Tumor immune escape is an important aspect of tumor development that ensures tumor progression. Tumor cells produce soluble factors that modify microenvironment, attract various immune cells and drive their differentiation to immunosuppressive phenotype. In this study using various markers of tumor stroma cells we investigated immunosuppressive phenotype of esophageal squamous cell carcinoma (ESCC) (Fig. 4).
The main cell population we have studied is composed of TAMs. Like other immune effector and regulatory cells, macrophages demonstrate high degree of functional versatility and express different surface markers, and secretable factors(11). The role they play in the tumor immune escape depends on their phenotype. Macrophages can be boldly divided into two main subgroups: “classically activated” or M1, and “alternatively activated” or M2. M1 are pro-inflammatory and are thought to exert antitumor effects through production of IL-12, IL-23, and reactive oxygen and nitrogen species(12). M1 are not considered to be immunosuppressive, however existence of mixed M1/M2 phenotype of TAMs prompted us to analyze M1 marker – iNOS. We found that this marker is not suitable for TAM analysis, since its expression was observed solely in tumor cells in a small number of samples. Interestingly iNOS expression correlated with the stage of disease, high expression was found in tumors of stages III–IV, compared to low expression at stages I-II. No prognostic value of iNOS was determined. iNOS expression in esophageal cancer is poorly studied. No significant correlation with the clinical parameters of the tumor and iNOS expression was found in the study by Jin et al(13), the absence of expression difference between tumor and normal tissue of esophagus was also reported(14). Our results, however, suggest that deeper investigation of iNOS in esophageal tumors will reveal its diagnostic and/or prognostic value.
M2 are usually considered to be able to suppress antitumor properties of M1 TAMs and modulate tissue remodeling by producing matrix metalloproteinases, transglutaminases and extracellular matrix components(15) and various cytokines and growth factors(16). In most of the tumors TAMs have M2 phenotype that are considered to be tumor supporting ones(17). All tumor associated macrophages independent on their phenotype, seem to express CD68. As subtype-specific marker of M2 CD163 is frequently used(16). However there are several other markers like CD204, CD206 or Stabilin-1 that can be used to detect type 2 macrophages. We examined the relationship between TAM density, and clinical characteristics and outcomes in 48 patients who had undergone resection of esophageal cancer. We demonstrated that out of all markers studied, only CD206 correlated with the histological grade of the tumor. No other correlations were found.
There are contradictory literature data regarding prognostic value of M2 number in the tumor. In most of the cases high number of M2 TAMs correlates with poor prognosis, since these macrophages promote vascularization, invasion and metastasis in many cancer types(18). In our study, the number of CD163 + M2 correlated with a good prognosis of esophageal cancer (HR = 0,4447, p = 0,0456*). In contrast to our results Hu et al demonstrated correlation of stromal CD163 + TAMs with poor prognosis of esophageal cancer, however if CD163 + cells were counted in tumor nests only, as well no difference in patients survival was found in that study(19). This differences in results clearly indicates the importance of the way how and in which areas of the tumor TAMs are analyzed.
Since the total amount of macrophages is a highly important criteria, there is an urgent need for a macrophage marker that allow clear identification of the cell. We selected PU.1 as such a marker. PU.1 is a transcription factor regulating hematopoietic differentiation pathways(20). Upon lineage differentiation and maturation, PU.1 is expressed at varied levels in mature blood cells, with higher levels found in macrophages than B cells(21). In our study expression of PU.1 showed strongest correlation with CD68, and a staining pattern indicating that the cells stained for PU.1 are CD68 positive macrophages. Taking into account nuclear staining PU.1 will be more suitable for precise cell quantification.
In the present study, we also explored the impact of TILs on the clinical significance in ESCC. It was demonstrated that high numbers of TILs is a marker of good prognosis and longer survival in ESCC. Particularly the presence of T cells (CD3+) and T cell subpopulations (e.g., CD4+, CD8+, CD103+) were established to be markers of a good prognosis(22). CD8 + T cells can recognize tumor‑associated antigens as major histocompatibility complex (MHC) class I molecules on the cancer cell surface and lyse cancer cells. Therefore, the presence of CD8 + T cells in the tumor is considered as a host immunoreaction and is associated with a better prognosis in a variety of cancers. However opposite results are also reported, where high levels of CD8 + T cells in the tumor are associated with a poor prognosis(23). In our study we found no significant correlations of CD3 + and CD8 + cells and clinical features of the tumor. As well analysis of prognostic value of T-cells in general and cytotoxic T-cells did not reveal statistically significant differences.
Another T-cell type that has diagnostic and prognostic value in different types of cancer is regulatory T-cells, expressing FOXP3. FOXP3 is a member of the forkhead/winged-helix family of transcription factors that is critically involved in the development and function of Tregs(24). Several studies demonstrate that FOXP3 + Tregs infiltrating tumor suppress CD8 + T cells to maintain immunological tolerance, associates with advanced tumor growth and poor prognosis in several types of malignant tumors(25–27). In contrast, other studies have shown that tumor FOXP3 expression is a favorable prognostic factor for breast cancer(28, 29). In the case of esophageal cancer high numbers of FOXP3 + cells was reported to be an indicator of poor(30) as well as good(31) prognosis. In our study we demonstrated that high number of FOXP3 + cells is associated with good prognosis in the analysis of overall survival (HR = 0.5407, p = 0.0462).