Background
Oral squamous cell carcinoma (OSCC), which accounts for approximately 2 % of total new cancer cases, is the most common type of oral cancer [
1]. Despite recent advances in our understanding and in the treatment of other types of cancer, the five-year survival rate after diagnosis of OSCC remains low at approximately 50–60 % [
2]. The survival rate of patients with early-stage OSCC is higher than that of advanced patients, exceeding 70 % [
3]. Therefore, early detection of OSCC is indispensable for improving prognosis.
Oral leukoplakia is a premalignant lesion of the oral mucosa that is characterized by a circumscribed thickening of the mucosa covered by whitish patches [
4]. Although hospital-based follow-up studies have shown that between <1 % and 18 % of oral premalignant lesions will develop into oral cancer, a certain clinical subtype of leukoplakia with epithelial dysplasia has been shown to be at an increased risk for malignant transformation [
5]. However, histological assessment of epithelial dysplasia has also demonstrated that not all lesions that show dysplasia will develop into oral cancer, and some will even regress [
5]. Therefore, the development of other methods for predicting the malignant potential of premalignant lesions has been proposed. Recent studies have examined the molecular profiles of oral premalignant lesions in terms of the risk for malignant transformation [
6]. Genetic alterations and molecular abnormalities have been identified in oral premalignant lesions. A loss of heterozygosity (LOH) at chromosome 9p and 3p and the absence of p19, a tumor-suppressor protein, are frequently observed in oral premalignant lesions [
7,
8].
Although genetic alterations in epithelial cells are essential for the development of premalignant lesions, recent studies have shown that the nature of the tumor microenvironment and circumjacent stromal cells, including infiltrated immune cells, can significantly modify the outcome of these alterations [
9,
10]. Numerous studies have demonstrated that tumor-associated macrophages (TAMs) initiate and promote tumorigenesis in many types of solid tumors [
11‐
13], and a strong correlation between an abundance of TAMs and poor prognosis has been demonstrated in breast, prostate, cervical, and bladder cancers [
11]. However, contrary to their tumor promoting function, TAMs that infiltrated colon and lung cancers have been associated with a better prognosis in patients [
14‐
18]. Analysis of the phenotypes of the infiltrated TAMs revealed that the TAMs involved in poor patient prognosis share many common features with alternatively activated macrophages or M2 macrophages, which express high levels of the scavenger receptors CD163 and CD204, high levels of the chemokines CCL17, CCL22 and CCL24, and low levels of IL-12 [
12,
19]. In contrast to alternatively activated macrophages, the TAMs associated with a better patient prognosis share a phenotype with classically activated macrophages or M1 macrophages, which express HLA-DR, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) [
17,
18]. These lines of evidence indicate that the functional competence of macrophages is heterogeneous and that the functional properties are acquired and modified in response to changes in the tumor microenvironment [
12,
13].
Previous studies have observed the increased infiltration of mononuclear cells in oral premalignant lesions and OSCC [
20‐
24]. We and others have previously observed an increased number of TAMs during the progression of OSCC, and this number positively correlates with the histopathological grade of OSCC and poor prognosis in OSCC patients [
25‐
29]. These results suggest that TAMs participate in the progression and development of OSCC. Although the phenotypes of TAMs in various types of solid tumors have been extensively characterized, the phenotypes and functional properties of the TAMs that infiltrate premalignant lesions of solid tumors remain to be determined. This study aimed to examine TAM density in oral leukoplakia, a premalignant lesion of the oral cavity, and to characterize the macrophage phenotype (M1 or M2). We also investigated the involvement of infiltrated T cells that contribute to the induction of macrophage phenotypes.
