Contributed equally
The growing importance of antitumour immunity by cancer immunotherapy has prompted studies on radiotherapy-induced immune response. Previous studies have indicated that programmed cell death-1 ligand (PD-L1) expression is regulated by DNA damage signalling. However, PD-L1 up-regulation after radiotherapy has not been fully investigated at the clinical level, particularly in the context of expression of DNA repair factors. The present study examined the correlation of mRNA expression between PD-L1 and non-homologous end joining (NHEJ) factors using The Cancer Genome Atlas database analysis. Among NHEJ factors,
The success of immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1)/programmed cell death-1 ligands (PD-L1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has highlighted the important role of antitumour immunity in cancer treatment (
Previous studies have suggested stimulation of local and systemic immune responses after radiotherapy; for example, radiotherapy promotes the induction of damage-associated molecular patterns and tumour-associated antigens as well as the release of exosomes containing DNA fragments that activate dendritic cells (
To date, multiple pathways regarding the regulation of PD-L1 expression in tumours have been suggested. Microsatellite instability induced by defects in mismatch repair (
The present study examined the correlation of mRNA expression between PD-L1 and NHEJ factors using The Cancer Genome Atlas (TCGA) dataset analysis.
Normalised RNA sequences of
A total of 75 patients with cervical squamous cell carcinoma (median age, 62 years; range, 32–87 years) who met the following criteria were eligible for the present retrospective study: i) Pathologically-confirmed newly diagnosed cervical cancer by pathologists who were not associated with the present study; ii) treated with definitive platinum-based chemoradiotherapy (CRT group) or radiotherapy alone (RT group) at Gunma University Hospital between August 2009 and November 2013; iii) staged as IB1-IVA according to the FIGO classification 2008 (
All patients received definitive radiotherapy in combination with external body radiotherapy (EBRT) and intracavity brachytherapy (ICBT). EBRT typically used 10 MV X-rays with 4-field irradiation. The most common EBRT dose and fractionation was 50 Gy in 25 fractions (2 Gy/fraction, once per day, five times weekly). EBRT was performed using a combination of total pelvic irradiation (20–40 Gy) followed by a central shielded irradiation at a 3-cm width. The pelvic field was expanded to the metastatic area in patients with paraaortic lymph node metastases. Patients with lymph node metastases received an additional boost of 6–8 Gy in three to four fractions. ICBT was performed once a week with concurrent central shielding EBRT. EBRT was not performed on the day of ICBT administration. Three-dimensional image-guided brachytherapy was performed on all patients using a high-dose-rate 192Ir remote after loading system (microSelectron® Digital; Elekta Instrument AB). The prescription dose for each ICBT was determined to cover 90% of the high-risk clinical target volume, using a total dose of 6 Gy. Bulky and/or asymmetrical tumours were treated using additional interstitial irradiation. ICBT was typically performed four times. Concurrent chemotherapy was given to 64.0% (48/75) of the patients. According to the Japan Society of Gynecologic Oncology (JSGO) Guidelines 2017 for the Treatment of Uterine Cervical Cancer, cisplatin was usually administered weekly at a dose of 40 mg/m2 (
PD-L1 expression in tumour cells and Ku80 expression in the nuclei of biopsy specimens excised from the cervical squamous cell carcinoma pre-RT and 10 Gy-RT were evaluated using immunohistochemical staining. Biopsied samples were fixed in 10% buffered formalin for 24 h at room temperature and then dehydrated, degreased and paraffin-embedded. Paraffin embedded sections (4-µm thick) were dewaxed in xylene at room temperature and rehydrated using a graded ethanol series. Endogenous peroxidase activity was blocked using a 10-min incubation in 0.3% hydrogen peroxide. After blocking by 10% goat normal serum (cat. no. 5425; Cell Signalling Technology, Inc.) for PD-L1 and Ku80 sections with a 20 min incubation at room temperature, the sections were incubated overnight with primary antibodies at 4°C. The sections were incubated with a commercially available biotin-streptavidin immunoperoxidase kit [Histofine; SAB-PO (rabbit); cat. no. 424032; Nichirei Biosciences, Inc.; Nichirei Corporation] for 20 min at room temperature. Then, the sections were incubated for 5 min with diaminobenzidine at room temperature. The following antibodies were used: Monoclonal anti-PD-L1 antibody (1:100; clone E1L3N rabbit IgG; cat. no. 13684; Cell Signalling Technology, Inc.) and monoclonal anti-Ku80 antibody (1:200; clone C48E7, and rabbit IgG; cat. no. 2180T; Cell Signalling Technology, Inc.). The quality of the tumour samples was carefully evaluated and validated independently by two pathologists who are co-authors of the present study.
