were classified as grade B and 5 cases were classified as grade C.

A total of 100 cases were diagnosed with liver cirrhosis. The maximum tumor diameter was >5 cm in 55 cases and ≤5 cm in 59 cases. There were 80 cases presenting with a single tumor and 34 cases presenting with multifocal tumors (Table III). Among patients with multifocal tumors, the maximum diameter of multiple tumors ranged between 0.8 and 7.5 cm, with a median of 3.4 cm. A total of 20 patients had two tumors, 8 patients had three tumors, 3 patients had four tumors, 2 patients had five tumors and 1 patient had more than five tumors (seven tumors). The levels of AFP before TACE treatment were <200 ng/ml in 54 cases, while 60 patients exceeded these levels. After TACE treatment, AFP levels in 99 cases were <200 ng/ml and 15 cases remained at levels of ≥200 ng/ml. A total of 59 patients responded well to TACE, while the remaining 55 patients responded poorly. The end date of the follow-up was April 2020, with a median of 23.5 months (range, 3–82 months). A total of 37 patients died and the overall mortality rate was 32.5% (Table III).

To further verify the effects of TACE treatment on the immune function of patients, the immune status of patients was examined before and after TACE treatment. The levels of CD3⁺, CD4⁺/CD8⁺, PD1 and PD-L1 were selected as indices to evaluate immune function (Table IV, Fig. 1). Before patients underwent TACE treatment, the mean values of PD1 and PD-L1 mRNA expression levels were 2.496 and 2.853, respectively. After TACE treatment was performed, the mean values of PD1 and PD-L1 mRNA expression levels were 4.312 and 3.11, respectively. CD4⁺/CD8⁺ significantly decreased after TACE treatment (P<0.001). Notably, the relative mRNA expression levels of PD1 were significantly upregulated (P<0.001). The mRNA expression levels of PD-L1 were also increased after TACE treatment; however, this difference was not significant (P=0.083). In addition, there was no significant difference observed in CD3⁺ levels (P=0.167).

To further clarify the association between the therapeutic effects of TACE and PD1/PD-L1 expression, the mRNA expression levels of PD-L1/PD1 were detected in healthy donors and patients, which were divided into well or poor response to TACE groups (Fig. 2A and B). Compared with healthy subjects, the expression levels of PD-L1/PD1 in patients with HCC were significantly increased (P<0.001). The mRNA expression levels of both PD-L1 (Fig. 2A) and PD1 (Fig. 2B) in patients with HCC with poor TACE response were higher compared with those in patients with satisfactory TACE response (all P<0.001). Kaplan-Meier curves were plotted to analyze the survival of patients with high and low expression levels of PD1/PD-L1. According to the average mentioned in Table IV, patients were divided into two groups for further analysis. Before patients underwent TACE treatment, PD1 mRNA levels >2.5 and PD-L1 mRNA levels >3.0 were regarded as high PD1/PD-L1 expression levels, respectively. After TACE treatment was performed, PD1 mRNA expression >4.0 and PD-L1 mRNA expression >3.0 were considered as high PD1/PD-L1 expression levels, respectively. There was no statistical difference in survival prognosis between the high expression PD1 (P=0.761)/PD-L1 (P=0.221) group and the low expression group before TACE treatment (Fig. 2C and D). However, after TACE treatment, PD-L1 expression could be used to predict the prognosis of patients with HCC. After TACE treatment, compared with high PD-L1 expression, high PD-L1 expression significantly indicated improved prognosis (P=0.017), while the expression level of PD1 (P=0.371) could not predict the prognosis before and after TACE treatment (Fig. 2E and F).