Introduction
Patients suffering from gastroesophageal reflux disease (GERD) often present with a condition of chronic inflammation. In some GERD patients, reflux causes a replacement of the squamous epithelium of the esophagus by simple columnar epithelium of intestinal type. This is usually accompanied by goblet cells, which is called Barrett’s esophagus (BE). It has been proposed that these mucosal alterations further progress to dysplastic lesions and ultimately to esophageal adenocarcinomas (EAC) [
1]. The incidence of EACs is rising, and despite new multimodal therapies, only 30% of the patients are alive after five years. One reason for poor survival rates is loco-regional recurrences, which have been described in one-third of the patients after curative resection [
2‐
4]. Hence, there is an urgent need for new effective treatments. Immune therapies are currently being investigated, but have not yet entered routine clinical practice [
5].
Published data regarding the role of the immune system in the development of EACs have been conflicting [
6‐
9]. Indeed, most studies on esophageal cancer analyze esophageal adenocarcinoma and esophageal squamous cell carcinoma together. However, both entities demonstrate different risk factors, behave in a different biological way and are consequently treated differently [
4,
10,
11]. Because of these clear distinctions, we aimed to analyze histopathologically confirmed EACs in association with Barrett’s esophagus. We focused on CD4
+ T cells which orchestrate the immune system by producing a variety of different cytokines and are known to play a key role in several malignancies. Furthermore, a beneficial effect of inflammation in EACs is supported by recent studies showing that a high infiltration of CD3
+ and CD8
+ T cells correlates with a better prognosis [
9,
12,
13]. However, a detailed characterization of pro-inflammatory CD4
+ T cell subsets is missing. Regulatory FOXP3
+ cells (T
REG) are one subtype of CD4
+ T cells. These are usually associated with a poor prognosis in most malignancies [
14]. Interestingly, increased numbers of T
REG were found in EACs, although their impact on prognosis remains contradictory [
8,
13]. One key cytokine product of regulatory T cells is interleukin (IL) 10 [
15,
16]. However, the role of IL-10 in tumorigenesis and cancer progression is still controversial and its effect in EAC is unknown [
17,
18]. Other studies also indicate that a variety of cytokines play a role in EAC [
7‐
9,
13]. Kavanagh et al. found elevated levels of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and IFN-γ in addition to increased levels of anti-inflammatory IL-10 in the supernatant of EAC biopsies compared to healthy controls, indicating a mixed pro- and anti-inflammatory environment in esophageal adenocarcinomas [
7]. In addition, the role of IL-22
+ and IL-17
+ T cells in esophageal adenocarcinomas is still unclear. IL-22 and IL-17 are produced by CD4
+, gamma delta and innate lymphoid cells. Since CD4
+ T cells are the main source of IL-22 and IL-17, we focused on investigating these cells. Interestingly, several studies have reported a contribution of the latter cytokines to both wound healing and cancer development especially in the context of chronic intestinal inflammation [
19‐
22]. Given that EACs are also usually accompanied by chronic inflammation, we aimed to further characterize the role of IL-22 and IL-17 in this type of tumor.
Immune checkpoint inhibitors, which promote the immune response, have become a promising therapeutic target in cancer. For example, negative costimulatory signals mediated by cell surface proteins such as the programmed cell death protein 1 (PD1) with its programmed cell death ligand 1 or 2 (PDL1 or PDL2) or the cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibit the activation of T cells and thereby play a central role in anti-tumor immunity in many malignancies [
23]. Therefore, the use of immune checkpoint inhibitors is also currently being tested in EACs. Despite these studies, however, the expression profile of these immune checkpoints in EACs is still unclear [
24‐
27]. Before considering the routine use of immune therapies in EACs, it is thus essential to understand the prognostic effect of immune cells, their cytokines and surface receptors. These data could then also form the basis to identify patients, who are most likely to benefit from these immune therapies. This was the other aim of our study.