All immunostaining images were obtained using the light microscope Leica DM4000 B (Leica Microsystems, GmbH) equipped with a ×20 objective lens. Expression of cytosolic PD-L1 was measured using public domain software ImageJ v1.53a (National Institutes of Health) as follows. In 32-bit colour images, which are commonly used in computers, each pixel has a red, blue and green signal intensity from 0 to 255. Brown, which is composed of a higher red than blue signal, represents PD-L1 labelled by antibody in the immunohistochemical image. The nucleus is shown in blue and the background is shown in white, which consists of high red, blue and green signals. Therefore, brown is the only colour that has much higher red compared with blue signal in the microscopy images. The captured images were split into red, blue and green channels. The PD-L1 signal was enhanced by subtracting the blue signal from the red signal, taking advantage of the fact that all the subtracted values are returned as zero when the subtracted value is zero or less. The mean of the signal intensities in three areas of the tumour tissues were quantified. PD-L1 change was calculated as PD-L1 intensity (10 Gy-RT) subtracted by PD-L1 intensity (pre-RT). Furthermore, the percentages of tumour cells with cell-surface staining for PD-L1 were recorded and expressed as a tumour proportion score (TPS). If the TPS was 1–50%, the sample was classified as PD-L1 1+, whereas samples with TPS >50% were classified as PD-L1 2+. The population of Ku80-positive cells were measured using an opened source software QuPath (v0.1.2; Queen's University Belfast; Northern Ireland). The tumour tissue area was targeted to detect Ku80-positive cells and the Ku80 positivity was quantified.
HeLa cells were obtained from the American Type Culture Collection and cultured in Eagle's Minimum Essential Medium (FUJIFILM Wako Pure Chemical Corporation) with 10% foetal calf serum (Sigma Aldrich; Merck KGaA) at 37°C. siRNA transfection was performed using DharmaFECT (GE Healthcare Dharmacon, Inc.). siRNA was added to suspended HeLa cells after trypsinisation (the final concentration of siRNA is 16 nM). Non-targeting siRNA was used as negative control. The siRNA oligonucleotides used are listed in
For immunoblotting, cells were harvested at 48 h after transfection with ×1 Sample Buffer [50-mM Tris, 2% sodium dodecyl sulphate, 6% glycerol, 1% (w/w) 3-mercapto-1,2-propanediol and 0.008% bromophenol blue] following PBS wash. Harvested samples were boiled at 95°C for 5 min and sonicated using a Q55 Sonicator Ultrasonic Homogeniser (QSonica LLC; power amplification 15% for 5 sec, twice) (
Cells were exposed to 10 Gy of irradiation then incubated for 48 h prior to flow cytometry analysis. Harvested cells were washed and collected with ice-cold 1 mM EDTA-phosphate-buffered saline and then stained with anti-PD-L1 antibodies (clone E1L3N rabbit IgG; cat. no. 13684; Cell Signalling Technology, Inc.) for 20 min on ice. The fluorochrome used was APC. Dead cells detected by propidium iodide (Sigma-Aldrich; Merck KGaA) were excluded from the analysis. Flow cytometry analysis was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific, Inc.). Mean fluorescence intensity (MFI) of the PD-L1 was calculated as: MFI (PD-L1)-MFI (isotype control).
