Background
Lung cancer remains the most common cancer type worldwide and it is the leading cause of cancer death. The tumor microenvironment comprises a wide variety of cells including malignant and nonmalignant populations [
1]. Crosstalk between tumor cells and other tumor-associated cells may lead to either inhibition of tumor formation or enhancement of tumor growth and progression, and this double-edged sword characteristic of many tumor-infiltrating immune cells, such as macrophages, T cells, and dendritic cells, has been recognized [
2‐
4].
Macrophages are particularly abundant among tumor-infiltrating innate and adaptive immune cells and are present at all stages of tumor progression. The tumor microenvironment determines the behavior of cancer. It is known that the tumoricidal activity of macrophages may vary in different tumor compartments. Experimental murine models and clinical studies indicate that tumor-infiltrating macrophages generally play a pro-tumorigenic role [
5]. In early pre-invasive lesions, tumor cells release chemokines to attract macrophages as well as other inflammatory cells into the tumor stroma [
6]. Many substances secreted by macrophages in the tumor stroma may directly stimulate the proliferation, migration, and metastasis of tumor cells [
7].
Macrophages are particularly heterogeneous in phenotype and function, and this is one of the most important characteristics of these cells. Based on a particular physiologic or pathologic situation, macrophages can be polarized into different phenotypes: pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. M1 macrophages are tumoricidal and their derived cytokines have the ability to kill pathogens. M2 macrophages are pro-angiogenic, and participate in wound healing where they downregulate inflammatory response to promote connective tissue remodeling [
8]. Defining and differentiating distinct pro-tumoral and anti-tumoral subsets of macrophages remain challenging. However, it is already clear that in the absence of M1 macrophage-orienting signals, M2 macrophages promote tumor cell proliferation in vitro and in experimental murine models [
9].
Previous studies demonstrated conflicting evidence regarding the significance of macrophages in cancer. Early studies reported that in colorectal tumors, infiltrating macrophages have pro-inflammatory properties, play an anti-tumor role, and are associated with good prognosis [
10,
11]. However, other clinical studies have shown that in many tumors such as lung, cervical, ovarian, esophageal, breast carcinoma, and melanoma, macrophages are considered to be anti-inflammatory and are linked to poor prognosis. [
12]. After recruitment to tumor site, exposure to tumor microenvironment-derived factors such as cytokines, growth factors, and hypoxia polarize macrophages phenotype from tumoricidal to tumorigenic. Loss of tumor-infiltrating macrophages cytotoxic ability and pro-inflammatory cytokines production represent substantial barriers to immune clearance of solid tumors [
12].
Macrophages play an important role in tumor growth and progression as they produce a large quantity of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and interferon-γ (IFN-γ). IL-10 is commonly regarded as immunosuppressive, anti-inflammatory, cytokine that favors tumor escape from immune surveillance. However, some authors indicated some immunostimulating properties of IL-10 [
13‐
15]. On the one hand, IFN-γ may inhibit tumor-induced angiogenesis, while on the other, IFN-γ can promote tumor growth through proliferative and anti-apoptotic signals as well as escape of the tumor cells from recognition and cytolysis by NK cells [
16]. TNF-α can facilitate the generation and maintenance of anti-tumor immune responses through the activation of NK cells and CD8
+ T cells [
17]. Furthermore, TNF-α can directly affect tumor cells by increasing lysosomal enzymes and inducing apoptosis [
18]. However, TNF-α also can contribute to chronic inflammation and promote tumor formation, growth, and metastasis [
17].
The role of macrophages and cytokines in non-small cell lung cancer (NSCLC) remains controversial. While clearly implicated in inhibiting tumor growth with consequent tumor regression, macrophages have also been demonstrated to have pro-tumor functions resulting in tumor progression. Moreover, a number of cytokines have been described as possessing dual roles in NSCLC [
19]. Further studies are needed to examine macrophage functions in NSCLC under different conditions and to relate this to patient response to treatment and prognosis. Therefore, in this study, we aimed to evaluate serum cytokine levels and tumor islet- and stroma-infiltrating macrophages (M1 and M2) and analyze the associations with NSCLC patients’ survival.