Based on all of this, we investigated the hypothesis that an anti-inflammatory environment might facilitate tumor development and tumor growth not only in EAC, but also in its non-malignant healthy tissue close to the resection margin leading to early recurrences and poor patient survival. We found an increased infiltration of CD4+ T cells with an accumulation of FOXP3+ IL-10+ secreting T cells in EAC, but a decreased proportion of pro-inflammatory CD4+ IL-17+ and IL-22+ T cells. Interestingly, the non-malignant tissue of EAC patients showed a similar immunological pattern. Accordingly, anti-inflammatory markers were identified as potential prognostic markers indicating worse survival especially in the unaltered peritumoral mucosa independent of other histopathological parameters as shown by multivariate analysis.
Materials and methods
Patient samples
Retrospective patient samples were collected from the biobank, and only patients with confirmed Barrett’s esophagus-associated EAC and without neoadjuvant treatment were selected. Samples were obtained from the carcinomas and from peritumoral histomorphologically unaltered esophageal tissue close to the proximal resection margins. For further patient data, see Supplementary Table 1. Esophageal control samples were obtained from healthy volunteers, who underwent routine gastroscopy due to reflux symptoms. The histomorphology of all samples was evaluated using HE staining. Eight samples of the peritumoral tissues were excluded due to infiltration of cancer cells or missing esophageal tissue.
Isolation of immune cells
Lymphatic cells were isolated from healthy human esophageal mucosa, EAC tissue and its corresponding unaltered tissue close to resection margins. Samples were obtained freshly after esophagectomy from patients diagnosed with EAC. Tissues were washed with PBS. For isolation of intraepithelial lymphocytes (IEL), the esophageal tissue was incubated in HBSS containing 1 mM dithioerythritol (DTE), followed by a dissociation step using 1,3 mM EDTA for 20 min at 37 °C, respectively. To isolate lamina propria lymphocytes (LPL), the tissue was further cut into small pieces and minced with a scalpel. The remaining tissue was incubated for 45 min at 37 °C on a shaking incubator in HBSS (with Ca2+ and Mg2+) with collagenase (1 mg/ml) and DNase I (10 U/ml), and supernatant was collected. Leukocytes were further enriched by Percoll gradient centrifugation (GE Healthcare). IEL and LPL were collected and pooled.
Flow cytometry
Human fluorochrome-conjugated antibodies, anti-CD127, anti-CD4, anti-CD3, anti-CD11c, anti-Siglec-8, anti-IL17A, anti-IFNγ, anti-TNF-α, anti-IL22, anti-FOXP3, anti-IL10 all were purchased from BioLegend. Additional anti-human CD3 and anti-human CD4 were used from BD Biosciences. Anti-human IL-22BP antibody (clone 87554) and IgG2B isotype control were obtained from R&D Systems. To identify dead cells, 7-AAD staining (BioLegend) was performed. For extracellular staining, isolated hematopoietic cells from esophageal tissues were incubated for 20 min at 4 °C. Cells were acquired on a LSRII Fortessa flow cytometer (BD). Data were analyzed with FlowJo software (Treestar).
RNA analysis
Total RNA was extracted from esophageal tissue using TRIzol reagent (Invitrogen). The high-capacity cDNA synthesis Kit (Applied Biosystems) was used for cDNA synthesis. Real-time PCR was performed using the Kapa Probe Fast qPCR Master Mix (Kapa Biosystems) on the StepOnePlus system (Applied Biosystems). For probes used, see Supplementary Table 2. Relative RNA expression was normalized to HPRT and calculated using the 2−∆∆Ct method. The mRNA expression levels of HPRT across groups were not statistically different (see Fig. S1).
