Abstract
Background
Endometrial cancer (EC) is the sixth most common cancer in women. A rising age-standardized incidence rate of EC has been observed globally. Tumor-associated neutrophils (TANs) have been demonstrated to be a “double-edged sword” in human cancers. We aimed to study the role of CCL20 and neutrophil infiltration in human EC in the study.
Methods
Differential expression genes (DEGs) associated with chemokines in EC compared with normal samples were analyzed based on The Cancer Genome Atlas (TCGA)-Uterine Corpus Endometrial Carcinoma (UCEC) database, and DEGs associated with neutrophil infiltration were screened out as candidate genes. Through clinical samples, we verified the expression of CCL20 in EC tissues. Gene set enrichment analysis (GSEA) was performed. Through a supernatant co-culture system, flow cytometry and immunofluorescence, we studied the effect of CCL20 on neutrophils. The correlation of CCL20 and neutrophil infiltration with clinicopathological parameters and survival rates in EC patients was analyzed based on TCGA-UCEC database. It was validated through Gene Expression Omnibus (GEO) database and local samples.
Results
CCL20 was screened out to be highly expressed and associated with neutrophil infiltration in EC. CCL20 was associated with immune-related signaling pathways including NOD-like receptor, TNF, NF-kappa B and IL-17. Through a supernatant co-culture system, we examined that EC cell-derived CCL20 enhanced cell viability and programmed death-ligand 1 (PD-L1) expression in neutrophils. Data from TCGA database showed that CCL20 level was negatively correlated with International Federation of Gynecology and Obstetrics (FIGO)-stage and Histopathological Grade, and associated with good prognosis in EC patients. Local tissue study demonstrated CCL20 was overexpressed in EC tissues and the expression level of CCL20 was negatively correlated with FIGO-stage in EC patients.
Conclusions
CCL20 is an important biomarker in human EC, which is highly expressed and is strongly correlated with neutrophil infiltration in EC. Moreover, high level of CCL20 and neutrophil infiltration are associated with favorable prognosis in patients with EC. Our findings suggest that CCL20 could be potentially used for prognostic prediction and treatment in human EC.
Keywords: Tumor-associated neutrophils (TANs), CCL20, immune microenvironment, endometrial cancer (EC)
Highlight box.
Key findings
• CCL20 was over-expressed in human endometrial cancer (EC), negatively correlated with International Federation of Gynecology and Obstetrics (FIGO)-stage and histopathological grade, and associated with good prognosis in EC patients. High level of CCL20 was associated with neutrophil infiltration in EC and enhanced CCL20 level in EC cells activated cell viabilities of neutrophils. Tumor-derived CCL20 promoted programmed death-ligand 1 (PD-L1) expression in neutrophils.
What is known and what is new?
• CCL20 performs different roles in different human cancers. In EC, CCL20 facilitates RANK/RANKL-induced epithelial-mesenchymal transition, enhancing cell migration and invasion; CCL20 upregulation via PGF(2α)-F-prostanoid receptor signaling promotes cell proliferation in endometrial adenocarcinoma. However, the role of CCL20 on tumor immune microenvironment of human EC remains unclear.
• This study demonstrated that CCL20 was associated with neutrophil infiltration in EC and enhanced CCL20 levels in EC cells activated cell viabilities of neutrophils. Neutrophils could be recruited to EC tissues by CCL20 secreted by EC cells and predicted better prognosis in EC patients.
What is the implication, and what should change now?
• CCL20 was an important biomarker in human EC, which could be potentially used for prognostic prediction and treatment in human EC.
• We examined that tumor-derived CCL20 promoted PD-L1 expression in neutrophils. However, the exact roles and deep mechanisms of PD-L1 in neutrophils of human EC should be further studied.
