Background
Esophageal cancer is one of the leading causes of cancer-related death worldwide. Globally, approximately half of all cases occur in China [
1]. Despite of great advancements in early detection, precision diagnosis and combination therapy, the overall 5-year survival rate of esophageal cancer is still unsatisfactory [
2]. Evasion of immune surveillance is an important hallmark of cancer, acquired during tumor initiation and development. Dysfunction or exhaustion of T lymphocytes in tumor microenvironment has been recognized as a key mechanism to the pathogenesis of human malignant diseases [
3]. Notably, Immunotherapy aimed at restoring anti-tumor activity of T lymphocytes has become a pillar of cancer therapy [
4].
Natural killer (NK) cells are the main cells that constitute innate immunity and play an important role in the anti-tumor immune surveillance [
5]. Many studies have shown that the number of infiltrating NK cells in tumor tissues is significantly related to the prognosis of cancer patients, including esophageal cancer [
6,
7]. NK cells within tumor microenvironment are often impaired by many different mechanisms, such as reduced numbers, imbalances between activating and inhibitory receptors, and immunosuppressive cytokines [
8]. Recently, dysfunctional NK cells are characterized by surface expression of co-inhibitory receptors [
9]. It has been reported that programmed cell death protein 1 (PD-1) on NK cells indicates poor survival of esophageal cancer and blockade of PD-1 signaling restores NK cell function [
7,
10].
Besides PD-1, T-cell immunoglobulin domain and mucin domain-3 (Tim-3) is another potential exhaustion marker induced by chronic infections or cancers. Tim-3 was first discovered on Th1 cells and exhibited functions as a co-inhibitory receptor that down-regulates the activity of tumor infiltrating lymphocytes (TIL) in different types of cancer [
11‐
13]. Blockade of Tim-3 signaling restores TIL functions in vitro and in vivo [
14]. Later, Tim-3 has also been found on the surface of innate immune cells, including dendritic cells, macrophages, and NK cells [
15]. Importantly, high Tim-3 expression on innate immune cells may mediate suppressive responses [
16]. Early research suggests that Tim-3 functions as an inducible receptor on human NK cells to enhance IFN-γ production in response to galectin-9 [
17]. However, later studies have shown that Tim-3
+ NK cells from cancer patients produce lower levels of IFN-γ and are functionally exhausted [
12]. Recent studies reported that a high percentage of Tim-3
+ NK cells was associated with poor prognosis in patients with gastric cancer and lung adenocarcinoma [
18,
19]. Furthermore, Tim-3 blockade can increase the antitumor activity of NK cells from melanoma patients [
20]. However, the relationship between Tim-3 expression on NK cells and human esophageal carcinoma is not well understood.
In this study, we characterized the phenotypes and functions of NK cells from esophageal carcinoma in human and mice. We found that Tim3+ NK cells were functionally defective and correlated with poor prognosis in esophageal cancer patients. Mechanistically, Tim-3 was induced by tumor necrosis factor-α (TNF-α) through NF-κB signaling pathway. These findings indicate Tim-3 as a potential prognostic marker and a promising therapeutic target in esophageal cancer.
Materials and methods
Esophageal cancer patients
Blood and tissue samples were collected from 52 patients with untreated esophageal cancer in the First Affiliated Hospital of Zhengzhou University between September 2016 and April 2018. The demographic information of patients was summarized according to their age, gender, tumor site and pathological stage (Additional file
1: Table S1). Blood samples from healthy donors were obtained from the local blood bank.
Isolation of lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. The Tumor Dissociation Kit and the gentleMACS Dissociator (Miltenyi Biotec) were used for TIL isolation from tumor tissues according to the manufacturer’s instructions. Cell suspensions were then isolated by Ficoll-Hypaque to remove red cells and dead cells. The fresh TIL were immediately used for flow cytometry analysis or sorting.