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed and paraffin-embedded sections of healthy esophageal tissue, tissue with Barrett’s metaplasia, Barrett’s esophagus containing low-grade (LGD) and high-grade dysplasia (HGD) and early stage (pT1) esophageal adenocarcinomas. An experienced pathologist reviewed the slides for confirmation of morphological changes. DAKO, Monoclonal Mouse Anti-Human CD4, Clone 4B12; eBioscience, Anti-human Foxp3 Purified, Clone 236A/E7 and Abcam, Anti-PD1 antibody (NAT105) ab52587 were used for staining. The sections were counterstained with hematoxylin. CD4, FOXP3 and PD1 presence was determined in a blinded fashion by measuring four fields of visions in the area of interest and calculating the mean.
Statistical analysis
Statistical analysis was performed with GraphPad Prism® Software (GraphPad Software, San Diego, CA, USA). To compare the groups, the two-sided Mann–Whitney test was used. Significance for Kaplan–Meier curves was calculated by log-rank test. For multivariate survival measurements, statistical analysis was performed using SAS for Windows, version 9.4 (SAS Institute Inc., Cary, NC). Baseline characteristics are given as mean ± standard deviation (SD) and count (proportion) for continuous and categorical factors, respectively. The effect of different variables on time to cancer-related death was investigated using a Cox proportional hazards model. Prior to multivariate survival analysis, the significance was evaluated in a preliminary test using a Cox model. Patients who died within 30 days after surgery were excluded from survival analyses. All analyses were complete case analyses, and therefore, the model was based on 34 patients. Statistical significance was defined as p < 0.05.
Discussion
EAC is a disease with poor survival rates that still lacks an effective systemic treatment option. This is one of the few studies, which focused on patients whose immune response was not altered because of chemotherapy. This is because we obtained samples from a unique cohort of patients that did not receive any neoadjuvant therapy. Pro- and anti-inflammatory cytokines have both been described in EACs. However, their effect on patient survival had not yet been investigated. Moreover, we analyzed the immune system also in the histologically unaltered esophageal tissue close to the oral resection margin.
Our data indicate a strong influence of IL-10 on patient survival in EACs irrespective of other clinical or histopathological parameters. In gastric cancer patients, increased mRNA levels of
IL10 were also associated with worse survival in multivariate analysis [
18]. Of note, we found that in the unaltered peritumoral tissue,
IL10 was a stronger predictor of survival compared to EACs. In line with the latter finding, Fitzgerald et al. observed increased levels of IL-10 in the distal parts of BE. In these parts of BE, the least inflammation was reported and four of the six EACs occurred later on [
28]. In addition, an in vitro analysis detected increased levels of IL-10 in the supernatant of Barrett’s mucosa and EACs compared to healthy controls [
7]. These data underline the immunosuppressive function of IL-10 and support our findings regarding the prognostic relevance of IL-10 even if no malignancy has occurred.
Supporting the hypothesis of a predominantly anti-inflammatory environment in EACs, we also found more CD4
+CD127
lowFOXP3
+ cells in EACs and in its unaltered peritumoral tissue and less effector T cells. We did not only identify a significant increase in FOXP3
+ cells in late stage EACs, but also in early stage EACs and prior to malignant mucosal changes. Somja et al. also reported a significant increase in FOXP3
+ cells during the dysplasia–carcinoma frequency in Barrett’s esophagus [
29]. Hence, CD4
+CD127
lowFOXP3
+ cells, which are usually classified as regulatory T cells (T
REG), might contribute to tumor development by suppressing pro-inflammatory cytokines. In line with the anti-inflammatory role of T
REG, a reduced number of CD4
+ CD45RO
+ memory T cells were observed in EACs [
7]. In addition, Rauser et al. showed a positive correlation between high density of CD45RO
+ T cells and disease-free survival in stage I–IIa patients [
12]. Stein et al. also demonstrated that a high amount of intratumoral tissue-infiltrating lymphocytes (TIL) and a high grade of total inflammation were beneficial for the prognostic value in EACs [
13]. However, Zingg et al. failed to show a prognostic effect on the number of TILs [
8]. When specifically looking at FOXP3
+ cells, only Stein et al. were able to find a positive correlation between high infiltration of FOXP3
+ T cells and patient survival. In our cohort, elevated
FOXP3 expression levels correlated with worse patient survival in the peritumoral group after R status was taken into account. Taken together, our findings suggest that IL-10 and FOXP3
+ cells are an evidence of an anti-inflammatory response to a chronic inflammation, which can be detected in other cytokines such as IFN-γ and TNF-α. Hence, IL-10 and FOXP3
+ T cells might serve as a potential prognostic biomarker in BE and EAC. However, further studies are warranted to test this hypothesis.