Introduction
Endometrial cancer (EC) ranks as the sixth most prevalent malignancy among women worldwide, accounting for 420,242 diagnoses and 97,704 deaths in 2022 (1). Compared with data from 2020, both the incidence and mortality of EC have risen (2). A global increase in the age-standardized incidence rate of EC has also been documented (3). Although diagnoses have increased across all age groups, a notable doubling in cases has been reported among women under 40 years of age; in the United States, this group comprises 4.2% of all low-grade EC diagnoses (4). Regarding treatment, the standard surgical approach involves hysterectomy combined with bilateral salpingo-oophorectomy. For early-stage EC, surgery aims to excise visible tumors, assess potential microscopic metastases, and determine tumor stage to guide decisions on adjuvant therapy (5). EC is recognized as an immunogenic tumor, with active immune engagement observed in both benign and malignant endometrial tissues (6,7). Therefore, the introduction of immunotherapy might change the treatment framework and provide more possibilities for the prognosis in EC patients.
Inflammation occurs in almost all forms of cancer even at the early stages of tumor development. Tumors release danger signals that stimulate local secretion of inflammatory cytokines and chemokines, thereby recruiting inflammatory leukocytes into the neoplastic sites. Generally, immune cells can exert either anti-tumor or pro-tumor effects, rendering them a “double-edged sword” within the tumor microenvironment (TME) (8). For many years, tumor-associated neutrophils (TANs) were predominantly considered immunosuppressive (9,10) and associated with poorer clinical outcomes (11-13). However, emerging evidence indicates that neutrophils can also eliminate tumor cells through the release of active elastase (14), nitric oxide synthase (15), or reactive oxygen species (ROS) (16). Additionally, neutrophils may exhibit an anti-tumor immune phenotype by enhancing interferon-mediated immunostimulation (17) or promoting autologous T cell responses (18). Previous studies suggest that the neutrophil-to-lymphocyte ratio in peripheral blood may serve as a discriminative marker for EC (19), and elevated ratios are positively correlated with myometrial invasion in EC (20). These findings underscore the significant role of neutrophils in EC progression. Nevertheless, the concrete mechanism of neutrophils underlying the progression of EC is still poorly understood. It is a significant therapeutic challenge for us to understand how to harness the power of these effectors and the underlying mechanisms. We present this article in accordance with the MDAR and TRIPOD reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0307/rc).
Methods
Bioinformatics analysis
Gene sequencing data and the corresponding clinical parameters of EC patients were collected from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Samples without complete clinical information or with a survival time less than 13 days were excluded. A total of 539 EC samples and 35 normal control samples were included in this study. In addition, GSE17025 from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was downloaded and involved in this study. We collected 81 chemokine-related protein-coding genes from the GeneCards website (https://www.genecards.org/). Differential expression gene (DEG) analysis was performed using the DESeq2 package in R (version 4.3.2) and the ggplot2 package in R. Immuno-Oncology Biological Research (IOBR) through R was used for immune cell infiltration analysis. The receiver operating characteristic (ROC) curve was used to evaluate the capacity of CCL20 to predict neutrophil infiltration in EC. Gene set enrichment analysis (GSEA) was performed based on clusterProfiler and c2.all. v2023.2.Hs.symbols.
Cell culture and siRNA transfection
EC cell lines Ishikawa and RL95-2 were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) at 37 ℃ in 5% CO2 as recommended. The culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. siRNA transfection was performed using Lipofectamine™ 2000 Reagent (Invitrogen). The siRNA sequences were as follows: si-CCL20#1: CCGTATTCTTCATCCTAAA; si-CCL20#2: CTTGGGTGAAATATATTGT; si-NC: UUCUCCGAACGUGUCACGUTT.
Clinical samples
In this study, 11 pairs of paraffin-embedded EC and adjacent normal endometrial tissues, and another 24 cases of paraffin-embedded EC tissues were collected at the Department of Pathology in the First Affiliated Hospital of Anhui Medical University (Hefei, China). These tissues were from patients who underwent surgical resection between 2020 and 2023. The clinicopathological parameters of these EC patients were collected for correlation analysis. This work was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This work was approved by the Institutional Review Board of Anhui Medical University (No. 83220486) and the informed consent was taken from all patients.
Enzyme-linked immunosorbent assay (ELISA)
We measured the concentration of CCL20 in the supernatant of EC cells using the Human MIP-3α ELISA Kit (BBI Life Sciences Corporation, Shanghai, China; Cat. No. D711016-0096).