The correlation between Ku80 positivity and PD-L1 signal intensity was evaluated using Spearman's rank correlation coefficient. Clinical outcomes were calculated using the Kaplan-Meier method, and statistically significant differences were confirmed using the log-rank test. Mann-Whitney U test was used to compare Ku80 positivity in pre-RT samples and classification of PD-L1 expression. MFI in flow cytometry analysis was compared using unpaired Student's two-tailed t-test. Results were shown as mean ± error bars, which represent the standard error of three samples in the experiment. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS (v24; IBM Corp.).
TCGA datasets were used in the present study to investigate the relationship of gene expression between PD-L1 and DNA double-strand break (DSB) repair factors in tumour cells. Our previous study reported that tumour specimens harbouring mutations in NHEJ genes (Ku70/80) exhibit greater PD-L1 expression after irradiation in several cancer types (
Our previous study reported that PD-L1 expression is up-regulated in cervical squamous cell carcinoma specimens after being treated with 10 Gy of radiotherapy (
To confirm radiation-induced PD-L1 up-regulation in a Ku80-depleted cervical cancer cell line
The present study revealed that
Our previous studies reported that DNA damage signalling induced by irradiation or oxidative damage up-regulates PD-L1 expression at the transcriptional level (
The present study revealed that Ku80-depleted HeLa cells also expressed greater PD-L1 after irradiation. Together with the findings of TCGA analysis, which demonstrated that low
To date, multiple pathways regarding PD-L1 expression after DNA damage have been suggested, such as cytosolic sensors cGAS/RIG-I transducing immune-activating signal releasing interferon stimulate genes following the recognition of cytosolic DNA/RNA released from the nucleus (
The present study quantified the signal intensity of PD-L1 using software. Although the signals from inside the cell or the cell surface were unable to be differentiated, which seems to be important to detect functional PD-L1 on cell surface, the software approach has advantages in measuring an interval scale of their expression. PD-L1 expression was also examined using a manual classification method scored by cell-surface staining for PD-L1. Notably, the present study obtained similar results using both methods. Thus, it was hypothesized that this computational measurement could be a useful tool to quantify PD-L1 expression; however, it must be carefully accessed depending on the type of targets.
Limitations of the present study included the limited number of clinical samples. As there are multiple regulators of PD-L1 expression (
Recent radiotherapy technologies are highly developed and enable radiation oncologists to target cancer using high-precision radiotherapy, while also avoiding irradiation to the surrounding normal tissue. These technological developments increase the importance of controlling tumour cells outside the irradiation field for long-term survival of patients (
Notably, the situation of radiotherapy, such as dose fractionation and radiation quality, affects immune responses. For example, hypofractionated radiation at 8–12 Gy per fraction activates the cGAS/STING pathway more effectively compared with a higher single dose of ≥20 Gy (
The present study demonstrated that PD-L1 expression is induced by radiotherapy and can be affected by the expression levels of Ku80 prior to radiotherapy. The present study did not reveal any differences in Ku80 positivity with patient outcomes, which may be because of the multiple functions of Ku80 that are involved in both tumour immunity and DNA repair (
The authors thank Dr Yuya Yoshimoto (Fukushima Medical University, Fukushima; Gunma University, Maebashi, Japan) for his assistance in performing the immunohistochemical analysis. The authors thank Mr Koji Isoda (Gunma University, Maebashi, Japan) for his technical assistance in performing the immunohistochemical analysis.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
TK, YM, HS, TBMP, YU, KO, SK, KS, HI, HY and AS performed the experiments and analysed the data. TK, TBMP, HS and AS wrote the manuscript. TK, YM, SN and KO coordinated the clinics, carried out the treatment and participated in the patient follow-up. SG, TN and TO contributed conception and design, and reagents/materials/analysis tools and gave final approval for the manuscript version to be published. TK and HS confirm the authenticity of all the raw data. HS and TO supervised the study with the support from AS. All authors have read and approved the final manuscript.