Furthermore, we found an increase in
CTLA4 expression in peritumoral as well as in tumoral samples of EAC patients. This datum sustains our finding that an anti-inflammatory environment characterizes EACs. Unexpectedly, in multivariate analysis,
CTLA4 expression only correlated with patient survival in the unaltered peritumoral group. In patients with ESCC, increased CTLA4 levels have been implicated to be associated with shorter overall survival in multivariate analysis [
30]. Hence, patients with increased
CTLA4 expression in EACs might benefit from a CTLA4 blockade.
Another important receptor for suppressing T cell activation is PD1. The PD1/PD-L pathway plays an important role in the regulation of the immune cells against tumor cells [
23]. Derks et al. reported PD1
+ infiltrating lymphocytes in 59.8% of the investigated EACs. They were also able to demonstrate a correlation with T stage and grading [
25]. In our study, relative
PD1 expression levels did not significantly differ between EAC and controls. However, we found an increase in PD1
+ T cells in high-grade dysplasia and in EACs themselves. In addition, increased
PD1 levels demonstrated to be worse for overall survival in the peritumoral group. Our findings emphasize PD1 as a potential target in EACs for immune therapies. Accordingly, in one of the first studies testing the treatment of PD1-blockade as third line therapy in advanced gastric or gastroesophageal junction cancer, a significant benefit for PD1-blockade was found [
31]. However, it is becoming evident that not only the presence of PD1, but rather the local and systemic composition of inflammatory cells reacting to the PD1/PD-L pathway is important for the response to the anticancer immune therapy [
32,
33].
In Barrett’s esophagus, increased expression levels of IFN-γ and TNF-α have been reported and TNF-α seems to increase along the metaplasia–dysplasia–carcinoma sequence [
6,
34]. However, we did not find any significant differences between RNA expression levels of IFN-γ and TNF-α, nor did we find any significant changes in the relative amount of infiltrating CD4
+ IFN-γ
+ and CD4
+ TNF-α
+ T cells between all groups. This confirms the findings of a recent study, in which no significant differences in the relative amounts of CD4
+ IFN-γ
+ and CD4
+ TNF-α
+ cells were reported in EACs [
7]. Notably, we found that
IFNγ expression levels in peritumoral tissues showed to be worse for patients’ prognosis. Usually,
IFNγ expression is associated with a superior outcome in most cancer entities, such as colorectal cancer [
35]. The underlying reason is currently unknown. However, our results could be explained by findings of other studies, which have reported an induction of PD-L1 expression by IFN-γ leading to a potent immune escape in certain malign cells [
36,
37], thus adding to an anti-inflammatory environment.
