IL-6 and IL-8 secreted by tumour cells impair the function of NK cells via the STAT3 pathway in oesophageal squamous cell carcinoma

Fresh peripheral blood was collected from healthy volunteers with an average age of 29 years. Fresh peripheral blood, autologous adjacent tissues, and tumour tissues were obtained from ESCC patients during surgery at the First Affiliated Hospital of Southwest Medical University. None of the patients had received radiotherapy or chemotherapy before sampling, and individuals with autoimmune diseases, infectious diseases, or multi-primary cancer were excluded. According to the tumour-node-metastasis (TNM) classification system published by the International Union Against Cancer (Edition 7), the clinical stages of tumours were determined. The study was approved by the Ethics Committee of the First Affiliated Hospital of Southwest Medical University, and prior written informed consent was obtained from each patient.

Fresh tumour and adjacent tissues were used for the isolation of tissue-infiltrating lymphocytes. Paired fresh tumour and adjacent tissue samples were cut into small pieces, washed with 4 °C PBS, ground for an hour with a tissue grinder (Wei Ning biological company), and then filtered through the grinder to obtain single cell suspensions.

A total of 5 mL of lymphocyte separation solution (Tianjin Haoyang Biology Company) was added to 15 mL centrifuge tubes; 650 g of tissue was centrifuged for 20 min; the second layer of milky white lymphocytes was carefully moved to another 15 mL centrifugation tube (Thermo); and the collected lymphocyte cells were re-suspended.

A total of 10⁶ cells were suspended in 100 μl of PBS, and suitable amounts of fluorescently labelled antibodies were added and incubated at room temperature for 30 min. Cells were analysed by flow cytometry (BD Biosciences), and data were analysed using FlowJo software (TreeStar). The antibodies used for flow cytometry are described in Additional file 1.

Fresh ESCC tissues were brought to the laboratory in sterile frozen containers containing 5% iodine volts solution within 2 h. Appropriate amounts of sterile PBS solution were added, and the tissues were soaked for 15 min. Small scissors were used to cut off the necrotic parts of the tissue. The small tissue pieces were digested for an hour in Hank’s enzyme-free dissociation buffer (containing 1 mg/mL type IV collagenase, 1 mg/mL hyaluronidase, and 0.25 mg/mL DNase) at 37 °C for 1 h, and the primary oesophageal squamous carcinoma cells were collected. The cells were grown in RPMI1640 (Welgene) supplemented with 10% FBS.

All animal experiments were approved by the Animal Ethical and Experimental Committee of Southwest Medical University. Nude mice were purchased from the ChongQing TengXin company. A total of 10⁶ ESCC#1 or ESCC#2 cells were implanted into the left armpit of nude mice. On the 21st day after implantation, the mice were sacrificed, and the tumours were taken for H&E and IHC staining.

Peripheral blood NK cells were purified by a Human NK Cell Enrichment Set-DM kit (BD) and then expanded with an NK Cell Robust Expansion kit (Stemery) according to the manufacturer’s instructions. The purity of NK cells was > 90%, and the number of cells was increased 500–1000-fold. The primary oesophageal squamous carcinoma cell supernatant was collected and cultured with NK cells for 72 h. According to each group, anti- IL-8 or anti- IL-6 was added (Additional file 1) or not. Finally, the NK cell surface receptor expression levels, proliferation and cytokine expression levels were detected by flow cytometry.

Antibody chip experiments were conducted by Shanghai Youningwei Biotechnology Co. Ltd., Art. No: ARY005B.

Formalin-fixed and paraffin-embedded tissue sections with a thickness of 4 mm were dewaxed in xylene and a graded alcohol series, then hydrated and washed in phosphate-buffered saline (PBS). After pretreatment in a microwave oven, endogenous peroxidase was inhibited by 3% hydrogen peroxide in methanol for 20 min, followed by avidin-biotin blocking using a biotin-blocking kit (Dako, Germany). Slides were then incubated with IL-6 or IL-8 antibodies overnight in a humid chamber followed by incubation with rabbit secondary antibodies. The slides were developed with the Dako liquid 3,3′-diaminobenzidine tetrahydrochloride (DAB) + substrate chromogen system.