The present study was approved by the Institutional Review Board for clinical trials of Gunma University (approval no. HS2020-015; Maebashi, Japan). The protocol is described on the hospital website, and subjects were provided the opportunity to opt-out; therefore, no additional consent was required from patients.
Not applicable.
The authors declare that they have no competing interests.
breast invasive carcinoma
cervical squamous cell carcinoma and endocervical adenocarcinoma
colon adenocarcinoma
double-strand break
head and neck squamous cell carcinoma
immune checkpoint inhibitors
lung adenocarcinoma
mean fluorescence intensity
non-homologous end joining
skin cutaneous melanoma
stimulator of IFN genes
The Cancer Genome Atlas
tumour proportion score
uterine corpus endometrial carcinoma
ataxia telangiectasia and Rad3-related
checkpoint kinase 1
DNA-dependent protein kinase, catalytic subunit
X-ray repair cross-complementing protein 4
DNA ligase 4
Correlation between PD-L1 gene expression and DNA double-strand break repair factors. Volcano plots indicating the correlation between PD-L1 expression and central non-homologous end joining factors expression levels. Mutation statuses were provided by The Cancer Genome Atlas project were downloaded from the Genomic Data Commons Data Portal. BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; HNSC, head and neck squamous cell carcinoma; LUAD, lung adenocarcinoma; SKCM, skin cutaneous melanoma; UCEC, uterine corpus endometrial carcinoma; DNA-PKcs, DNA-dependent protein kinase, catalytic subunit; XRCC4, X-ray repair cross-complementing protein 4; LIG4, DNA ligase 4.
Correlations between PD-L1 expression and Ku80 positivity in cervical squamous cell carcinoma after treatment with radiotherapy. (A) Scatter plot showing the relationship between PD-L1 expression (pre-RT and 10 Gy-RT) and Ku80 positivity using ImageJ and QuPath, respectively. (B) Scatter plot showing the relationship between PD-L1 changes comparing pre-RT and 10 Gy-RT and Ku80 positivity. SRCC, Spearman's rank correlation coefficient; PD-L1, programmed cell death-1 ligands; RT, radiotherapy.
Representative images of immunohistochemical staining. The upper three images show specimens from the same patient with 91.0% Ku80 positivity (relatively high) and low PD-L1 induction following 10 Gy-RT. The lower three images show specimens from another patient with 5.9% Ku80 positivity (relatively low) and high PD-L1 induction following 10 Gy-RT. Scale bar, 50 µm. PD-L1, programmed cell death-1 ligands; RT, radiotherapy.
PD-L1 expression is enhanced after irradiation of Ku80-depleted HeLa cells. (A) Immunoblotting of Ku80 in HeLa cells transfected with scrambled siCont RNA or siKu80. (B) Flow cytometry histogram of PD-L1 expression after 10 Gy irradiation of HeLa cells. Cell-surface PD-L1 expression was examined at 48 h after 10 Gy irradiation. (C) Depletion of Ku80 enhanced the up-regulation of cell-surface PD-L1 expression after irradiation. Error bars represent the standard error of three samples in the experiment. *P<0.05. si, short interfering; cont, control; PD-L1, programmed cell death-1 ligands; IR, irradiation; MFI, mean fluorescence intensity.
Characteristics of patients (n=75) with cervical squamous cell carcinoma.
Characteristics | Value |
---|---|
Observation period, months | |
Median (range) | 63 (8–120) |
Age, years | |
Median (range) | 62 (32–87) |
Treatment, n (%) | |
RT alone | 27 (36%) |
Concurrent CRT | 48 (64%) |
FIGO stage 2008, n (%) | |
IB | 11 (15%) |
II | 31 (41%) |
III | 31 (41%) |
IVA | 2 (3%) |
Lymph node metastasis in pelvis, n (%) | |
Positive | 36 (48%) |
Negative | 39 (52%) |
Para-aortic lymph node metastasis, n (%) | |
Positive | 6 (8%) |
Negative | 69 (92%) |
TCGA analysis.