Discussion
The presence of tumor-infiltrating immune cells is evidence of a host response against the tumor. Previous reports have shown that macrophages in the tumor stroma secrete several growth factors and proteases involved in angiogenesis, thereby enhancing cancer progression. Contrarily, tumor islets-infiltrating macrophages produce cytotoxic cytokines, which may protect against tumor progression [
24]. In this study, we aimed to examine serum cytokines, tumor islet- and stoma-infiltrating M1 and M2 macrophages and to analyze the prognostic value of these cells and cytokines in NSCLC patients’ survival.
There are limited data comparing infiltration of M1 and M2 macrophages in the lung tissue between lung cancer patients and control subjects therefore we investigated and compared M1 and M2 macrophages in NSCLC and control patients. We used the total number (in tumor islets and stroma) of M1 and M2 macrophages while analyzing the control and NSCLC groups. A study of colon cancer performed by Sickert et al. showed that the number of macrophages was increased in the tumor tissue compared with the normal mucosa [
25]. In agreement with the data from this study, our results revealed that the number of M1 and M2 macrophages was significantly higher in the tumor tissue than in the lung tissue from the control group. There are some hypotheses elucidating mechanisms, which can cause the increased macrophages count in the tumor tissue. One of them asserts that macrophages are derived from circulating monocytes and are recruited to the tumor site by monocyte chemotactic protein-2 (CCL2), a chemotactic factor. CCL2 is acknowledged as the major factor responsible for recruiting circulating monocytes from the blood to a variety of mouse and human tumors. CCL2 is produced by tumor cells and the associated stromal cells [
1,
26]. The other hypothesis states that tumor cells and the associated stromal cells produce additional chemokines and various growth factors that are involved in monocyte recruitment to inflammatory sites and differentiation [
27].
It is known, that solid tumors are composed of two discrete but interdependent compartments: islets (malignant cells) and stroma (the supportive framework of a tumor tissue). The tumor stroma basically consists of the non-malignant cells of the tumor such as fibroblasts, mesenchymal cells, immune cells, vasculature with endothelial cells, and the extracellular matrix [
28]. The importance of accurate assessment of inflammatory cell microlocalization within both tumor islets and surrounding stromal components was highlighted in a study by Welsh et al., who demonstrated that the distribution of macrophages in tumor islets and stoma can impact prognosis [
29]. In our study, predominant infiltration of M1 and M2 macrophages in the tumor stroma compared with the tumor islets was observed, and similar findings have been noted previously in other studies of NSCLC [
30‐
32]. Moreover, in our study, M2 macrophages predominated over M1 macrophages in the tumor stroma. The majority of macrophages tend to accumulate in poorly vascularized hypoxic sites. Hypoxia or cytokines produced because of hypoxia attract macrophages to hypoxic tumor areas [
33]. During tumor progression and when hypoxia in the tumor increases, macrophages display defective production of inflammatory cytokines and progressively acquire pro-tumoral M2 functions [
34]. Also, tumor cells may switch macrophages to the M2 phenotype by releasing chemokines and polarizing cytokines, thus supporting their own escape from destruction.
Interestingly, we observed a greater number of M1 macrophages in the tumor stroma in NSCLC patients with lymph node metastasis compared with patients without lymph node metastasis. Ma et al. found that patients with lymph node metastasis had statistically significantly lower M1 macrophage density in the tumor islets than patients without lymph node metastasis, suggesting that tumor growth/progression might influence the distribution of M1 macrophages in the tumor microenvironment [
31]. Carus et al. demonstrated that the density of macrophages in the tumor islets as well as in the stroma was significantly elevated in patients with regional lymph node metastases compared with patients without them [
35]. In contrast, Zhang et al. found that M2 macrophages were more strongly correlated with lymph node metastasis than M1 macrophages [
32]. Inflammatory cells including macrophages in the tumor stroma can express vascular endothelial growth factor and then induce peritumoral lymphangiogenesis and lymph node metastasis [
32]. Moreover, in the tumor stroma, macrophages can produce proteases, such as matrix metalloproteinases (MMP), plasmin, and urokinase-type plasminogen activator that regulate matrix digestion. Proteases can degrade extracellular matrix and thus favor tumor cell invasion. Enhanced expression of MMP-2 was detected in several tumors and it strongly correlated with tumor stage and lymph node status [
36].