NK cells were purified from PBMC of healthy donors and esophageal cancer patients using MACS NK kit (Miltenyi Biotec). Tim-3+ NK cells and Tim-3− NK cells were sorted from TIL by MoFlo XDP cytometer (Beckman Coulter) and then used for gene expression analysis from small number of cells.
Flow cytometry
For surface phenotype assessment, cells were stained with fluorochrome-conjugated primary antibodies for 30 min at 4 °C. An isotype control was performed for each sample. For cytokine detection, cells were stimulated with PMA and ionomycin (Sigma) in the presence of a protein transport inhibitor brefeldin A (BioLegend) for 6 h, and then stained with surface fluorochrome-conjugated primary antibodies. After fixing with 4% paraformaldehyde and permeabilizing with 0.1% saponin, cells were finally stained intracellularly with specific antibodies against human cytokines. Samples were stained with 7-AAD viability staining solution (BioLegend) to exclude dead cells and analyzed with a FACSCanto II cytometer (BD Biosciences). Details of the antibodies were summarized in Additional file
1: Table S2.
RNA extraction and quantitative PCR
Total RNA was extracted from tissues using RNAiso Plus (TakaRa) according to the manufacturer’s instructions. Total RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific), and cDNA was synthesized by using PrimeScript RT reagent Kit with gDNA Eraser (TakaRa). Quantitative PCR (qPCR) reactions using specific primers and SYBR Green qPCR Master Mix (Roche) were performed in an Mx3005P qPCR System (Agilent Technologies). For detection of small cell samples, a one-step qPCR reaction was performed using CellsDirect One-Step qRT-PCR kit (Invitrogen). The sequences of PCR primers was listed in Additional file
1: Table S3.
TNF-α treatment of human NK cells in vitro
Purified human NK cells were suspended in modified GT-T551 medium (TaKaRa) supplemented with 10% FBS (HyClone), 200 IU/mL recombinant human IL-2, 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma). NK cells were treated with recombinant human TNF-α (Peprotech), NF-κB inhibitor (Selleck) or TNF-α inhibitor (Shanghai Saijin) for 48 h, respectively. Surface Tim-3 expression on NK cells was then evaluated by flow cytometry.
Murine esophageal carcinoma model
Male C57BL/6 mice (7-week-old) were purchased from Beijing Vital River Laboratory Animal Technology and randomly divided into two groups: control (n = 10) and 4-NQO (n = 18). The carcinogen 4-NQO (Sigma) was dissolved in propylene glycol (Sigma) at 5 mg/mL and diluted in the drinking water to 100 µg/mL. After 12 weeks of 4-NQO treatment, mice were followed by sterile distilled water for another 16 weeks before sacrifice. Esophageal tissues were used for histopathology and qPCR analysis, respectively. Spleens were obtained from each animal for lymphocyte analysis by flow cytometry.
Statistical analysis
All statistical analyses were performed by using the Graph-Pad Prism 7 software. The significance of variation between groups was evaluated using Student’s t-test. Data are presented as the mean ± SEM from at least three independent experiments. Differences were considered to be statistically significant when p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).
Discussion
NK cells and CD8+ T cells play a crucial role in eradicating cancer cells, and inhibition of their functions is a key mechanism of tumor immune escape. T cell exhaustion has been extensively studied in different types of cancers, while the dysfunction of NK cells remains largely unknown. In this study, we detected Tim-3+ NK cells in esophageal cancer patients and tumor-bearing mice, and aimed to explore Tim-3-mediated NK cell dysfunction and the potential relevance with clinical prognosis.