Quantitative real-time polymerase chain reaction (RT-qPCR)
The extraction of total RNA from paraffin tissues was performed using FFPE RNA Kit (Omega, USA). RNA samples were reverse transcribed to cDNA and RT-qPCR was performed using the HiScript III RT Super Mix for qPCR (Vazyme, Nanjing, China) and the Taq SYBR® Green qPCR Premix (BestEnzymes, Lianyungang, China) according to the manufacturer’s instructions. With normalization to β-actin, CCL20 expression levels were calculated using the 2−ΔΔCt method after repeated in triplicate. The primer sequences were as follows: CCL20-forward: 5'-TGCTCCTGGCTGCTTTGATG-3', CCL20-reverse: 5'-AGTCAAAGTTGCTTGCTGCTTC-3'; β-actin-forward: 5'-TCTCCCAAGTCCACACAGG-3', β-actin-reverse: 5'-GGCACGAAGGCTCATCA-3'.
Neutrophil isolation and stimulation
Neutrophils were isolated from the peripheral blood of three healthy male volunteers after abtaining informed consent for each experiment. The detailed process was described in former study (21). The isolated neutrophils were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 2% FBS and 1% penicillin-streptomycin at 37 ℃ in 5% CO2 as recommended. Neutrophils (2×105) were stimulated with 100 ng/mL recombinant human CCL20 (PEPROTECH, Cat No. 300-29A) or supernatant from Ishikava, RL95-2 cells, respectively.
Flow cytometry assay
The viability of neutrophils treated with supernatant from EC cell lines or recombinant human CCL20 was assessed according to the method described in a previous study (21). The viability of neutrophils was quantified using Annexin V Apoptosis detection kits (Best Bio, Shanghai, China).
Immunofluorescence staining
Neutrophils were processed following the experimental method described in former study (21). Neutrophils smeared onto a poly-lysine-coated sterile glass slide were fixed in 200 µL of 4% paraformaldehyde (1 hour) and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) (15 minutes). Blocking was then performed with PBS containing 1% bovine serum albumin (BSA) (30 minutes), followed by overnight incubation at 4 ℃ with anti-programmed death-ligand 1 (PD-L1) antibody (1:300, Proteintech, Cat No. 66248-1-Ig) in PBS-1% BSA. After three washes, the cells were incubated with Alexa Fluor-conjugated secondary antibody (1:300, Beyotime, Cat No. 329705) (1 hour), and finally with 4,6-diamino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) in the dark (5 minutes). PD-L1 expression was examined under a confocal laser scanning microscope 880.
Statistical analysis
Each experiment in this study was performed for at least 3 times, and the figures and tables were the average results. Unpaired two-tailed t-test was used to examine the differences in the expression of cytokine-related genes between EC and normal samples and in flow cytometry and immunofluorescence analyses. The correlation between CCL20 expression and neutrophil abundance was analyzed by Spearman correlation analysis. Chi-square test and one-way ANOVA were used to analyze the correlation between CCL20 expression/neutrophil-abundance and clinicopathological parameters in EC patients. Kaplan-Meier curves were used to analyze the overall survival (OS) rates in EC patients, and log-rank test was used. R (version 4.3.2) and GraphPad Prism 9.5 were used for plotting, and SPSS 26 was used for statistical analysis. P<0.05 was considered to be statistically significant.
Results
CCL20 was over-expressed and related with neutrophil infiltration in EC
To find out potential chemokines associated with neutrophil infiltration in EC, we collected data based on TCGA database and firstly analyzed DEGs in EC compared with normal tissues based on 81 chemokine-related genes. As shown in Figure 1A, the expression levels of 25 chemokine-related genes showed significant differences between EC and normal tissues, 9 of which were higher and 16 of which were lower in EC tissues. The 6 genes (CCL20, CCL18, CCR8, CCR3, CXCL5, CXCL17) with | log2 fold-change (FC) | >3 were selected for further study. Concordantly, the expression levels of these 6 genes were all significantly higher in EC compared with normal tissues (Figure 1B). Correlation analysis showed CCL20, CCR8, CCR3, CXCL5, CXCL17 were positively correlated with neutrophil infiltration in EC, and the correlation coefficient between CCL20 expression and neutrophil infiltration was highest (r=0.4916, P<0.0001) (Figure 1C). Therefore, CCL20 was over-expressed and correlated with neutrophil infiltration in EC.