Discussion
Macrophages are one of the major cellular components in the tumor microenvironment, and macrophages have been considered to be crucial to tumor development [
13,
37]. Although a number of studies have reported the phenotypes and properties of these macrophages (i.e., TAMs) in various human solid tumors, including oral squamous cell carcinoma, the phenotypes of TAMs in the premalignant lesions of these solid tumors remain to be determined. Specifically, changes in the phenotypes and functional properties of TAMs in premalignant lesions during tumor development have not yet been characterized. To this end, we examined dissected specimens from 30 patients who underwent biopsy and received a diagnosis of oral leukoplakia, an oral premalignant lesion, by immunohistochemical analysis of several macrophage and T cell markers. The results demonstrated that although CD163 has been considered a M2 macrophage marker in many solid tumors [
38‐
41], the CD163
+ macrophages in oral leukoplakia appear to possess an M1 phenotype characterized by the expression of IFN-inducible gene products. Furthermore, infiltrated CXCR3
+ and CCR5
+ Th1 cells, a major IFN-producing cell type, were also observed in the tumor microenvironment. These results suggest that the infiltrated Th1 cells, which produce IFN, affect the phenotype of CD163
+ macrophages in oral premalignant lesions.
The macrophages were classified as M1 (classically activated) and M2 (alternatively activated) macrophages based on the expression of macrophage gene products, including receptors, cytokines, and effector molecules, induced by classical macrophage-activating stimuli such as Th1-derived IFN or the Th2-derived anti-inflammatory cytokines IL-4 and IL-13 [
12,
19,
42]. M1 macrophages produce large amounts of pro-inflammatory cytokines, reactive oxygen intermediates and reactive nitrogen intermediates, such as nitric oxide (NO), which contribute to the anti-tumor activity of macrophages [
12]. In contrast, M2 macrophages have been suggested to contribute to angiogenesis, tissue remodeling, and tumor progression by inducing the expression of mannose receptors, scavenging receptors, angiogenic factor such as vascular endothelial growth factor (VEGF), and low levels of pro-inflammatory cytokines [
12]. Although the M1/M2 concept of macrophage polarization helps explain the functional properties of macrophages in various infectious and immunological diseases, recent accumulated evidence has shown that the infiltrated TAMs in human solid tumors appear to consist of a heterogeneous population [
40,
43]. TAMs in human cutaneous SCC appear to consist of a mixed subpopulation of CD163
+ cells that express M1 markers, M2 markers or both M1 and M2 markers [
43]. In agreement with this previous study, we have also demonstrated that CD163
+ cells express an M1 marker in oral premalignant lesions. Related to these findings, we and others have previously reported that CD163
+ macrophages are the major TAMs in OSCC and that an increased number of CD163
+ macrophages correlates with a poor prognosis [
27,
28,
44,
45]. Our preliminary double-labeling immunofluorescence data for CD163 and STAT1 shows that the CD163
+ TAMs in OSCC also coexpress STAT1. These results suggest that CD163
+ TAMs with the M1 phenotype persist in the tumor microenvironment from the premalignant to the malignant stage. The functional heterogeneity of the CD163
+ TAMs that express M1 markers in OSCC in terms of antitumor or protumor potency remains to be determined. CD163
+ TAMs need to be further characterized to better understand the role of TAMs in the progression of OSCC.