To further characterize the pro-inflammatory environment, we investigated the IL-22/IL-22BP axis and IL-17A
+ cells in EACs. Several immune cells, including innate lymphoid cells and CD4
+ T cells, produce IL-22. IL-17
+ and IL-22
+ which are involved in both wound healing and tumor development through activation of STAT3 [
20,
21]. In addition, it has been shown that IL-22 induces DOT1L in colon epithelial cells leading to the expression of
Nanog,
Sox2 and
Pou5F1. The latter genes cause stem cell-like characteristics in colon cells, which might contribute to tumor growth [
38]. In contrast, IL-22 also bears anti-tumor effects since blocking of IL-22 in early tumor initiation stages during DSS colitis leads to a reduced tumor burden. However, when blocking IL-22 in later stages, a pro-tumorigenic effect was observed [
19]. These data are in line with a recent study indicating that IL-22 is protective against early genotoxic events in the colon [
39]. Hence, multiple factors determine the biological function of IL-22. In addition, IL-22 binding protein has been described as a inhibitory soluble receptor of IL-22, which is expressed by a subset of dendritic cells [
40,
41]. The balance of the IL-22-IL-22BP axis seems to be critical for tumorigenesis in the colon [
19]. IL-17
+ T helper cells are thought to be involved in immunoregulation and primarily secrete IL-17A, IL-17F and IL-22. However, their precise role is still under debate [
42]. In squamous cell carcinomas of the esophagus (ESCC), IL-17
+ infiltrating cells seem to be anti-tumorigenic and were reported to positively correlate with a good patients’ prognosis [
43,
44]. In the progression from healthy tissue toward EACs, increased IL-17
+ cells have been found [
45]. Here, we describe that IL-22
+ and IL-17
+ effector T cells were reduced in EAC and low
IL22 expression was associated with worse survival in univariate analysis. The reduction in IL-17A
+ cells in EACs is in line with a finding from Kavanagh et al. [
7] who also reported a reduced proportion of CD4
+ IL17
+ cells in EAC compared to normal controls. The findings of reduced effector T cells emphasize the dominance of an anti-inflammatory environment in EACs. However, IL-22 has also been reported to function in cell regeneration on mucosal barriers. We observed elevated
IL22 expression and IL-22
+ cells in the peritumoral tissue compared to tumor tissue. Hence, one might speculate that in the unaltered peritumoral tissues, cytokine-mediated repair mechanisms of the mucosa are still intact, while in EACs, reduced IL-22 leads to impaired cytokine-mediated cell regeneration. We also found a reduced
IL22RA1 expression in tumor tissue. Since IL22RA1 is especially expressed on barrier surfaces, low expression levels could be due to replacement of the physiological esophageal mucosa by cancer cells. In addition, Gronke et al. very recently showed that stem cells deprived of IL-22 and exposed to carcinogens were able to escape apoptosis, had more mutations and were more likely to give rise to colon cancer [
39]. Thus, these data indicate a possible imbalance of IL-22 signaling axis in EACs, which might accelerate tumorigenesis and impair wound healing.
We found that the anti-inflammatory environment in the microscopically unaltered peritumoral tissue had a stronger impact on patient survival compared to tumoral tissue. One explanation could be the surgery itself since all cancer tissue is resected with safety margins to achieve an oncologic resection. However, the remaining tissue close to resection margins might already contain an anti-inflammatory environment and thus be prone to early recurrences. Hence, it might strongly influence overall survival. In addition, the similarity of the immunological profile in the unaltered peritumoral tissue compared to the malignant tissue was surprising. These data indicate a potential immunological field effect around EACs, which might also contribute to tumor progression in already existent EACs and might lead to tumor development or local recurrence despite radical resection.
In conclusion, we show that CD4+CD127lowFOXP3+ T cells infiltrate EACs, while CD4+ IL-22+ and IL-17+ effector T cells are reduced. FOXP3+ T cells can be found even prior to malignant mucosal changes. This might indicate a shift from a chronic pro-inflammatory environment toward an anti-inflammatory environment during EAC development. We for the first time describe an anti-inflammatory field effect in Barrett’s esophagus-associated cancers that might contribute to early local recurrences in the unaltered mucosa. Further, an association of CTLA4 and PD1 expression with reduced survival was found. In addition, we demonstrated a strong IL-10 expression in EACs, which negatively impacts survival independently of other clinical or histopathological parameters. Thus, our data indicate that IL10, CTLA4 and PD1 expression could serve as a biomarker to identify patients, who might benefit from closer surveillance and immunotherapy using checkpoint inhibitors.