The study has been approved by the appropriate institutional committee and have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The institutional review board of The First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, Zhejiang, China) approved the present study. Written informed consent was obtained from all individual participants included in the study.
Formalin-fixed paraffin-embedded tissue of 61 patients with locally advanced hypopharyngeal cancer treated with (chemo)radiotherapy and surgery at the First Affiliated hospital, College of Medicine, Zhejiang University between 2008 and 2017 was collected, among them, 14 patients were subsequently excluded due to the unavailable tumor samples or missing clinical data. Excluded criteria: Patients with distant metastasis at diagnosis, patients who received preoperative targeted therapy, immune therapy or chemotherapy, patients with more than one malignancy.
All patient datas were reviewed for the following baseline characteristics: gender, age, primary tumor histologic subtype, tumor location, clinical stages. RT parameters were also recorded, including total dose, dose per fraction, time between RT and sample resection.
OS data for patients were obtained by outpatient service or telephone. OS was determined from the date of diagnosis to the death of patients. A follow-up examination was performed every month during the first year, every three months during the second year, and every six months after the third year. In addition to routine physical examinations, patients underwent laryngoscopy, cervical CT or magnetic resonance imaging (MRI), or whole body PET/CT.
Immunohistochemical analysis and evaluation
we analysed the specimens of pre-RT and the post-RT. The expression of PD-L1, Glut-1, and the numbers of CD4+ and CD8+ T cells in the tumor tissues were detected by immunohistochemistry in 47 cases of hypopharyngeal cancer, Formalin-fixed paraffin-embedded specimens were obtained from the predominant lesions in each subject. Immunohistochemical staining was performed to detect expression of PD-L1(1:100 dilution; catalog no: 66248-1-Ig, Proteintech), Glut-1(dilution, 1:100; catalog no: ab14683; Abcam, Cambridge, UK) on tumor cells, and CD4+ T cells(1:50 dilution; catalog no: DF6451, Affinity), CD8+ T cells(1:200 dilution; catalog no: 66868-1-1, Proteintech) in the tumor specimens. Serial sections (4µm) subjected to immunohistological staining were fixed with 3% H2O2, and treated with antigen retrieval solution for 15 min. Monoclonal antibody incubated the sections for 30 min at 36‒38°C, followed by secondary antibody (K5007, Dako) incubation for 15 min at 20‒25°C. The final reaction product was developed by exposure to 0.03% diaminobenzidine, and the nuclei were counterstained with hematoxylin.
We scored the percentage of tumor cells with PD-L1 positive stained, increasing by 5% increments, and used a semiquantitative scoring method to evaluate PD-L1 expression, high PD-L1 expression was defined at a minimum of 10% of stained cells.
The staining intensity of Glut-1 was classified as no staining, weak, moderate, and strong intensity for 0, 1, 2, or 3 scores, respectively. The percentage of stained cells was classified as follows: 0‒25% stained cells for 1 score, 26‒50% stained cells for 2 score, 51‒75% stained cells for 3 score, and >75% stained cells for 4 score. The Glut-1 expression was assessed semi-quantitatively using the product of these scores (intensity × percentage of stained cells): 0‒5 points = negative expression and 6‒12 points = positive expression.
To evaluate CD4+, CD8+ T cells infiltration in tumor, we counted the numbers of CD4+ and CD8+ T cells in a selected hotspot under 400×magnification and selected the median number of CD4+, CD8+ T cells as the cut-off point for CD4+ and CD8+ T cells density.
Two experienced pathologists calculated the staining intensity and the percentage of stained cells independently.
Cell culture and reagents
FaDu cell line was purchased from the Cell Research Institute of Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium (Sigma, USA), supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin (Gibco, USA) and 10% heat-inactivated FCS, at 37°C in a 5% CO2 atmosphere.