TAMs acquire functional competence in response to various cytokines and mediators encountered within the tumor microenvironment [
46]. Tumor-associated immune cells, as well as tumor cells themselves, are the major sources of mediators that affect the functional properties of TAMs. A mouse tumor model of mammary carcinomas demonstrated that IL-4-expressing CD4
+ T cells indirectly promote the invasion and metastasis of mammary adenocarcinomas by promoting the protumor function of TAMs [
47]. The importance of Th2 cytokines, including IL-4, IL-10, and IL-13, in the regulation of the protumor functions of TAMs has also been demonstrated in human lung adenocarcinomas [
40,
48]. However, the role of infiltrated T cells in the polarization of TAMs in premalignant lesions of human solid tumors is not completely understood. In the present study, we evaluated the relationship between infiltrated T cells and the polarization of TAMs in oral leukoplakia and found a positive correlation between the numbers of CD4
+ T cells and CD163
+ macrophages. Intriguingly, the infiltrated CD4
+ T cells in oral leukoplakia consisted of CXCR3
+ and CCR5
+ Th1 cells, a major IFN-producing cell type, and CCR4
+ Th2 cells were rare in the lesion. Consistent with the increased infiltration of Th1 cells and STAT1
+ cells, the expression of an IFN-inducible gene product [
35] was also increased in the lesion. These results suggest that the tumor microenvironment of oral leukoplakia creates a Th1-dominated microenvironment that polarizes TAMs toward the M1 phenotype. Our double-labeled immunofluorescence analysis demonstrated that CD163
+ macrophages coexpressed active STAT1 (pSTAT1). Thus, it is highly likely that the infiltrated Th1 cells modulate the phenotype of TAMs in oral premalignant lesions.
The recruitment of CXCR3
+ Th1 cells is mediated by IFN-inducible chemokines such as CXCL9 and CXCL10 [
49], which are produced by a variety of cell types, including epithelial cells, fibroblasts, and macrophages, in response to IFNs [
50,
51]. IFNs and TNF or CD40 ligand synergistically induce the expression of these chemokines [
52,
53]. Although the initial triggering molecules that induce these chemokines are unknown, epithelial cells in the dysplastic lesion, which are continuously stimulated by carcinogens and are genetically altered, may produce these chemokines. In fact, our immunohistochemical analysis demonstrated that CXCL9 was present in the subepithelial lesion of leukoplakia. After the recruitment of CXCR3
+ Th1 cells, the secretion of IFN could further skew the oral premalignant lesions toward a Th1-dominated microenvironment. Intriguingly, a previous proteomic analysis of OSCC revealed that the IFN signaling pathway is significantly enhanced in OSCC lesions and that the expression of IFN-inducible gene products, including STAT1, was up-regulated [
54]. Taken together, these results suggest that the persistent IFN-stimulating environment from the premalignant to malignant lesion may allow tumor cells to acquire resistance to the antitumor responses of IFNs via cancer immunoediting [
55]. IFN-induced M1 macrophages have been shown to act as important effectors during cancer immunoediting in a mouse tumor model [
56]. Further
in vivo studies using animal models of OSCC are needed to explore the functional role of IFN-stimulated M1 macrophages in the progression of malignant transformation.
We histopathologically graded the biopsy specimens of oral leukoplakia based on the WHO classification [
30] and explored the relationship between the histological grading and the levels of infiltrated immune cells. Significant increases in CD163
+ macrophages (Fig.
1) and intraepithelial CD4
+ T cells (Fig.
2b) were observed in moderate dysplasia compared to samples without dysplasia (Fig.
1). The immunohistochemical analysis showed that CD163
+ macrophages were mainly distributed in the subepithelial region. Although the etiological roles of CD163
+ macrophages and intraepithelial CD4
+ T cells in the development of dysplasia are unclear, CD163
+ macrophages may contribute to the infiltration of intraepithelial CD4
+ T cells. Because intraepithelial lymphocyte migration is accompanied by fragmentation of the basement membrane [
57], CD163
+ macrophages may secrete matrix metalloproteinases (MMPs) that degrade the basement membrane [
58]. A loss of basement membrane components has been correlated with the invasive potential of malignant epithelial neoplasms [
59]. CD163
+ macrophages and the infiltration of T cells into the epithelial lesion may contribute to the early architectural disturbance of the epithelium during the development of dysplasia.
Competing interest
The authors declare that they have no competing interests.
Authors’contributions
KM and SH participated in the collection of biopsy specimens and carried out the immunohistochemistry staining and quantification. MH carried out the double-immunofluorescence staining. JS performed statistical analyses. YO designed and coordinated the studies, participated in data analysis, and drafted the manuscript. All authors critically reviewed and accepted the final version of the manuscript.