Lung cancer is a malignant tumor with the highest morbidity and mortality worldwide (1), and non-small cell lung cancer (NSCLC) accounts for 85% of cases (2). With the application of low-dose spiral computed tomography screening, the detection rate of early-stage lung cancer has improved, but many patients are still in an advanced or locally advanced stage at the time of diagnosis (3), which has a poor prognosis and survival rate. Therefore, it has always been the goal of clinicians to find specific biomarkers for earlier diagnosis and evaluate their prognostic value for lung cancer.
Butyrophilin (BTN) was first found in milk and is a type I transmembrane protein of an immunoglobulin (Ig) superfamily (4). The BTN family of genes, located at 6p22.1, is a group of major histocompatibility complex (MHC) related genes, which encode type I transmembrane proteins containing two extracellular Ig domains and an intracellular B30.2 domain. The three subfamilies of the human BTN gene are located in the MHC I region, and can be divided into BTN1, BTN2 and BTN3, which each comprise seven genes, including BTN1A1, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2 and BTN3A3 (5). The basic structure of the extracellular domain of the BTN family is the same as the members of the co-stimulatory and coinhibitory molecules family (6). Therefore, BTN family molecules are considered to be B7 family-related proteins. The receptor-ligand interactions between members of the B7 family are widely involved in immune regulation, such as the interactions between B7.1 (CD80), B7.2 (CD86) and T-cell surface receptors CD28 and CTLA-4, which initiate T-cell activation and inhibition signals, respectively. Programmed death receptor-ligand 1 (PD-L1/CD274), PD-L2 (CD273) and its receptor PD-1 (CD279) are T-cell coinhibitory molecules (7-9). The BTN and B7 families share the same origin (10), suggesting that the BTN family may have similar immunomodulatory functions. Previous study had shown that the BTN3A1 protein plays a key role in the activation of γδ T cells. By targeting the BTN3A1 receptor, the immunosuppression of αβ T cells can be achieved and γδ T cells can be activated cooperatively (11). In addition, BTN family members have many other functions and are widely involved in the occurrence of various human diseases. The rs1979 locus of the BTN3A2 gene is most significantly associated with schizophrenia. Abnormal expression of BTN3A2 affects the balance of neuronal excitatory and inhibitory synaptic transmission activity, which increases the risk of schizophrenia (12). The expression levels of BTN1A1, BTN2A2, BTN3A3 change significantly in inflammatory bowel disease (13). In addition, the BTN3A subfamily has been confirmed to be expressed on the surface of a variety of tumor cells (10,14). Recent studies have shown that specific single nucleotide polymorphisms in BTN3A3 and BTN3A2 increase the risk of ovarian cancer and gastric cancer, respectively (15,16). The expression of BTN3A3 can predict the overall survival (OS) of patients with gastric cancer who are treated with fluorouracil-based chemotherapy (17). In the tumor microenvironment (TME) of breast cancer, the LSECtin protein located on the surface of tumor-associated macrophages can promote the stemness of tumor cells by binding to BTN3A3 receptors on the surface of breast cancer stem cells (18).
BTN family members have diverse functions, which are related to the immunomodulation, initiation and progression of tumors. BTN3A3 is the membrane protein A3 of the third BTN subfamily. Compared with other BTN family members, there are few studies on BTN3A3, and research is still in the initial stage. In particular, the expression of BTN3A3 in NSCLC and its possible function have not been reported so far. Therefore, in-depth study of the expression of BTN3A3 in NSCLC, and its influence on the tumor’s biological behavior with expression changes and its potential role in the TME has great significance in understanding the occurrence and development mechanisms of NSCLC and the interactions between tumor and immune cells.
We present the following article in accordance with the REMARK reporting checklist (available at http://dx.doi.org/10.21037/atm-21-163).
