Abstract
Background: Regulatory T-cells (Tregs) are a T-cell subpopulation with suppressive capacities, which are specifically attracted by C-C motif chemokine 22 (CCL22). Treg infiltration of tumors is associated with a poor prognosis in many patients. We aimed to investigate whether CCL22 is expressed in human breast cancer and whether its presence is associated with Treg infiltration. Materials and Methods: Eighty paraffin-embedded human breast cancer samples were stained for CCL22 and for the Treg-specific transcription factor forkhead box P3 (FOXP3). Expression was evaluated in a semi-quantitative manner. Results: FOXP3+ cells infiltrated 50% of the breast tumors. Moreover, Treg infiltrated 93% of the tertiary lymphoid structures. CCL22 expression positively correlated with FOXP3+ cell infiltration into the tertiary lymphoid structures. Conclusion: Our results demonstrate that CCL22 expression correlates with infiltration by FOXP3+ cells. Interestingly, Treg presence negatively correlated with positive nodal status. In addition to their unfavorable role as mediators of evasion from antitumor immune response, Tregs might also have a beneficial role by reducing inflammation thereby limiting early tumor growth and spreading.
Worldwide, breast cancer is the most common invasive cancer in women, representing 23% of cancer diagnosed in women in 2012 (1). The same year, breast cancer resulted in 1.38 million diagnosed cases and 458,000 deaths worldwide (1). Survival rates in the developed world are high, between 80% and 90% of the women being alive 5 years after initial diagnosis (1, 2). However, since the 1970s the number of cases worldwide has significantly increased, possibly due to modern lifestyle, better detection, and longer lifespan, while the situation in developing countries remains problematic (3). Tumor development is a complex biological process that depends on acquired genetic abnormalities as well as the interplay between tumor cells, stromal cells and host immune cells (4). The role played by the immune system in cancer has been intensively studied and several studies have linked lymphocytic infiltrate in cancer tissues with a better outcome (5, 6). These infiltrating immune cells can participate in elimination of tumor cells and thus help control tumor growth (7). Consequently, acute tumor-directed immune responses involving cytotoxic T-lymphocytes appear to protect against tumor development, whereas immune responses involving chronic activation of humoral immunity, infiltration by T-helper type 2 cells (Th2), and pro-tumor-polarized innate inflammatory cells result in the promotion of tumor development and disease progression. Breast tumor is invaded by a diverse assortment of immune cells composed of both innate (myeloid) and adaptive (lymphoid) lineages (4). Innate immune cells, including macrophages, granulocytes, mast cells, dendritic cells and natural killer cells, normally represent the first line of defense against pathogens and foreign agents. These cells can locally secrete soluble factors such as cytokines, chemokines, bioactive mediators, and matrix-remodeling proteins that recruit additional leukocytes from the circulation.
Adaptive immune cells, involving cluster of differentiation 8 (CD8) cytotoxic T-lymphocytes and CD4 helper T-lymphocytes can be recruited by secreted soluble factors. They undeniably represent an attempt to eradicate transformed tumor cells. Unfortunately after a phase of equilibrium, the tumor is often eventually able to escape this immune response (8). In parallel, a growing body of literature has focused on the role played by regulatory T-cells (Tregs) because of their possible implication in this escape phenomenon. Tregs normally function to protect tissue from autoimmune diseases by suppressing self-reactive cells, including CD8 cytotoxic T-lymphocytes, B-cells and natural killer cells (9-11). In breast cancer, the dual role of Tregs, as assessed by forkhead box P3 (FOXP3) positivity, and CD8 cytotoxic T-lymphocyte infiltration has been described (12). In cancer tissues, the presence of high numbers of FOXP3+ T-cells predicts worse relapse-free survival and decreased overall survival. The C-C motif chemokine 22 (CCL22) can specifically attract Tregs and plays a role in the establishment of an immune privilege, associated with reduced overall survival in ovarian cancer (13, 14). The aim of this study was to analyze the CCL22 production in patients with breast cancer and draw a parallel with FOXP3+ cell infiltration and clinical parameters.