Significantly higher numbers of total and tumor stroma-infiltrating M1 and M2 macrophages in smoking patients compared with non-smokers with NSCLC were documented in our study. It is known that tobacco smoke stimulates the infiltration of the damaged tissue by a variety of inflammatory immune cells, including neutrophils, macrophages, CD4
+, CD8
+, and B cells and infiltration of dendritic cells and natural killer cells at smaller numbers [
37]. In agreement with these data, our previous study showed a greater number of total and tumor stroma-infiltrating CD4
+ and CD8
+ T cells in smoking NSCLC patients compared with non-smokers [
38]. Macrophages accumulate in the areas of lung destruction; therefore, their numbers are increased in the lungs of healthy smokers and individuals with COPD. Moreover, exposure to cigarette smoke also changes the macrophage phenotype by deactivation of M1 polarization and induction of M2 polarization. Besides the phenotypic changes, cigarette smoke significantly reduces the phagocytic function of macrophages [
39].
Macrophages are a major component of inflammatory infiltrate of various tumors and infiltration by these cells has been reported to be associated with an unfavorable outcome in several kinds of cancers including breast cancer [
40], melanoma [
41], endometrial cancer [
42], and gastric cancer [
43]. In contrast to other solid tumors, macrophages inhibit the progression of colon cancer [
44,
45] and are associated with better prognosis in prostate cancer [
46]. Moreover, previous studies have documented controversial results regarding the role of macrophages in NSCLC patients’ survival. Chen et al. noted that macrophages were negatively associated with survival in the NSCLC patients [
47]. Toomey et al. and Kawai et al. found no association between the macrophage number and NSCLC prognosis [
24,
48]. Furthermore, Welsh et al. found that the macrophage density in the tumor islets was positively associated with patient survival [
29]. Dai et al. reported that the total number of tumor-infiltrating macrophages did not predict prognosis, but macrophages in the tumor islets were positively associated with survival, and macrophages in the tumor stroma were negatively associated with survival [
49]. This in turn suggests that there may be differences in macrophage distribution and function in different types of cancers, and also this may be because of an antagonistic impact of M1 and M2 macrophage phenotypes on tumor progression. Therefore, a predictive value can be reduced when two macrophage populations are pooled together. However, macrophage phenotypes are not stable. Previous in vivo studies have reported that an activated macrophage phenotype can change over time. For example, during tumor progression, the macrophage phenotype changes from classically to alternatively activated [
50].
Similarly to our study, Ohri et al. performed a study of the distribution of M1 and M2 macrophages in NSCLC. They found that the number of M1 macrophages in the tumor islets was associated with an improved prognosis; however, M1 macrophages in the tumor stroma did not impact NSCLC prognosis [
51]. In contrast, Ma et al. published a study in which M1 macrophages in tumor islets as well as tumor stroma were associated with better NSCLC patient prognosis [
31]. However, in their study, unlike our results, no effect of M2 macrophage infiltration on prognosis was observed. Our study results showed that a higher number of total tumor-infiltrating M2 macrophages were associated with unfavorable NSCLC prognosis. Similar results were presented in a study by Zhang et al. [
32], where they used iNOS as a marker of M1 macrophages and CD163 as a marker of M2 macrophages, as in our study. M2 macrophages are proposed to be pro-tumorigenic [
9,
52,
53]. Contrary to the putative pro-tumorigenic effect, a few reports showed, that the presence of M2 macrophages correlated with a good prognosis in colorectal cancer [
54,
55]. Further investigation as to whether this is because of their biological activity or a co-operative interaction with M1 macrophages is required [
51]. However, the direct effect of macrophages on patients with lung cancer is unclear. Distinctions between studies might be associated with the examination of different lung cancer histological subtypes or different tumor stages. Furthermore, associated comorbidities
, including the presence or absence of COPD, patients’ demographic characteristics such as smoking status may also contribute to these differences.