Patients with primary NSCLC who were treated in the Department of Thoracic Surgery of Beijing Chest Hospital affiliated with Capital Medical University from January 2013 to December 2015 were included in this study. Frozen and paraffin-embedded tissue blocks of primary tumor specimens were obtained for experiments. Specific inclusion criteria were: (I) first diagnosis of NSCLC; (II) positive histopathological diagnostic results; (III) chemotherapy and radiotherapy not performed before surgery. The exclusion criteria are: (I) insufficient tumor tissue; (II) complicated with other malignant tumors; (III) incomplete clinical or follow-up data. Tumor staging was according to the 8th edition of the American Joint Commission on Cancer (AJCC) staging system (19,20), and the subtypes of NSCLC were classified according to the WHO guidelines (21). Patients in the study were followed up for survival and the OS was calculated according to the time period between the date of operation and the date of death or the last follow-up. This study was approved by the Ethics Committee of Beijing Chest Hospital (No. YJS-2021-010). All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). We used frozen and paraffin-embedded tissue blocks of primary tumor specimens, which belongs to the category of exemption from informed consent.
RNA preparation and quantitative real-time polymerase chain reaction (rt-qPCR)
RNA extraction reagent (RNAprep Pure Tissue Kit, RNAprep Pure, DP431) from the TianGen Company (China) was applied to extract the total RNA from NSCLC and adjacent tissues following the manufacturer’s instructions. First-strand cDNA synthesis was performed using Fast Quant RT Kit With gDNase (KR106; TianGen Co., Beijing, China). rt-qPCR was performed with a Roche Light Cycler 480 system, using QuantiNova SYBR Green PCR Kit (QIAGEN). The primer for BTN3A3 were as follows: forward 5'-GCCCTCTTCAAACCTGCGG-3' and reverse 5'-AGGACACAGTAACGCCATTCA-3'. Primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were as follows: forward 5'-TCAAGAAGGTGGTGAAGCAGG-3' and reverse 5'-GCGTCAAAGGTGGAGGAGTG -3'. The primers were synthesized by Sangon Biotech Company (Shanghai, China). All PCR tests were repeated at least three times.
Cells were lysed by RIPA buffer (P0013B, Beyotime, Shanghai, China), and the protein concentration was determined by BCA Protein Quantification Kit (PC0020, Solarbio, Beijing, China). After separation by 12% SDS polyacrylamide gel, the corresponding proteins were moved to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010, Billerica, MA, USA). Primary antibodies specific to BTN3A3 (dilution 1:5,000, ab251692, abcam, Shanghai, China) were applied, β-actin (dilution 1:5,000, 20536-I-AP, proteintech, Chicago, USA) was used as the control.
Cell culture and transfection
Normal human bronchial epithelial cell line (16HBE), human lung adenocarcinoma cell lines (A549, H2009 and H1975) and human lung squamous cell carcinoma cell lines (H226, SK-MES-1 and H1703) were preserved in the Central Laboratory and the Department of Cellular and Molecular Biology of Beijing Chest Hospital. The cells were cultured in RPMI-1640 complete culture medium (EallBio, China) at 37 °C in a 5% CO2 incubator. The small interfering RNA (siRNA) transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The knockdown effect was validated by rt-qPCR and western blot. Sequences of siRNA used for transfection were as follows: BTN3A3 siRNA sense: 5'- GCAACAACCAAUCAGAACCAUTT-3', BTN3A3 siRNA antisense: 5'- AUGGUUCUGAUUGGUUGUUGCTT-3'. BTN3A3 siRNA negative control (NC) sense: 5'- UUCUCCGAACGUGUCACGUTT-3', BTN3A3 siRNA-NC anti sense: 5'- ACGUGACACGUUCGGAGAATT-3'.
Scratch wound assay
The blank control group (mock), the siRNA negative control group (NC) and the siRNA knock-down group (siRNA) cells in logarithmic growth phase were seeded in 6-well plates and cultured overnight in RPMI-1640 complete medium. After being scratched with plastic tips, the cells were cultured in serum-free medium. Wound closure was viewed at 48 h.
Transwell chambers with 6.5-mm aperture were purchased from Corning (NY, USA). Three groups of cells were seeded in the upper chambers and 10% fetal bovine serum (FBS)-RPMI-1640 was added to the lower chambers. After culture for 24 h, 4% paraformaldehyde was used to fix for 20 min, and gentian violet was used to stain for 20 min. Finally, the number of migrated cells was counted under a light microscope.