Materials and Methods
Patient data. Tissue samples from 80 formalin-fixed and paraffin-embedded breast neoplasia biopsies from patients referred to the Gynecology Unit at Department of Obstetrics and Gynecology, Ludwig Maximilians University of Munich from 1991 to 2000, were included in this study. All cases had been previously characterized in terms of histology (grade, tumor size and lymph node infiltration by tumor cells) at the Department of Pathology, Ludwig Maximilians University of Munich (Table I). Patients fulfilling the inclusion criteria (i.e. adequate routinely fixed biopsy material, no history of previous malignancy) were chosen on the basis of equal distribution among certain clinical pathological findings such as lymph node involvement, histopathological type (invasive ductal), and as far as possible estrogen/progesterone receptors status. All tumor samples were classified by the TNM system (15). The histological classification was determined according to a modification of the Elston and Ellis grading proposed by Bloom and Richardson (16). The hormone receptor status was evaluated by immunohistochemistry. The tumor was classified as hormone receptor-positive in cases with positive staining in ≥10% of the tumor. The age of the patients ranged between 54 and 95 years. All of the patients were postmenopausal. Sections of 3 μm were assessed.
All material was sampled for diagnostic purposes and research was carried out in accordance with the legal requirements concerning confidential medical communication as well as the data protection act. Consequently, consulting the Ethics Committee of the Medical School, Ludwig Maximilians University of Munich, and written informed consent from the patients prior to participation in the study was not required.
Immunohistochemistry. Paraffin wax-embedded tissue slide samples were deparaffinized in xylol for 20 min, washed in 100% ethanol and then incubated in methanol/H2O2 (3%) for 20 min. After rehydration in an alcohol gradient to distilled water, the slides were placed in a pressure cooker containing sodium citrate buffer (pH=6.0) and heated for 5 min. Slides were washed twice in phosphate buffer solution (PBS) and blocked using blocking solution (Zytomed, Berlin, Germany) for 20 min. Each slide was separately incubated with a polyclonal rabbit primary antibody to human CCL22 (500-P107; Peprotech, Hamburg, Germany) diluted 1/200 in PBS, or a monoclonal mouse primary antibody to human FOXP3 (ab10563; Abcam, Cambridge, UK) diluted 1/300 in PBS. Incubation of the sections with the primary antibodies lasted for 16 h at 4°C. Subsequently, sections were washed twice in PBS before incubation with post-block reagent (Zytomed, Berlin, Germany) for 20 min. Finally, slides were washed in PBS and then incubated with the horseradish peroxidase-polymer (Zytomed, Berlin, Germany) for 30 min. Staining was performed using 3,3-diaminobenzidinesubstrate solution (Dako, Glostrup, Denmark) for 180 sec. Counterstaining was carried out with Mayer's hemalaun for 2 min. Finally, sections were washed in tap water for 5 min and then dehydrated in an ascending alcohol series and washed in xylol. Slides were cover-slipped with Eukitt quick-hardening mounting medium (Sigma-Aldrich, St Louis, MO, USA).
Positive controls were carried out with human tonsil sections. Negative controls were performed by replacement of the primary antibody and alternative incubation of the slides with IgG rabbit or mouse control antibodies (Biogenex, San Ramen, CA, USA).
The distribution of antigen expression was scored as following: 0: no staining; 1: few isolated CCL22/FOXP3+ cells; 2: moderate CCL22/FOXP3+ cell expression/infiltration; 3: strong CCL22/FOXP3+ cell expression/infiltration; 4: extreme CCL22/FOXP3+ cell expression/infiltration. Additionally, the intensity of CCL22 expression was evaluated as 0: none; 1: weak; 2: moderate; 3: strong, and multiplied with the distribution score to obtain the final scoring. Two independent investigators examined the sections using a Leitz Diaplan microscope (Leitz, Wetzlar, Germany). The concordance between investigators was almost 95%; in cases of different staining evaluation, both investigators re-evaluated slides until an agreement was reached (~5% of all cases).
Statistical analysis. Correlation of staining for the two antigens and their correlation with grading, age, tumor size, receptor status and nodal status were evaluated with the statistical program R (Version 0.98.1028; RStudio, Inc., Boston, MA, USA). The Mann–Whitney U-test was used for evaluation of two independent groups. Values with p<0.05 were considered statistically significant. Data are presented using the mean and standard deviation.