A cytotoxicity assay was performed using stimulated NK cells as effector cells. NK cells were suspended at 1 × 10⁶ cells/mL in RPMI1640 medium without phenol red. K562 cells (Central Laboratory of Southwest Medical University) were used as the target cells. K562 cells were suspended at 5 × 10⁴ cells/mL and co-cultured with effector cells (50 μL/well each) for 4 h on a 96-well microplate at 37 °C with 5% CO₂. The effector to target (E:T) cell ratio was 20:1. A colorimetric-based lactate dehydrogenase (LDH) assay (Cytotoxicity Detection Kit^PLUS; Switzerland) was used, and cytotoxic activity was calculated according to the manufacturer’s instructions. Cell-mediated cytolysis was converted to percent specific lysis (%SL) using the following formula: %SL = [(experimental LDH release-spontaneous LDH release)/(maximum LDH release-spontaneous LDH release)]× 100.

IL-6 and IL-8 staining scores were evaluated semi-quantitatively and graded for both intensity (absent or weak 1; moderate, 2; strong, 3) and extent (percentage of positive cells: < 25%, 1; 25–50%, 2; > 50%, 3). The intensity and extent scores were multiplied to obtain a comprehensive score of 0–9 for each specimen. Composite scores of 0–3 were defined as negative protein expression, scores of 4–6 were defined as weakly positive expression, and scores of 7–9 were considered strong positive expression.

Total proteins were extracted using RIPA lysis buffer (Beyotime) according to the manufacturer’s protocol. Forty micrograms of total proteins were separated on 10% SDS-PAGE gels. The proteins were transferred to PVDF membranes. The membranes were incubated with primary specific antibodies (Additional files 2 and 3) at 4 overnight and then incubated with HRP-conjugated secondary antibodies (Beyotime) for 1 h, and signals were detected with an electrogenerated chemiluminescence (ECL) detection reagent (Amersham Life Science, Piscataway). Relative target protein expression levels were normalized to GAPDH and visualized using ImageJ software.

To evaluate the potential roles of NK cells in human oesophageal squamous cell carcinoma, we first analysed the percentage of NK cells in the peripheral blood, tumour tissues and matched adjacent tissues of 35 healthy volunteers or 52 patients (Fig. 1a). The characteristics of T cell and NK cell lymphocyte gates were defined by forward (FSC) and side scatter (SSC) dot plots. The results of the flow cytometry analysis revealed that the percentage of CD3-CD56+ NK cells in patient peripheral blood increased significantly compared with that in healthy volunteer peripheral blood, while CD3+ T cells displayed inverse percentages (p < 0.05) (Fig. 1b). Moreover, the percentage of NK cells and T cells in ESCC tissues was significantly higher than that in matched adjacent tissues (p < 0.05) (Fig. 1b). Convincing evidence has revealed that CD56^dim NK cells are mainly distributed in peripheral blood for immune surveillance and that CD56^bright NK cells are mainly located in tissues for immunomodulation via cytokine secretion [12]. Intriguingly, we observed that the CD56^dim NK cells were the predominant subtype in tumour tissues, which was consistent with the subtype content in peripheral blood but different from that in adjacent tissues (Fig. 1c). This finding suggests that the tumour-infiltrating NK cells may originate in the peripheral blood. Altogether, these results show that the percentage of tumour-infiltrating NK cells is increased in the peripheral blood and tissues of patients with oesophageal squamous cell carcinomas.