Sequences of GLUT-1 and PD-L1 entire coding regions were obtained from GenBank, and primers were designed using ClustalX and the Omega 2.0 software. The high-purity total RNA rapid extraction kit was purchased from Generay (Cat No: GK3016, Batch: 1703G01), the reverse transcription kit HiScript-II Q RT SuperMix for qPCR was purchased from Vazyme (Cat No: R222-01, Batch: 7E092G6), PrimeScriptTM RT reagent Kit was purchased from TaKaRa (Cat No: RR037A, Batch: AK5302-1), qPCR reagent ChamQ SYBR Color qPCR Master Mix was purchased from Vazyme (Cat No: Q411-02, Batch: 7E092H6). The quantitative PCR instrument was CFX connect Real-Time PCR System. PVDF membrane was purchased from Millipore (Cat No: IPVH00010, Batch: K5JA5013L).
Tumor cell line irradiation
To determine whether GLUT-1 is a key factor involved in the mediation of tumor immune mircoenvironment by radiotherapy, we inhibited GLUT-1 expression using GLUT-1 siRNA. GLUT-1 siRNA was purchased from GenePharma Co. Ltd. (Shanghai, China). The sequences were: sense, 5’-GGAAUUCAAUGCUGAUGAUTT-3’; antisense, 5’-AUCAUCAGCAUUGAAUUCCTT-3’. We performed the GLUT-1-siRNA transfection when the cells reached 50% conﬂuence. The FaDu cell and siRNA-GLUT-1 FaDu cell were both seeded at a density of 10,000–20,000 per 25 cm2 and were divided into 4 goups respectively: control group, 24h, 48h, 96h group. Tumor cells were subjected to radiation after resting overnight except the control group. RT was taken using an X-ray generator (22.7 mA, 120 kV, variable time; GE Inspection Technologies, Germany) with a single dose of 10 Gray on day 1. The tumor cells of 24h, 48h, 96h group were harvested at 24h, 48h and 96h after the radiotherapy respectively, PD-L1 and GLUT-1 expression on tumor cells were analyzed. The control group tumor cells were harvested and analyzed on day2.
Reverse transcription polymerase chain reaction (RT-PCR)
The PCR primers used were as follows: GLUT-1 sense, 5′-GTCAACACGGCC TTCACTG-3′, GLUT-1 antisense, 5′-GGTCATGAGTATGGCACAACC-3′ (111 bp), PD-L1 sense, 5′-TTACAGCAGCCAGACGATCA-3′, PD-L1 antisense, 5′-CCCTGC AGTAGGTTTCTGCT-3′(233 bp). GAPDH sense, 5′-TGTTGCCATCAATGACCCCTT-3′, GAPDH antisense, 5′-CTCCACGACGTACTCAGCG-3′ (202bp). The specific steps are the same as described previously. To calculation differential gene expression, the 2-ΔΔCt formula was used.
Tumor cells were lysed in Radio Immunoprecipitation Assay (RIPA) lysis solution and were separated by gel electrophoresis and transferred to membranes. We blocked the membranes with 5% non-fat dry milk in TBST and soaked in the primary antibody buffer at 4°C overnight (PD-L1 1:800 dilution(Proteintech, Chicago, IL, USA, Art No: 66248-1-1g), (GLUT-1 1:800 dilution (Proteintech, Chicago, IL, USA, Art no: 20960-1-AP). We soaked the membranes in secondary antibody buffer and incubated at room temperature for for 2h. Enhanced chemiluminescence was used to visualize the proteins and then the proteins were exposed to X-ray film. Protein expression was analyzed semi-quantitatively using the ChemiDoc XRS+ System (Bio-RAD, USA).
SPSS software (ver. 22.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Categorical variables were assessed by χ2 or Fisher’s exact tests. Correlation analyses were performed using Spearman’s rank analysis. Changes in PD-L1, Glut-1 expression and CD4+, CD8+ T cells before and after RT were tested with Student’s t-test. The Kaplan-Meier method and log-rank test were used to calculate survival curves and compare the results. The Cox proportional hazards regression model was used for multivariate analysis. P values <0.05 were considered to indicate statistical significance.