We observed significantly higher levels of serum IL-10 and IFN-γ as well as TNF-α concentration in NSCLC patients compared with the control group. These findings are in agreement with results from other studies, reporting raised serum levels of TNF-α and IL-10 in NSCLC patients compared with healthy volunteers [
56,
57]. Interestingly, a study by Martin et al. reported opposite results showing diminished levels of TNF-α and IFN-γ in NSCLC patients compared with the control group [
58].
It is well known that different macrophage types vary in their functions and, consequently, the cytokines that they secrete [
59]. M1 macrophages express high levels of pro-inflammatory cytokines such as TNF-
α, IL-12, and IL-23 and low levels of IL-10. In contrast, M2 macrophages secrete a series of anti-inflammatory molecules such as IL-10, TGF-
β, arginase1, plus low levels of IFN-
γ, IL-12 and IL-23. The phenotype of macrophages depends on the cytokines produced to support macrophage differentiation [
60]. The interaction between macrophages and cytokines was also found in our study as there was a significant correlation between serum cytokine concentration and the number of macrophages in different compartments of lung cancer. Our study results showed that TNF-α correlated with M1 macrophages in stroma as well as the total number of M1 macrophages and M2 macrophages in tumor islets. Interestingly, we found that the IFN-γ serum concentration correlated with M2 macrophages in islets. These findings suggest that M2 macrophages might play a dual role in carcinogenesis and we hypothesized that M2 macrophages in tumor islets might produce pro-inflammatory cytokines with anti-tumorigenic features. Also this may be because of the production of IFN-γ by other tumor-infiltrating immune cells.
The tumor microenvironment often directs macrophage polarization from the M1 phenotype to the M2 phenotype. In a murine model, blocking of IL-10 receptor was found to promptly trigger a shift in tumor-infiltrating macrophages from the M2 to the M1 phenotype [
61]. IL-10 may inhibit the release of INF-γ, which induces M1 macrophage activation [
62]. Ohtaki et al. reported that IL-10 was significantly associated with the number of tumor stroma-infiltrating M2 macrophages in patients with lung adenocarcinoma [
63], whereas we observed a negative correlation between IL-10 and M1 macrophages in the stroma and total number of M1 macrophages. Our results suggest that IL-10 may impact a diminished M1 macrophage number in the tumor microenvironment.
Enewold et al. reported that the TNF-α serum concentration was associated with worsened NSCLC prognosis [
64], while other authors did not find such associations [
56,
65]. In agreement with these studies, we also did not observe any associations between TNF-α serum levels and NSCLC patients’ survival. Martin et al. reported that a decreased serum IFN-γ level was associated with reduced NSCLC survival [
58]. Contrary to this study, we found that serum IFN-γ level had no prognostic significance. Previous studies showed that decreased serum IL-10 in patients with NSCLC could be linked to poor prognosis [
15,
66]. However, an elevated serum IL-10 level was also associated poor survival [
67]. Moreover, our previous study did not show any associations between the IL-10 level and NSCLC patients’ outcome [
38]. Thus, our findings and previous studies support the idea that cytokines might play a dual role in carcinogenesis.
Our results suggest that, even in early NSCLC stages, while macrophages with anti-inflammatory and pro-tumorigenic features predominated in tumor stroma, a small proportion of M1 macrophages possessing inflammatory and anti-tumorigenic features predominated in the tumor islets, and these phenotypes were related to NSCLC prognosis.