Figure 1.
CCL20 was over-expressed and related with neutrophil infiltration in EC. (A) Volcanic map showing chemokine related DEGs in EC compared with normal tissues based on TCGA database. (B) The mRNA levels of 6 candidate genes (CCL20, CCL18, CCR8, CCR3, CXCL5, CXCL17) in EC compared with normal tissues based on TCGA database. (C) Correlation analysis of the expression of 6 candidate genes (CCL20, CCL18, CCR8, CCR3, CXCL5, CXCL17) with neutrophil infiltration in EC based on TCGA database. ****, P<0.0001. CCL18, C-C motif chemokine ligand 18; CCL20, C-C motif chemokine ligand 20; CCR3, C-C motif chemokine receptor 3; CCR8, C-C motif chemokine receptor 8; CXCL5, C-X-C motif chemokine ligand 5; CXCL17, C-X-C motif chemokine ligand 17; DEG, differential expression gene; EC, endometrial cancer; FC, fold change; FDR, false discovery rate; TCGA, The Cancer Genome Atlas.
CCL20 expression and signaling pathway analysis in EC
Based on GEO data (GSE17025), we confirmed that the expression level of CCL20 was significantly higher in EC compared with normal tissues (Figure 2A). Moreover, we collected 11 pairs of local EC and adjacent normal tissues and examined the mRNA level of CCL20 through RT-qPCR. As shown in Figure 2B, the mRNA level of CCL20 was dramatically higher in 9/11 EC compared with respective adjacent normal tissues. As shown in the GSEA analysis, CCL20 was associated with immune-related signaling pathways including NOD-like receptor, TNF, NF-kappa B and IL-17 (Figure 2C).
Figure 2.
CCL20 expression and signaling pathway analysis in EC. (A) The mRNA level of CCL20 in EC compared with normal tissues based on GEO data (GSE17025). (B) The mRNA level of CCL20 in 11 pairs of local EC and adjacent normal tissues as examined by RT-qPCR. (C) Single gene GSEA analysis of CCL20 in EC. ns, no significance; *, P<0.05; **, P<0.01; ****, P<0.0001. CCL20, C-C motif chemokine ligand 20. EC, endometrial cancer; GEO, Gene Expression Omnibus; GSEA, gene set enrichment analysis; RT-qPCR, quantitative real-time polymerase chain reaction.
Tumor-derived CCL20 activated neutrophils
As examined by ELISA, the concentrations of CCL20 in the supernatant of EC cells Ishikava and RL95-2 were dramatically higher compared with control medium (Figure 3A). Neutrophils from healthy volunteers were treated with supernatant of Ishikava/RL95-2 cells and control medium, and a flow cytometry assay showed that the viability of neutrophils obviously increased after treatment with supernatant of Ishikava/RL95-2 cells compared with control (Figure 3B). Moreover, recombinant human CCL20 (rhCCL20) treated neutrophils showed increased cell viability compared with control (Figure 3C). In addition, small interfering RNAs (siRNAs) were employed for CCL20 knock down in Ishikava and RL95-2 cells (Figure 4A). Concordantly, neutrophils treated with supernatant of CCL20-siRNA-transfected Ishikava/RL95-2 cells showed decreased viability compared with respective control neutrophils treated with supernatant of NC-siRNA-transfected Ishikava/RL95-2 cells (Figure 4B,4C). Therefore, EC cell-derived CCL20 enhanced cell viability of neutrophils.
Figure 3.
EC cell derived CCL20 activated neutrophils. (A) CCL20 protein level in the supernatant of EC cells Ishikava and RL95-2 compared with control medium (RPMI 1640) was examined by ELISA. (B) Viabilities of neutrophils treated with supernatant of Ishikava and RL95-2 cells compared with control medium (RPMI 1640) were examined by flow cytometry assay. (C) Viabilities of neutrophils treated with recombinant human CCL20 (rhCCL20) compared with control medium (RPMI 1640) were examined by flow cytometry assay. Data is represented as mean ± standard deviation of three independent experiments. ***, P<0.001. ****, P<0.0001. CCL20, C-C motif chemokine ligand 20; EC, endometrial cancer; ELISA, enzyme-linked immunosorbent assay; NEU, neutrophil; RPMI, Roswell Park Memorial Institute.