Gene | Study | Correlation | Approximation formula | Spearman's rho (r) | P-value |
---|---|---|---|---|---|
COAD | Negative | y=−0.001536×+32430 | -0.079 | 0.070 | |
BRCA | Negative | y=−0.003198×+34244 | -0.005 | 0.869 | |
SKCM | Negative | y=−0.01340×+62900 | -0.157 | 0.360 | |
LUAD | Negative | y=−0.04393× +181872 | -0.236 | 8.88×10−16 | |
HNSC | Negative | y=−0.01328×+136161 | -0.053 | 0.232 | |
UCEC | Negative | y=−0.009837×+30808 | -0.397 | 2.21×10−5 | |
CESC | Negative | y=−0.0433×+177708 | -0.151 | 0.008 | |
COAD | Negative | y=−0.003319×+38104 | 4.81×10−4 | 0.991 | |
BRCA | Positive | y=0.0009455×+26915 | -0.025 | 0.374 | |
SKCM | Negative | y=−0.01050×+71586 | -0.229 | 0.179 | |
LUAD | Positive | y=0.001316×+118475 | -0.154 | 1.87 ×10−7 | |
HNSC | Negative | y=−0.01467×+165072 | -0.108 | 0.016 | |
UCEC | Positive | y=0.001272× +10171 | -0.090 | 0.353 | |
CESC | Negative | y=−0.008341×+141397 | -0.110 | 0.054 | |
COAD | Positive | y=0.01038×+25588 | -0.108 | 0.014 | |
BRCA | Positive | y=0.003123×+27447 | 0.047 | 0.098 | |
SKCM | Negative | y=−0.01111×+45835 | -0.171 | 0.320 | |
LUAD | Positive | y=0.01127×+117308 | -0.069 | 0.019 | |
HNSC | Negative | y=−0.03491×+137903 | -0.035 | 0.432 | |
UCEC | Negative | y=−0.008411×+16815 | -0.371 | 7.98×10−5 | |
CESC | Negative | y=−0.1111×+163343 | -0.074 | 0.198 | |
COAD | Positive | y=0.1244×+19506 | 0.069 | 0.116 | |
BRCA | Positive | y=0.03546×+26440 | 0.077 | 0.007 | |
SKCM | Positive | y=0.5896×+763.2 | 0.280 | 0.098 | |
LUAD | Positive | y=0.1271×+111566 | 0.062 | 0.036 | |
HNSC | Positive | y=0.3040×+87953 | 0.175 | 8.04×10−5 | |
UCEC | Negative | y=−0.02123× +14814 | -0.233 | 0.014 | |
CESC | Negative | y=−0.1882×+137753 | 0.142 | 0.012 | |
COAD | Positive | y=0.07266×+25129 | 0.022 | 0.615 | |
BRCA | Positive | y=0.08249×+23007 | 0.078 | 0.006 | |
SKCM | Positive | y=0.08564×+33814 | 0.102 | 0.552 | |
LUAD | Positive | y=0.1574×+109242 | 0.099 | 0.001 | |
HNSC | Positive | y=0.08293×+110697 | 0.097 | 0.029 | |
UCEC | Negative | y=−0.02302× +14377 | -0.009 | 0.924 | |
CESC | Negative | y=−0.07419×+122540 | -0.029 | 0.605 |
COAD, colon adenocarcinoma; BRCA, breast invasive carcinoma; SKCM, skin cutaneous melanoma; LUAD, lung adenocarcinoma; HNSC, head and neck squamous cell carcinoma; UCEC, uterine corpus endometrial carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; DNA-PKcs, DNA-dependent protein kinase, catalytic subunit; XRCC4, X-ray repair cross-complementing protein 4; LIG4, DNA ligase 4.