Colony formation test
Three groups of cells in logarithmic growth phase were prepared for cell suspension, seeded in 6-well plates and cultured in 10% FBS-RPMI-1640 medium at a density of 1,000 cells at 37 °C in a 5% CO2 incubator for 2 weeks. The culture was terminated when the clonal cell cluster became visible by naked eye. Four percent paraformaldehyde was used to fix for 20 min, and gentian violet was used to stain for 20 min. Clone formation rate = (number of clones formed/number of cells inoculated) ×100%.
CCK-8 proliferation assay
The CCK-8 cell proliferation detection kit was purchased from EallBio Company (China). Three groups of cells in logarithmic growth phase were prepared for cell suspension, seeded in 96-well plates and cultured in 10% FBS-RPMI-1640 medium at a density of 8,000 cells at 37 °C in a 5% CO2 incubator for 24, 48, and 72 h. Next, 10 μL of CCK-8 reagent was added 2 h before each detection time point and incubated without light for 2 h. The optical density (OD) was measured at a wavelength of 450 nm.
Tissue microarray construction
Paraffin-embedded tissue blocks of primary tumor specimens were used to construct tissue microarray (TMA) sections. Importantly, the blocks had to contain enough tumor tissue for the TMA construction. Duplicate 1.5-mm tissue cores were randomly taken from the tumor area in the paraffin-embedded tissue blocks. TMAs containing the tissue cores were then cut into 4-μm sections for multiplex immunofluorescence staining.
Multiplex immunofluorescence staining and analysis
To identify the tumor cells expressing BTN3A3 and other immune markers in the TME, multiplex immunofluorescence staining was performed using Tyramide Signal Amplification Plus Fluorescence kits (PANO 6-plex IHC Kit, Panovue, Beijing, China). Different primary antibodies were sequentially applied, followed by secondary antibody incubation and tyramide signal amplification. Between each step, the slides were washed with TBST (Tris-buffered saline + Tween 20) solution buffer three times for 3 min each time. The TMA slides were treated with microwave heat-antigen retrieval with citric acid buffer (pH 6.0) after each TSA operation. Nuclei were stained with 4'-6'-diamidino-2-phenylindole after all the antibodies had been labeled. The stained slides were scanned by the Vectra system (PerkinElmer), and the multispectral images were analyzed by InForm software 2.4.8 (PerkinElmer).
Differences in means for continuous variables were compared using Student’s t test, and the differences in categorical variables between groups were compared using the χ2 test. Survival curves were plotted using the Kaplan-Meier method. The log-rank test was used to identify the prognostic factors of OS. Univariate and multivariate Cox regression models evaluated the hazard ratio (HRs) with 95% confidence intervals (CI) for OS. The variables with statistical significance in the univariate analysis (P<0.05) were included in the multivariate analysis. Spearman's rank correlation was used to analyze the correlation of quantitative data. All statistical analyses were conducted with IBM SPSS Statistics (Version 23.0), and the survival curves were drawn by GraphPad Prism v.8.0. Bilateral P value <0.05 was considered to be statistically significant.
Detection of BTN3A3 expression in clinical specimens
A total of 75 patients were included in the study and their general clinical characteristics are shown in Table S1. Total RNA was extracted from the tumor and adjacent tissues of NSCLC patients for rt-qPCR detection. The results showed that the expression level of BTN3A3 in tumor tissues was significantly lower than in adjacent tissues, and the difference was statistically significant (P<0.001). The relative expression level of BTN3A3 in tumor and adjacent tissues is shown in Figure 1A.
Relationship between BTN3A3 expression and patients’ OS
BTN3A3 expression and survival time of the 75 patients was analyzed. According to the expression level of BTN3A3 in tumor tissues, the patients were divided into two groups: high expression (N=37) and low expression (N=38). The results showed that the OS of patients with low BTN3A3 expression was significantly shorter than for those with high BTN3A3 expression (P=0.040). Kaplan-Meier survival curves were plotted to represent survival in the two groups (Figure 1B).