Figure 4.
Diminished CCL20 decreased the viability of neutrophils. Ishikava and RL95-2 cells were transfected with si-CCL20#1, si-CCL20#2 or si-NC respectively. (A) CCL20 protein level in the supernatant was examined by ELISA. (B) Viabilities of neutrophils treated with supernatant of Ishikava si-CCL20#1/si-CCL20#2/si-NC cells were examined by flow cytometry assay. (C) Viabilities of neutrophils treated with supernatant of RL95-2 si-CCL20#1/si-CCL20#2/si-NC cells were examined by flow cytometry assay. Data is represented as mean ± standard deviation of three independent experiments. **, P<0.01. ***, P<0.001. ****, P<0.0001. CCL20, C-C motif chemokine ligand 20; ELISA, enzyme-linked immunosorbent assay; NEU, neutrophil; si-NC, small interfering RNA negative control.
Tumor-derived CCL20 promoted PD-L1 expression in neutrophils
To further study the role of tumor-derived CCL20 on neutrophils, we examined the expression of PD-L1 in neutrophils upon cell supernatant/rhCCL20 incubation. As shown in Figure 5A, the immunofluorescence staining demonstrated that Ishikava/RL95-2 supernatant and rhCCL20 incubation significantly increased the protein level of PD-L1 in neutrophils compared with control. Concordantly, knockdown of CCL20 with siRNAs in Ishikava and RL95-2 cells dramatically decreased PD-L1 expression in neutrophils under cell supernatant treatment. However, the decrease of PD-L1 expression in neutrophils was abolished by rhCCL20 addition (Figure 5B,5C). Therefore, tumor-derived CCL20 promoted PD-L1 expression in neutrophils.
Figure 5.
EC cell derived CCL20 promoted PD-L1 expression in neutrophils. (A) Neutrophils were treated with supernatant of Ishikava/RL95-2 cells, rhCCL20 or control medium (RPMI 1640) respectively, and PD-L1 levels in neutrophils were examined by immunofluorescence. (B) Neutrophils were treated with supernatant of Ishikava si-CCL20#1/si-CCL20#2/si-NC cells ± rhCCL20, and PD-L1 levels in neutrophils were examined by immunofluorescence. (C) Neutrophils were treated with supernatant of RL95-2 si-CCL20#1/si-CCL20#2/si-NC cells ± rhCCL20, and PD-L1 levels in neutrophils were examined by immunofluorescence. Data is represented as mean ± standard deviation of three independent experiments. ***, P<0.001. CCL20, C-C motif chemokine ligand 20; DAPI, 4,6-diamino-2-phenylindole; EC, endometrial cancer; NEU, neutrophil; PD-L1, programmed death-ligand 1; RPMI, Roswell Park Memorial Institute; si-NC, small interfering RNA negative control.
High expression of CCL20 and increased neutrophil infiltration indicated good prognosis in EC patients
Next, we analyzed the correlation of CCL20 expression with pathological features and survival rates in EC patients based on the TCGA database. As shown in Table 1 and Figure 6A, the expression level of CCL20 was negatively correlated with International Federation of Gynecology and Obstetrics (FIGO)-stage and histopathological grade in EC patients, and the expression level of CCL20 was significantly higher in Endometrioid EC compared with non-endometrioid EC. Kaplan-Meier analysis showed that the 3-year OS rate of EC patients with high CCL20 expression was dramatically higher compared with EC patients with low CCL20 expression (Figure 6B). Concordantly, based on GEO data (GSE17025), we determined that the expression level of CCL20 in grade 1/2 EC patients was significantly higher compared with Grade 3 EC patients (Figure 6C). As analyzed in local EC patients, the expression level of CCL20 was negatively correlated with FIGO-stage (P<0.05). However, there was no significant correlation between CCL20 expression and patients’ age, lymph node metastasis, or tumor distant metastasis (P>0.05) (Table 2). For neutrophil infiltration prediction analysis, ROC curves were plotted, and the area under the curve (AUC) value was 0.7373 (Figure 6D). In addition, neutrophil infiltration in grade 1/2 EC patients was significantly higher compared with grade 3 EC patients; but there was no significant difference of neutrophil infiltration in EC patients with different FIGO stages (Figure 6E). Kaplan-Meier analysis showed that the 3-year OS rate of EC patients with high neutrophil infiltration was dramatically higher compared with EC patients with low neutrophil infiltration (Figure 6F). Therefore, high expression of CCL20 and increased neutrophil infiltration indicated a good prognosis in EC patients.
