The potential role of microenvironment (ME) inflammation has been extensively investigated in carcinogenesis, tumor progression and potential implications for treatment across various malignancies, including Oral Squamous Cell Carcinoma (OSCC) (1, 2).

Moving to the onset and progression towards malignant transformation (MT) of Oral Potentially Malignant Disorders (OPMDs), the literature provides limited evidence and implications due to inflammatory infiltrate still needs to be fully elucidated (3). It would be valuable to understand whether the inflammatory ME has a role in promoting MT and whether its features and role are consistent among different OPMDs.

A band-like chronic inflammatory cell infiltrate in the superficial lamina propria (with a so-called lichenoid features) commonly accompanies premalignant and malignant oral lesions (4). Recently the potential role of the inflammatory infiltrate in promoting oral carcinogenesis has been highlighted and it has been hypothesized that OPMD-associated inflammation can represent a surrogate marker to diagnostically differentiate between Oral Epithelial Dysplasia (OED) and Oral Lichen Planus (OLP) and to predict MT (5).

Microenvironment is shaped by the dynamic interaction between inflammatory and non-inflammatory cells, along with a variety of mediators able to influence disease progression and the immune response (3, 6, 7). Evidences on the pivotal role of infiltrating macrophages (MΦ) in tumor development and progression lead to the establishment of the concept of Tumor Associated Macrophages (TAMs) (8–10). Moving to OPMDs, we are just laying the first foundations for a more in-depth knowledge of the potential role of the inflammatory infiltrate in OPMDs.

The present review aims to collect evidence from the literature about presence and characterization of MΦ infiltrate in OPMDs ME.

In normal oral mucosa, MΦ are present in the subepithelial connective tissue but in lower numbers compared to other immunocompetent cells (T lymphocytes and dendritic cells) and they are only occasionally observed in the epithelium. Among inflammatory cells, MΦ have garnered considerable interest in recent years since they seem to play a critical role in tumor development and progression, so that MΦ associated to tumor progression are commonly referred as Tumor-Associated Macrophages (TAMs) (10). While typically sparse in mucosal and submucosal tissues, MΦ increase in both OPMDs and OSCC. These MΦ originate from resident macrophage and circulating monocytes, which are recruited by signals from epithelial cells and the surrounding stroma. Dendritic cells (DCs) and MΦ represent a first line of innate immune surveillance, recognizing pathogens or tissue damage; by presenting antigens to T cells they initiate and regulate adaptive immunity (50). Data from the literature investigating the expression of DCs in OPMDs are contrasting: some studies link OL, PL or high-grade OED to reduced CD1a+ Langerhans cell density (30, 51, 52), while others report an increase (53). Another study found no difference between OL samples with and without OED (54).

The immunocompetent cells, especially the MΦ and lymphocytes, are likely the main source of cytokine synthesis. Of interest, MΦ are characterized by an ambivalent role due to their potential polarization into two distinct phenotypic profiles: M1, generally associated with pro-inflammatory and anti-tumor activity, and M2, linked to anti-inflammatory and pro-tumor functions.

Immunosuppressive tumor microenvironment (iTME) plays a key role in carcinogenesis, and some MΦ subsets are associated with iTME generation. However, the sub-population characterization of MΦ in oral carcinogenesis remains largely unclear (13).

TAMs display remarkable plasticity within the TME and may transform from one phenotype to another and they always present a mixture of M1-like and M2-like phenotypes (55).

Classically activated (M1) MΦ are induced in response to IFN-γ and lipopolysaccharides and they are characterized by the production of high levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin IL-1, IL-6, IL-12, IL-23 and inducible nitric oxide synthase (iNOS), which play a crucial role in their anti-tumor activity. Notably, iNOS plays a dual role: in early stages, it promotes inflammation and anti-tumor immunity, while in advanced tumors, it contributes to immune suppression and tumor progression. Alternatively activated (M2) MΦ mainly secrete molecules involved in processes such as angiogenesis, tissue remodeling, and tumor progression: IL-10, arginase (ARG), and transforming growth factor β (TGF-β) to suppress the inflammatory response and upregulating mannose receptors, scavenger receptors, and angiogenic factors like vascular endothelial growth factor (VEGF), while exhibiting low levels of pro-inflammatory cytokines (8). M1 TAMs can recruit CD8+ cytotoxic T cells, while M2 TAMs predominantly attract CD4+ T cells, particularly Tregs (56).