Expression of BTN3A3 in NSCLC cell lines
Total RNA was extracted from 16HBE and 6 NSCLC cell lines for rt-qPCR detection. The results showed that the expression level of BTN3A3 in the NSCLC cell lines was significantly lower than in the 16HBE cell line (P<0.05). In the three lung adenocarcinoma cell lines, the expression level of BTN3A3 in H2009 was significantly higher than in the H1975 and A549 cell lines (P<0.05), and in the three lung squamous cell carcinoma cell lines, the expression level was significantly higher in H226 than in the SK-MES-1 and H1703 cell lines (P<0.05) (Figure 2A).
Transfection with siRNA for knock down of BTN3A3 expression in NSCLC cell lines
The siRNA was transfected into the H2009 and H226 cell lines, and rt-qPCR was used to evaluate the efficiency of siRNA knockdown. The results showed that the expression level of BTN3A3 in the siRNA transfection group was significantly lower than in the mock and NC group (P<0.05) (Figure 2B). The total protein of the siRNA, NC, and mock group cells was extracted for western blot assay, and the expression of BTN3A3 protein in the two siRNA-transfected cell lines was significantly decreased (Figure 2C).
Effects of low expression of BTN3A3 on migration and invasion
The siRNA was transfected into the H2009 and H226 cell lines to verify the migratory and invading abilities of NSCLC cells. The results showed that the migratory ability of the two siRNA groups was significantly higher than that of the mock and NC group (P<0.05) (Figure 3A). And the invading ability of cells transfected with siRNA was significantly enhanced (P<0.05) (Figure 3B).
Effects of low expression of BTN3A3 on cell proliferation and clone formation
The siRNA was transfected into the H2009 and H226 cell lines to verify the proliferatory and cloning abilities of NSCLC cells. The results showed that the cloning ability of the two siRNA groups was significantly higher than that of the mock and NC group (P<0.05) (Figure 3C). And cell proliferation was significantly enhanced in the siRNA group (P<0.05) (Figure 3D).
Significance of BTN3A3, CD4, CD8, CD68, and FoxP3 expression with clinicopathological characteristics
Multiplex immunofluorescence staining was used to detect immune-related markers’ expression levels in TMA slides for the NSCLC patients. 88 patients were qualified to be analyzed and finally included in the study (Figure 4). According to the density of BTN3A3+ tumor cells, the 88 patients were divided into two groups. The results showed that in clinical I+II stage, the number of patients with high expression of BTN3A3 (31/44, 70.5%) were significantly higher than those with low expression (19/44, 43.2%), and the difference was statistically significant (P=0.010). Among the poorly differentiated tumors, the number of patients with low expression of BTN3A3 (22/44, 50.0%) were significantly higher than those with high expression (12/44, 27.3%), and the difference was statistically significant (P=0.029). The general clinical characteristics of these patients and detailed results are shown in Table S2. We also quantified the density of BTN3A3+ tumor cells and infiltrating immune cells, and analyzed their relationship with the clinicopathological characteristics of the patients. The results showed that the density of BTN3A3+ tumor cells was significantly higher in patients with earlier clinical stage (P=0.033) (Figure 5A) and with higher degree of tumor differentiation (P=0.048) (Figure 5B); The density of CD8+ T cells in the tumor area was significantly higher in patients with earlier clinical tumor T stage (P=0.035); The density of FoxP3+ T cells in the tumor area was significantly higher in the late clinical stage (P=0.030), whereas the density of CD8+ T cells in the stroma was significantly higher in patients with earlier clinical tumor T stage (P=0.030). Detailed results are shown in Table 1.
Correlation between density of BTN3A3+ tumor cells, CD68+ macrophages, and CD4+, CD8+ and FoxP3+ T cells
BTN3A3+ tumor cell density positively correlated with CD8+ T-cell density in the tumor area (r=0.265, P=0.012) (Figure 6A). CD68+ macrophage density in the tumor area positively correlated with CD4+ T-cell density in the tumor area (r=0.322, P=0.002) and CD68+ macrophage density in the stroma (r=0.447, P<0.001). CD4+ T-cell density in the tumor area positively correlated with CD8+ T-cell density in the tumor area (r=0.322, P=0.002), and with CD4+ T-cell density (r=0.573, P<0.001), CD8+ T-cell density (r=0.281, P=0.008), and FoxP3+ T-cell density (r=0.238, P=0.026) in the stroma. CD8+ T-cell density in the tumor area positively correlated with CD4+ T-cell density (r=0.224, P=0.036) and the CD8+ T-cell density (r=0.558, P<0.001) in the stroma. And the FoxP3+ T-cell density in the tumor area positively correlated with the FoxP3+ T-cell density in the stroma (r=0.352, P=0.001). Detailed results are shown in Table 2.