Table 1. Association of CCL20 expression with clinicopathological features in endometrial cancer patients based on TCGA database.
| Characteristics | n | Low expression of CCL20, n (%) | High expression of CCL20, n (%) | χ2 | P |
|---|---|---|---|---|---|
| Age, years | 0.250 | 0.62 | |||
| ≤55 | 111 | 41 (20.50) | 70 (18.77) | ||
| >55 | 462 | 159 (79.50) | 303 (81.23) | ||
| FIGO-stage | 6.135 | 0.01 | |||
| I–II | 406 | 250 (67.39) | 156 (77.23) | ||
| III–IV | 167 | 121 (32.61) | 46 (22.77) | ||
| Histopathological grade | 35.207 | <0.001 | |||
| Grade 1 | 89 | 15 (11.45) | 74 (29.84) | ||
| Grade 2 | 106 | 25 (19.08) | 80 (32.26) | ||
| Grade 3 | 185 | 91 (69.47) | 94 (37.90) | ||
| Histologic subtype | 18.472 | <0.001 | |||
| Endometrioid | 421 | 251 (68.02) | 170 (84.58) | ||
| Non-endometrioid | 149 | 118 (31.98) | 31 (15.42) |
CCL20, C-C motif chemokine ligand 20; FIGO, International Federation of Gynecology and Obstetrics; TCGA, The Cancer Genome Atlas.
Figure 6.
High expression of CCL20 and increased neutrophil infiltration indicated good prognosis in EC patients. (A) The mRNA level of CCL20 in EC patients with different FIGO-stages and histopathological grades based on TCGA database. (B) Kaplan-Meier survival curve showed the OS rates in EC patients with low- and high-CCL20 expression based on TCGA database. (C) The mRNA level of CCL20 in EC patients with different histopathological grades based on GEO data (GSE17025). (D) ROC curve analysis for neutrophil infiltration prediction by CCL20 in EC. (E) Neutrophil infiltration in EC patients with different FIGO-stages and histopathological grades based on TCGA database. (F) Kaplan-Meier survival curve showed the OS rates in EC patients with low- and high-neutrophil infiltration based on TCGA database. ns, not significant; *, P<0.05; ****, P<0.0001. AUC, area under the curve; CCL20, C-C motif chemokine ligand 20; EC, endometrial cancer; FIGO, International Federation of Gynecology and Obstetrics; GEO, Gene Expression Omnibus; NEU, neutrophil; OS, overall survival; TCGA, The Cancer Genome Atlas.
Table 2. Association of CCL20 expression with clinicopathological parameters in endometrial cancer patients based on clinical tissues.
| Characteristics | n | Low expression of CCL20, n (%) | High expression of CCL20, n (%) | χ2 | P |
|---|---|---|---|---|---|
| Age, years | 1.446 | 0.23 | |||
| ≤55 | 16 | 6 (35.29) | 10 (55.56) | ||
| >55 | 19 | 11 (64.71) | 8 (44.44) | ||
| FIGO-stage | 3.998 | 0.046 | |||
| I–II | 22 | 6 (42.86) | 16 (76.19) | ||
| III–IV | 13 | 8 (57.14) | 5 (23.81) | ||
| Lymph node metastasis | 1.151 | 0.28 | |||
| No | 25 | 10 (62.50) | 15 (78.95) | ||
| Yes | 10 | 6 (37.50) | 4 (21.05) | ||
| Distant metastasis | 1.177 | 0.28 | |||
| No | 27 | 11 (68.75) | 16 (84.21) | ||
| Yes | 8 | 5 (31.25) | 3 (15.79) |
CCL20, C-C motif chemokine ligand 20; FIGO, International Federation of Gynecology and Obstetrics.