Typically, NK cells sense the loss of major histocompatibility complex (MHC) class I molecules as a way to discriminate harmful cells. However, the induction of NK cell dysfunction by MHC class I-deficient tumor cells leads to tumor evasion from immune surveillances [
5]. Functional reversal of exhausted NK cells appears to be a great potential for the treatment of cancer [
9,
29]. PD-1 was reported as a marker of exhausted NK cells in digestive cancer patients, and blocking PD-1/PD-L1 signaling can restore the functions of NK cells [
10]. Recent studies have reported Tim-3 is an exhaustion maker for NK cells and blocking Tim-3 signaling can increase the antitumor activity of NK cells in lung adenocarcinoma and melanoma [
19,
20]. However, the role of Tim-3 in NK cells has not yet been well investigated in esophageal cancer. Here, we observed a significant increase of Tim-3 expression on NK cells from esophageal cancer patients in comparison with healthy donors. Notably, tumor-infiltrating Tim-3
+ NK cells exhibited signs of dysfunction. It has been reported that co-expression of PD-1 and Tim-3 was associated with functionally exhausted NK cells in colon cancer tissues [
5]. However, co-expression of inhibitory immune checkpoints was not predominantly observed among tumor-infiltrating NK cells in this study. Further studies are needed to define the function of NK cell subsets expressing different immune checkpoints.
Conventionally, NK cells are divided into two major subsets: CD56
bright and CD56
dim NK cells. Watanabe et al. reported the down-regulated CD16 and up-regulated CD56 molecules on NK cells of esophageal cancer patients, resulting in NK cell dysfunction [
30]. A prevalence of CD56
bright NK cells has also been observed in other solid malignancies, such as an increase of CD56
brightperforin
low NK cells in human breast and lung cancers [
31,
32]. Recently, it was reported that CD56
bright NK cells had markedly higher PD-1 expression relative to CD56
dim NK cells in Hodgkin lymphoma patients, indicating an exhausted NK cell subset to induce immune evasion [
33]. However, Pesce et al. showed that PD-1 expression is restricted to CD56
dim NK cell subset, representing terminally differentiated NK cells with impaired functions [
34]. To date, little is known regarding Tim-3 expression on NK cell subsets. Here, we observed that Tim-3 expression is more significantly up-regulated on CD56
bright NK cells from esophageal cancer patients, especially in tumor microenvironment. However, whether Tim-3 expression on different NK cell subsets has different functions needs to be further addressed.
We attempted to explore the mechanisms involved in Tim-3 expression of NK cells in esophageal cancer microenvironment. Ndhlovu et al. reported that peripheral NK cells expressed high levels of Tim-3 after stimulation with IL-2, IL-12, IL-15 or IL-18 in vitro [
35]. In this study, we identified for the first time that TNF-α could induce significant Tim-3 expression on NK cells from healthy donors in vitro. Whether this reflects the responsiveness of tumor-infiltrating NK cells remains to be determined. TNF-α is a multifunctional cytokine produced by activated immune cells, tumor cells or stromal cells in tumor microenvironment [
36‐
38]. The NF-κB cascade can be activated by inflammatory cytokines such as TNF-α. Our results showed that NF-κB inhibitor blocked Tim-3 expression on NK cells induced by TNF-α, suggesting that NF-κB mediates the expression of Tim-3. The remarkable association between Tim-3 and TNF-α in both human and murine esophageal tumor tissues further support the important role of TNF-α in Tim-3 expression on NK cells.
Accumulating evidence indicates the low density of NK cells in different forms of cancers such as melanoma, lung cancer, breast cancer, renal cell cancer and gastrointestinal tumors [
39‐
42]. The activating receptors NKp30 and NKp46 have been identified as predictive markers for a good prognosis in acute myeloid leukemia and metastatic prostate cancer [
43,
44]. However, the prognostic value of co-inhibitory receptors on NK cells has been very barely studied, especially in esophageal cancer. Our analyses of human cancer samples clearly indicate that Tim-3
+ NK cells are significantly associated with pathological parameters for poor prognosis of esophageal cancer. This implies that the dysfunction of NK cells may contribute to the progression of the disease in esophageal cancer patients. It would be of great interest to establish whether Tim-3
+ NK cells can serve as a predictive biomarker for the outcome of esophageal cancer patients.
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