Effects of clinicopathological features and TME on OS
The 88 patients were followed up for 60 months, during which 67 patients died, giving a mortality rate of 76.14%. The relationships between prognosis and clinicopathological features, TME immune markers (BTN3A3+ tumor cell density, tumor and stromal immune cell densities), radiotherapy and chemotherapy were analyzed. Univariate analysis showed that OS was significantly prolonged in patients aged ≤60 years (P=0.034), with earlier tumor T stage (P=0.005), without lymph node metastasis (P<0.001), with earlier clinical stage (P<0.001), without postoperative radiotherapy (P=0.049), with higher BTN3A3+ tumor cell density (P=0.041) (Figure 6B), and with higher CD8+ T-cell density (P<0.01) (Figure 6C) in the tumor area. The difference was statistically significant. Detailed results are shown in Table S3.
In this study, we used clinical tumor specimens to detect the expression of BTN3A3, and the results showed that its expression in tumor tissues was significantly lower than in adjacent tissues, which preliminarily indicates that a change in BTN3A3 expression may play a key role in the carcinogenesis and progression of NSCLC. The OS of patients with low expression was significantly shortened. We used siRNA to transfect NSCLC cell lines to knock down BTN3A3 expression. The results showed that low expression of BTN3A3 promoted the proliferation, migration and invasion of NSCLC cells. We further investigated the associations between tumor BTN3A3 expression, as well as that of CD4, CD8, CD68 and FoxP3, in the TME, and the clinicopathological characteristics and OS of 88 NSCLC patients using the multiplex immunofluorescence staining technique. We found that the expression of BTN3A3 was relatively high in the early clinical stage and well-differentiated tumors, suggesting that patients with low expression may have a more invasive phenotype. In addition, the density of BTN3A3+ tumor cells positively correlated with the density of CD8+ T cells in the tumor area, suggesting that the expression of BTN3A3 is closely related to immune cell infiltration and immune response. In addition, the expression level of BTN3A3 protein was associated with the patients’ prognosis, and low expression showed a poor OS. At present, there are few studies of the BTN3A subfamily, especially on BTN3A3 expression in NSCLC. This is the first time BTN3A3 has been studied at the level of gene and protein expressions, and we confirmed that low expression of BTN3A3 promotes the proliferation, migration and invasion of NSCLC cells. We quantitatively explored the relationship between BTN3A3+ tumor cells and immune cell infiltration in the TME, and our results indicated that BTN3A3 may play a potential role in the evaluation of long-term prognosis of NSCLC patients. Our study provides a new clinical basis for improving our understanding of the mechanism of occurrence and development of NSCLC and finding specific prognostic indicators and therapeutic targets.
Tumorigenesis is usually accompanied by aberrant alterations in the expression levels of genes involved in cell proliferation, such that the tumor cell growth is uncontrolled, and ultimately leads to tumor metastasis. To date, there are few studies of changes in the expression of BTN family genes, and how they are involved in the process of tumorigenesis. In an ovarian cancer study, Peedicayil et al. found that a single nucleotide polymorphism of BTN3A3 negatively correlated with the incidence of ovarian cancer (16). In a gastric cancer study, Pan et al. established an analytical model and confirmed that BTN3A3 can predict the OS of postoperative patients receiving fluorouracil chemotherapy (17). In a recent study, Liu et al. reported that LSECtin on the surface of tumor-associated macrophages directly interacts with the BTN3A3 on the surface of breast cancer cells, which promoted the stemness of breast cancer cells in the TME (18). Blocking the LSECtin-BTN3A3 signal axis may be a potential treatment for breast cancer. These results suggested that BTN3A3 may also play a key role in regulating the stemness of tumor cells in NSCLC, or mediating the escape of tumor stem cells through interaction with tumor-associated macrophages. On the other hand, during carcinogenesis, tumor formation and metastasis, the interaction between tumor cells and stromal cells (including immune cells, endothelial cells, fibroblasts, etc.) persists in the TME. Both antitumor immunity and tumor metastasis require the dynamic participation of a variety of immune cells. Therefore, the inclusion of more immune cells as the evaluation index of NSCLC would have important clinical implications for better prediction of tumor initiation, progression, metastasis and recurrence.