Discussion
Current research on polymorphonuclear neutrophils (PMNs) in cancer has increasingly indicated a pro-tumorigenic function for these immune cells, although evidence also points to their antitumor capabilities. Thus, TANs are often regarded as a “double-edged sword” in human malignancies. In this study, we demonstrated the ability of neutrophils to delay progression of EC. CCL20 was highly expressed in EC tissues and significantly associated with neutrophil infiltration as analyzed based on the TCGA database. Correlation analysis and Kaplan-Meier survival rate analysis suggested CCL20 expression level was positively correlated with neutrophil infiltration and patient OS rate. CCL20 level in the microenvironment was positively correlated with PD-L1 expression in neutrophils.
Chemokines play a fundamental role in leukocyte development, differentiation, and migration, and are also implicated in processes such as angiogenesis, wound healing, inflammatory disorders, and the advancement of malignancies. In different conditions, they may function as either anti-tumor or pro-tumor agents, contributing to the suppression of tumor immunity and modulation of cancer progression (22). CCL20, also known as Exodus-1, liver and activation-regulated chemokine (LARC) and macrophage inflammatory protein (MIP)-3α, is a member of the chemokine family within the cytokine classification (23,24). This chemokine exhibits context-dependent roles across various cancers. In colorectal cancer, CCL20 secreted by tumor cells recruits Treg cells to enhance chemoresistance via the FOXO1/CEBPB/NF-κB pathway (25). Similarly, CCL20 produced by HCC cells attracts CCR6+CD5+ B cells and stimulates angiogenesis, thereby supporting tumor growth (26). In cervical cancer, CCL20 predominantly expressed in the stroma is linked to infiltration of CD4+IL17+ cells and advanced FIGO stages (27). Conversely, CCL20 may also act as a tumor suppressor in certain contexts. As reported formerly, the proportion of CCR6+ and CCL20+ cells decreased as the increase of lesion severity (intraepithelial neoplasia to cervical cancer) (28). Another study reported reduced proportion of CD1a+ Langerhans cells and lower CCL20 expression from HPV(−) and HPV(+) normal cervical samples to carcinoma in situ and cervical cancer samples, suggesting that high-risk HPV-type lesions inhibits the expression of CCL20 via E6/E7 to facilitate immune evasion (29). Experimental studies in mice revealed that injection of Ad-CCL20-IL-15 into the tumors suppressed tumor growth in models of tumor-bearing mice (mouse colon cancer cells and melanoma cells); splenocytes treated with Ad-CCL20-IL-15 produced tumor-specific cytotoxic T cells that secreted IFN-γ and thus protected mice from challenge, highlighting its anti-tumor immune potential (30). In EC, CCL20 facilitates RANK/RANKL-induced epithelial-mesenchymal transition (EMT), enhancing cell migration and invasion (31). Additionally, CCL20 upregulation via PGF(2α)-F-prostanoid receptor signaling promotes cell proliferation in endometrial adenocarcinoma (32). Besides the direct effect of CCL20, we could not ignore the role of CCL20 on tumor immune microenvironment. For instance, compared to non-lymphocyte-rich hepatocellular carcinoma (LR-HCC), CCL20 is highly expressed in LR-HCC, where it correlates with improved prognosis and increased infiltration of CD8+ T cells (33). In this study, we demonstrated that neutrophils could be recruited to EC tissues by CCL20 secreted by EC cells and predicted better prognosis in EC patients.