Results
CCL22 expression in breast cancer. A total of 75% of the studied breast cancer cases expressed CCL22 protein (i.e., at least some isolated cells). Differential expression of CCL22 in the cytoplasm of cells in human breast cancer assessed by immunoperoxidase staining is presented in Figure 1. The expression was highly variable, from no expression at all to very strong and diffuse expression. CCL22-producing cells looked like macrophages or dendritic cells, according to previous descriptions of these cells as being capable of CCL22 production (17). In the vast majority of patients, positively stained cells were mostly localized in the periphery, but in about 40% of the cases, they also infiltrated the tumor core. Notably, massive leukocyte infiltrations were generally present in the vicinity of CCL22-producing cells. Expression of CCL22 by tumor cells was observed, but was mostly limited to a diffuse expression concerning few tumor cells.
FOXP3+ cell infiltration in breast cancer. In 50% of the studied breast cancer cases, lymphocytes expressed FOXP3 protein (i.e. at least some isolated cells) in the nucleus. Differential expression of FOXP3 in the nucleus of lymphocytes in human breast cancer assessed by immunoperoxidase staining is presented in Figure 2. The expression was highly variable, from no expression at all to a very strong and diffuse expression. FOXP3+ cells all displayed the morphology of small lymphocytes, with large nuclei and very scarce cytoplasm. They were all very similar in appearance to the Tregs we observed in human tonsils. Tregs were found dispersed between decidual stromal cells or in the vicinity of blood vessels and, when grouped, were frequently associated with lymphocyte infiltration. Moreover, in cases with massive leukocyte infiltration, Tregs were present among the infiltrating cells in 93% of patients. These massive lymphocyte infiltrations were present in 69% of the patients and all presented characteristics of tertiary lymphoid structures (TLS).
Clinical characteristics of the study population.
CCL22 expression is correlated with FOXP3+ cell infiltration of TLS. To further explore the role of CCL22 expression and FOXP3+ cell infiltration in breast cancer, we performed Spearman's rank correlation for CCL22 expression and FOXP3+ cell infiltration of the tumor core and FOXP3+ cell infiltration of TLS. We found CCL22 expression correlated with FOXP3+ cell infiltration, but only for cells recruited into the TLS (p=0.019) and not for cells recruited into the tumor core (p=0.434) as detailed in Table II. This result confirms the role of CCL22 expression in FOXP3+ cell infiltration into breast carcinoma.
Correlation of C-C motif chemokine 22 (CCL22) expression with regulatory T-cell (Treg) recruitment (Spearman linear regression). CCL22 staining was significantly positively correlated with Treg infiltration of tertiary lymphoid structures (TLS).
Correlation of forkhead box P3 (FOXP3)+ cell infiltration with clinical characteristics. FOXP3+ cell infiltration significantly negatively correlated with positive nodal status (Spearman linear regression).
FOXP3+ cell infiltration is correlated with nodal status. To better characterize the influence of FOXP3+ cell infiltration in breast cancer, we performed Spearman's rank correlation for FOXP3+ cell infiltration and other parameters, all available at the time of the surgery: higher grade as graded after pathological characterization, age at the onset of disease, size of resected tumor, and presence of infiltrating tumor cells in the local lymph node. The results are presented in Table III. We found no correlation between Treg infiltration and the clinical parameters considered, except for the nodal status as illustrated in Figure 3a. Women with nodal metastases had significantly less Treg infiltration in the primary breast tumor (p=0.019). We found no correlation between nodal status and CCL22 expression in the primary tumor (Figure 3b).
C-C Motif chemokine 22 (CCL22) production in breast cancer. Expression of the CCL22 protein in macrophage-like cells in human breast cancer was assessed by immunoperoxidase staining: Negative control (tonsil) (a); positive control (tonsil) (b); no expression (c); low expression (d); intermediate expression (e); high expression (f). All at magnification: ×80.
Discussion
The results obtained during this histological study give an insight into the role of CCL22 in the recruitment of Tregs to breast cancer. The expression of CCL22 protein correlated with the Treg infiltration. Despite the central role played by chemokines in the immunoediting process, quantification of chemokine expression and associated immune cell recruitment are not yet used in the clinic. Interestingly, we found a very high infiltration of Tregs in breast cancer. Moreover, Treg infiltration was positively associated with CCL22 expression and negatively associated with nodal status.