Next, we investigated the phenotype of NK cells in the peripheral blood and tissues. The expression of activating or inhibitory receptors on the surface of NK cells was determined by flow cytometry. In peripheral blood, the level of NKG2D, an activating receptor, was increased, and the level of NKG2A, a known inhibitory receptor, was significantly decreased in patients with ESCC compared with healthy volunteers (p < 0.05) (Fig. 2a and b), suggesting that the activity of NK cells was enhanced. In comparison with matched ESCC patient blood samples, lower percentages of activator receptors, such as NKp30, CD16, NKp46, NKG2D, and CD226, and higher percentages of inhibitory receptors, such as NKG2A, were observed in adjacent tissues and tumour tissues (p < 0.05) (Fig. 2a and b). Moreover, the expression of the activating receptors NKp30 and NKG2D in tumour tissues was lower than that in adjacent tissues, suggesting that the phenotype of NK cells could be modulated after infiltration into the tumour microenvironment. Next, we detected the expression of effector molecules in tumour-infiltrating NK cells and found that the expression of granzyme B was decreased in comparison with that in adjacent tissues or peripheral blood, but perforin expression was not significantly changed (Fig. 2c and d). In addition, we further detected KI67 expression, which is a marker of cell proliferation. The results revealed that Ki67 expression was significantly decreased in tumour-infiltrating NK cells, suggesting that the proliferation potential of NK cells was impaired. Taken together, these results demonstrate that the phenotype and function of tumour-infiltrating NK cells is impaired in tumour tissues of patients with oesophageal squamous cell carcinoma.

In addition, clinicopathological variables of 52 patients were collected; the percentage of tumour-infiltrating NK cells correlated with the G stage and TMN stage of ESCC. Although there was a certain trend in the correlation, it was not statistically significant due to the insufficient number of samples (Additional files 1, 2 and 3).

Primary tumour cells are one of the main constituents of tumour tissues and are reported to contribute to immune system suppression. For instance, tumour cells recruit Treg cells to tissues to suppress the activity of infiltrating immune cells or secrete multiple cytokines to sustain the immunosuppressive microenvironment to help tumour cells survive [18]. To uncover the underlying mechanism of the impairment of tumour-infiltrating NK cells in ESCC, we first sought to isolate primary ESCC cells in vitro. Our results showed that two primary cell lines were obtained, namely, ESCC#1 and ESCC#2 (primary cells of oesophageal squamous cell carcinoma#1 and primary cells of oesophageal squamous cell carcinoma#2), the morphology of which was homogeneous under the light microscope. Moreover, the two primary cell lines were both cancerous in tumorigenicity assays in nude mice (Fig. 3a and b). The pathologist confirmed that the two primary cell lines were moderately differentiated squamous cell carcinomas via H&E staining and IHC for CK5/6 and p63 (Fig. 3c). Next, we purified NK cells from peripheral blood and cultured them in vitro for 15 days. After co-culturing the NK cells with the ESCC#1 and ESCC#2 supernatants (Fig. 3d), we investigated whether the NK cell characteristics were altered. The results showed that the levels of the activating receptors NKG2D and NKp30 (Fig. 3e and f) and the levels of the effector molecule granzyme B (Fig. 4a and b) were suppressed in the ESCC#1 and ESCC#2 co-cultured supernatants in comparison with those in the RPMI1640 medium (NC) supernatant of healthy oesophageal cell lines KYSE150 and EC9706. After co-culturing with NK cells, apart from NKp30 and NKG2D, there was no significant difference between the KYSE150 and EC9706 groups in terms of other activating receptors or inhibitory receptors, even though EC9706 is a well-recognized invasive oesophageal squamous cell carcinoma strain. However, compared with that in the KYSE150 and EC9706 groups, the NKG2A expression level in the ESCC#1 and ESCC#2 groups was higher. These results indicate that primary ESCC cells impaired the activity and function of NK cells, and the effect was stronger than that of the cell lines.

Increasing evidence has revealed that tumour cells can modulate immune cells by secreting multiple cytokines. To address whether the primary ESCC cells inhibited the function of NK cells by secreting cytokines, we performed a cytokine array. The results revealed that the levels of five cytokines, IL-6, IL-8, CCL2, CXCL1, and CXCL10, were significantly changed between the cell lines and the primary cell lines (Fig. 4c). In addition, we noticed that the relative expression levels of IL-6 and IL-8 were both upregulated in the EC9706 group compared with those in the KYSE150 group. In addition, through IHC, we confirmed strong positive staining of IL-6 and IL-8 proteins in the xenograft tissues (Fig. 4d). Although numerous papers have reported that IL-6 and IL-8 can enhance the invasion and metastasis of solid tumours, few studies have discussed how IL-6 and IL-8 modulate the function of NK cells [20, 21]. To determine whether the ESCC cells impair the function of NK cells by secreting IL-6 and IL-8, 5 μg of IL-6 and IL-8 antibodies was added to the supernatant of ESCC#1 cells. The ESCC#1 group with added IL-6 and IL-8 antibodies displayed restored expression levels of NKp30, NKG2D and GranB in comparison with the co-culture group (Fig. 4e and f). Moreover, we confirmed that the cytotoxicity of NK cells was impaired after co-culturing with the supernatant of ESCC#1, which could also be restored by the addition of IL-6 or IL-8 antibodies (Fig. 4g).