Neutrophils, the most abundant myeloid cells in human blood, are recruited to sites of tissue injury and serve as the first line of defense against microbial infection. In this study, we found that neutrophils infiltration was negatively correlated with tumor grade and positively correlated with patient OS rate in EC patients. Neutrophils display remarkable functional complexity, with distinct states including angiogenic, inflammatory, and antigen-presenting phenotypes in human cancers (34), which meant tissue and phenotypic plasticity of neutrophils. For instance, neutrophils secrete IL-1 receptor antagonist (IL-1RA) to offset oncogene-induced senescence in pancreatic cancer cells (35). In anaplastic thyroid cancer, TANs undergo oxidative mitochondrial metabolism that sustain their viability while releasing NETs to enhance cancer cell proliferation (36). Although neutrophils are often implicated in cancer progression, substantial evidence also supports their antitumor roles. Human neutrophils can express TNF-related apoptosis-inducing ligand (TRAIL) to induce cancer cell death (37). Additionally, neutrophils expressing the receptor tyrosine-protein kinase MET exhibit direct killing of cancer cells through nitric oxide release upon hepatocyte growth factor (HGF) stimulation (38). Blaisdell et al. reported neutrophil-mediated antitumor effects in a murine uterine cancer model, where neutrophils disrupted cancer cell adhesion to the surrounding basement membrane, thereby inhibiting early-stage tumor growth; reduced hypoxia further enhanced their antitumoral activity (39). Leucine metabolism and subsequent histone H3K27ac modification could endow an antigen-presenting phenotype for neutrophils; these neutrophils could further invoke both (neo)antigen-specific and antigen-independent T cell responses; the antigen-presenting program was associated with favorable survival in most cancers (34). As for human endometrial carcinoma, it is reported that circulating neutrophil extracellular traps (NET) biomarkers could be used as prognostic markers in human cancers (40). In this study, we demonstrated that high level of CCL20 could recruit neutrophils in EC and high level of neutrophil infiltration predicted better prognosis in EC patients. Tumor-derived CCL20 enhanced the expression of PD-L1 in neutrophils, which might be related with tumor immunity and the lifespan of neutrophils. As reported, expression level of PD-L1 in neutrophils contributed to T-Cell Immunity suppression in breast cancer, hepatocellular carcinoma, gastric cancer, etc. (21,41,42).
PD-L1, a type I transmembrane protein with a size of 40 kDa, was identified as programmed cell death protein 1 (PD-1) ligands in 2000 (43). It is widely expressed in both non-lymphoid tissue and lymphoid tissue, in antigen-presenting cells (dendritic cells, macrophages, etc.) and all kinds of tumor cells as well as cancer cells (43,44). Tumor cells utilize immune evasion strategies by upregulating PD-1 and PD-L1, which suppress T cell activity (45). Furthermore, studies have shown that upregulation of PD-L1 can prolong the lifespan of neutrophils via PKM2/STAT1 during sepsis (46). This compels us to ponder the complex role of PD-L1, particularly its interplay with CCL20. However, we have not yet retrieved any literature reports investigating the relationship between PD-L1 and CCL20; nevertheless, studies demonstrated that a close connection between PD-L1 and integrin α5β1, which interacts with CCL20 to promote the differentiation of lung fibroblasts into myofibroblasts via TGF-β/Smad signaling (47). Studies have shown that PD-L1 protein on tumor cells can be transferred to platelets in a fibronectin 1, integrin α5β1, and GPIbαdependent manner, thereby exerting immunosuppressive effects in non-small cell lung cancer and colorectal cancer (48,49). Perhaps the lack of relevant literature provides us with a challenge that we should conduct related research, especially on the exact deep mechanisms of PD-L1 and CCL20 in EC.
Conclusions
In this study, we demonstrated that CCL20 was over-expressed in human EC, negatively correlated with FIGO-stage and Histopathological Grade, and associated with good prognosis in EC patients. High level of CCL20 was associated with neutrophil infiltration in EC and enhanced CCL20 level in EC cells activated cell viabilities of neutrophils. Tumor-derived CCL20 promoted PD-L1 expression in neutrophils. Therefore, CCL20 was an important biomarker in human EC, which could be potentially used for prognostic prediction and treatment in human EC.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
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 conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This work was approved by the Institutional Review Board of Anhui Medical University (No. 83220486) and the informed consent was taken from all patients.
Footnotes
Reporting Checklist: The authors have completed the TRIPOD and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0307/rc
Funding: This work was supported in part by grants from CSCO Tongshu Cancer Research Program (No. Y-tongshu2021/qn-0287) and The Key Program of Natural Scientific Research in Higher Education Institutions of Anhui Province (No. 2023AH050571).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0307/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0307/dss
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