The immune system is present and functional against the tumor in patients with breast cancer and may promote both humoral and cellular responses (18, 19). However, it is less clear how a tumor can avoid destruction by these cells and even turn the regulatory mechanisms of the immune system to its own advantage. One hypothesis is that the production of CCL22 that results in Treg recruitment allows the tumor to evade the early antitumor immune response (20).
On the other hand, it is well established that chronic infiltration of tissue by some innate immune cell types (e.g., immature monocytes, macrophages, mast cells or neutrophils) contributes to cancer development (21, 22). Indeed, a potential tumor-enhancing effect of adaptive immunity has already been observed after passive transfer of tumor-specific antibodies in vivo (23). Thus, CCL22 production by innate immune cells might also protect, for a time at least, from quick growth and spreading of tumor cells.
T-Cell infiltration into breast tumor has been extensively described and these cells can represent a very large proportion of the total immune cell infiltration (24). In proliferating tumors, the presence of T-lymphocytes might represent a good prognostic indicator (25). However, great variations in such infiltration were observed, particularly with regard to the Treg population, which might affect both disease progression and overall survival, sometimes leading to confusing results (12, 26, 27). These cells can be recruited from the periphery by CCL22 produced in the tumor, leading to the creation of an immunosuppressive milieu, favorable to tumor growth (13, 14). However, the activation and proliferation of Tregs in tumor might also play a role in tumor development (27). Because the antigen specificity of tumour infiltrating Tregs has not been established in humans, these cells may have opposing effects depending on the tumor microenvironment: they may be deleterious when they block antitumor effector T-cells, or beneficial when they reduce chronic inflammation (4).
Forkhead box P3 (FOXP3)+ cell infiltration in breast cancer. Expression of FOXP3 in the nucleus of lymphocytes in human breast cancer was assessed by immunoperoxidase staining: Negative control (tonsil) (a); positive control (tonsil) (b); no expression (c); low expression (d); intermediate expression (e); high expression (f). All at ×80 magnification.
We found no correlation between Treg infiltration and the clinical parameters considered here (namely grading, age, receptor expression and tumor size). An interesting finding was the negative correlation of Treg infiltration with nodal status. The detection of metastatic tumor cells present in lymph nodes is routinely used in the surgical management of breast cancer (28). In addition to metastatic cells, lymph nodes are also directly exposed to soluble factors released by primary tumor, such growth factor, and other cytokines (29, 30).
A low number or absence of Tregs in primary tumor could open the way to development of chronic inflammation, led by some innate immune cell types, therefore contributing to cancer development and spread to local lymph nodes. One limitation of this study is the absence of information on such an inflammatory condition, that should be further evaluated. Nevertheless, this work suggests that CCL22 could be evaluated as a target for tumor therapy, with respect to its implication in the recruitment of Tregs.
In summary, we found a positive correlation between CCL22 production and Treg infiltration in human breast cancer. Tregs were preferentially attracted into TLS, in the vicinity of the CCL22-producing cells. Interestingly, we found a negative correlation between Treg infiltration and nodal status, which could possibly result from the chronic inflammation in the primary tumor. Thus Treg, in addition to their unfavorable role as mediators of evasion from antitumor immune response, might also have a beneficial role, reducing inflammation and therefore limiting early tumor growth and spreading. The precise role of Treg in breast cancer development, however, should be better evaluated before their full prognostic value can be established. Tregs might have a more complex role in breast cancer than simply helping to establish an immunosuppressive milieu and patients could greatly benefit from better understanding of their role in the disease course.
Forkhead box P3 (FOXP3)+ (a) and C-C Motif chemokine 22 (CCL22) (b) score according to nodal status in women with breast cancer. FOXP3+ cell infiltration is reduced in women with positive nodal status. Boxplot with error bars indicating standard deviation. Mann–Whitney U-test. *p<0.05; n.s.: not significant.
Acknowledgements
The work presented here is part of the Ph.D. thesis of CPF and was supported by a Ph.D. stipend of the DFG (Graduiertenkolleg 1202). This work was presented at the 16th Hamburger Symposium on Tumor Markers, Hamburg (2014) under the title “CCL22 Expression and FOXP3+ Cell Infiltration in Human Breast Cancer.
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
The Authors declare no conflicts of interest with regard to this study.
- Received February 17, 2016.
- Revision received May 7, 2016.
- Accepted May 13, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