Tumor infiltrating lymphocytes (TILs) are a heterogeneous lymphocyte population that mainly exists in the TME, and are closely related to the antitumor immune response. TILs have significant effect on facilitating tumor immunity escape (22,23). CD8+ T cells are considered to be effector cells in the immune system that directly kill tumor cells. Studies have shown that infiltration of a higher number of CD8+ T cells is related to a better clinical outcome of various cancers (including hepatocarcinoma, esophageal cancer, colon cancer, and ovarian cancer, etc.) (24-27). In our study, we found that the density of BTN3A3+ tumor cells positively correlated with the density of CD8+ T cells in the tumor area, which may be one of the reasons for the better survival of patients with high expression of BTN3A3. However, whether the expression of BTN3A3 affects the function of CD8+ T cells, and how to the regulatory role plays out between them, need to be further studied. In addition to inhibiting CD8+ T cells, another mechanism for tumor cells’ immune escape is to recruit T regulatory cell (Tregs) into the TME (28,29). Tregs are a specific T-cell subset. Natural Tregs (nTregs) have mature functions, which depend on the expression of forkhead box transcription factor P3 (FoxP3) (30,31). nTregs functionally inhibit the activation or proliferation of a variety of immune cells, then suppresses the immune response, and finally promote tumor growth and metastasis. Therefore, many studies have confirmed that the existence of Tregs is negatively associated with tumor prognosis (32,33). In this study, we did not find a significant correlation between density of BTN3A3+ tumor cells and the density of FoxP3+ T cells in the tumor or stroma. On the one hand, it is considered that the expression of BTN3A3 in tumor cells is not an influencing factor for FoxP3+ T-cell infiltration. However, patients with low BTN3A3 expression show a late tumor stage and lower degree of differentiation, indicating that low expression of BTN3A3 and FoxP3+ T-cell infiltration may be differently involved in the regulation of tumor immunity escape. In addition, we did not find a relationship between the expression of BTN3A3 in tumor cells and CD4+ T-cell infiltration. Considering that CD4+ T cells comprise multiple heterogeneous cell subsets (34,35), different subsets may play various roles in the TME, so it is also unknown whether BTN3A3 is involved in the regulation of the different subsets of CD4+ T cells. Finally, the relationship between the expression of BTN3A3 and CD68+ macrophage infiltration has not been found; however, the latest study has preliminarily proved that the target of LESCtin protein expressed on macrophages is the BTN3A3 protein expressed on the surface of breast cancer stem cells, which could help us understand the function of BTN3A3 in the future, explore the mechanism of tumor metastasis, and provide an important reference for monitoring and evaluating the prognosis of NSCLC.
In conclusion, downregulated expression of BTN3A3 in NSCLC is related to invasive clinicopathological features and poor OS. Low expression of BTN3A3 promotes the proliferation, migration and invasion of NSCLC cells. In the TME, the expression of BTN3A3 in tumor tissue positively correlated with infiltration of CD8+ T cells, which may be an important factor affecting long-term survival. This study reveals a new suppressor gene involved in the carcinogenesis of NSCLC, which is expected to become a potential prognostic marker and therapeutic target for NSCLC patients.
We thank colleagues and members of the Pathology Department of Beijing Chest Hospital for their assistance with experiments.
Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at http://dx.doi.org/10.21037/atm-21-163
Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-21-163
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-21-163). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of Beijing Chest Hospital (No. YJS-2021-010). All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was taken from all the patients.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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(English Language Editor: K. Brown)