In vivo, a KYSE150 ESCC cell transplantation model was established in mice with thymic and NK cell defects. Our results revealed that after injecting normal humanized NK cells via the tail vein, the tumour size of BALB/c nude mice was significantly lower than that of mice injected with NK cells co-cultured with ESCC#1 supernatant; furthermore, the tumour size was restored by the addition of IL-6 and IL-8 antibodies (Fig. 5a and b). A similar result was observed in the lung metastasis model, indicating that the impairment of NK cells by ESCC was dependent on IL-6 and IL-8 (Fig. 5c and d). Taken together, these data suggest that ESCC cells impair the function of NK cells through IL-6 and IL-8 secretion.

The STAT signalling pathway plays an important role in the differentiation and maturation of NK cells. For instance, STAT1 can enhance IFN-γ production and the cytotoxicity of NK cells; phosphorylated STAT3 can impair tumour immune surveillance and promote tumour escape from immune control [16]. Increasing evidence has revealed that IL-6 promotes tumour development through the STAT3 signalling pathway [22, 23]. Currently, there are few studies on the relationship between IL-8 and STAT3. To investigate the underlying mechanism of IL-6/IL-8 regulation of NK function, the phosphorylation of STAT1, STAT2, STAT3 or STAT6 was detected in NK cells co-cultured with IL-6 or IL-8 for 2 h. The results showed that the level of p-STAT3 was increased in NK cells, and the level of p-STAT1 was slightly increased (Fig. 5e and f). Furthermore, we detected p-STAT3 levels in NK cells through western blotting and found that the supernatant of primary ESCC cells increased p-STAT3 levels in NK cells, which could be reversed by IL-6 or IL-8 antibody treatment (Fig. 5g and h). In addition, we used 100 ng/mL rIL-6 and rIL-8 to stimulate NK cells for 2 h and then detected p-STAT3 levels in NK cells through western blotting. The results showed that rIL-6 and rIL-8 treatment promoted STAT3 phosphorylation to varying degrees (Fig. 5i). Moreover, we further inhibited the phosphorylation of STAT3 using Stattic, an inhibitor of STAT3, and found that the phenotype and function of NK cells was restored (Fig. 6a and b). Taken together, these results suggest that the impairment of NK cells by primary ESCC cells is mainly dependent on IL-6- or IL-8-induced activation of STAT3 signalling.

To elucidate the correlation between IL-6 or IL-8 and clinical characteristics, we performed an IHC array to analyse IL-6/IL-8 expression in 103 pairs of tumours and matched adjacent tissues of patients with ESCC (Figs. 6c, d and 7a, b). We found that the number of IL-6- and IL-8-positive tumour cells was significantly increased in tumour tissues in comparison with matched adjacent tissues. Moreover, the number of IL-6- and IL-8-positive tumour cells positively correlated with TNM stage, and high IL-6 and IL-8 expression was found to indicate poor overall survival of ESCC patients when the medium value of all IL-6 and IL-8 percentages was used as a comparison point. In addition, the number of IL-6- and IL-8-positive tumour cells positively correlated with other advanced-stage clinical characteristics, including tumour invasion, lymphoid node and distant metastases and tumour size. IL-6 and IL-8 expression was not correlated with age or gender. Taken together, these results suggest that increased IL-6 and IL-8 expression is involved in ESCC progression and indicates a poor overall survival of patients with ESCC.