Different markers are associated with M1 or M2 polarization (57, 58) as shown in Table 1. It is known that the M2-like phenotype predominates in TAMs with an established tumor-promoting effect and M1-like phenotype are conventionally known for their anti-tumor functions, including the induction of inflammation and direct tumor cell attack. However, recent studies suggest that, under specific conditions, they may also contribute to tumor progression (59). Furthermore, TAMs expressing different markers have been observed to localize in different regions within the TME, and it could be related to their specific functions. Namely, CD163+ cells are distributed throughout the stroma, whereas CD204+ and CD206+ cells are predominantly concentrated near the tumor nest (60).

The recruitment of MΦ could be an early event in the carcinogenesis process, which could lead to the initial dense infiltration of both M1 and M2 MΦ. Therefore, an overall assessment could not be able to detect differences due to macrophage polarization rather than to the overall cell count. When comparing macrophage infiltration between different steps in the carcinogenesis process (e.g., OED and OSCC), an overall assessment based of a pan-macrophage marker (CD68) could not be able to differentiate such conditions (27). Additionally, since polarization exists on a spectrum rather than a strict dichotomy, using multiple markers increases accuracy. To accurately quantify the proportion of M1 and M2 MΦ, a combination of markers should be used (e.g., CD80/CD86 for M1, CD163/CD206 for M2).

Recent advances in techniques including scRNA-seq and spatial transcriptomics have made it possible to simultaneously obtain spatial organization information and transcriptome data, providing a comprehensive spatiotemporal perspective on gene expression within a specific tissue or throughout disease progression (61). A couple of studies applied such techniques on sampling from the same patient and simultaneously containing a normal region, OL harboring moderate-severe dysplasia (OED-OSCC) and OSCC (13, 15). This method is of interest as pairwise sampling allows to reduce heterogeneity when comparing sampling (62).

The aim of the present review is to improve knowledge of the role of MΦ in OPMDs; this implied the inclusion of heterogeneous studies where different OPMDs and different MΦ markers have been investigated. In addition to this, clear numerical data were not always available, making a meta-analysis approach unfeasible. Even with such limitations, a comprehensive analysis of these studies, including 1,573 samples from OPMDs or OSCC, reveals quite consistent results, describing differences (often significant) in MΦ infiltration between normal mucosa, OPMDs, OED, and OSCC. The present review, excluding in vitro studies, could have missed research investigating the mechanisms through which MΦ influence the immune microenvironment and contribute to carcinogenesis.

When considering tumor immunology, OPMDs could represent the equilibrium phase as defined by the concept of cancer immunoediting (26), but key inflammatory mediator(s) able to modulate the progression of OPMDs to OSCC have not yet been identified.

In the presence of OPMDs, MΦ may predominantly exhibit an anti-tumorigenic phenotype, aimed at counteracting MT, this could be consistent with an increasing M2 prevalence positively correlated with the grade of OED, while in earlier stages M1 have been reported to be present (35); CD163+ macrophages in oral leukoplakia co-express active STAT1 and suggest that the CD163+ macrophages possess an M1 phenotype in a Th1-dominated microenvironment. Consistently, RNA-seq and Gene Ontology analyses revealed in moderate-severe OED an inflammatory microenvironment primarily characterized by altered expression of genes related to immunosurveillance, lymphocyte infiltration, cytotoxic response, and surrogate markers of tumor-associated MΦ (5). This could align with a clinical scenario where OL does not progress to MT, at least in the short term, possibly due to MΦ polarized toward an anti-tumorigenic phenotype. If, as suggested by Mori, CD163+ and STAT1+ characterize MΦ infiltrating OL before transformation, this may indicate that MΦ are actively working to prevent malignant progression. The observed presence of infiltrated Th1 cells (CD4+ T cells CXCR3+ and CCR5+), producing IFN and affecting the phenotype of CD163+ MΦ in OL could be consistent with a ME trying to contrast the progression of OL. However, since Mori selected OL cases without considering their eventual transformation, the long-term clinical trajectory remains uncertain. Finally, the increased presence of CD163+ MΦ and intraepithelial CD4+ Th1 cells observed in the presence of moderate OED, compared to samples without dysplasia, could be associated with an increased immunogenicity of dysplastic keratinocytes (35).

Transforming OL showed an increased or decreased CD163+ MΦinfiltration in the epithelial or subepithelial compartment respectively, while no differences for CD11c+ MΦ infiltration were observed related to MT in OL (31). In transforming OL an increased infiltration of CD163+ MΦ and an M2 polarization (inferred based on the CD163/CD11c expression ratio) were observed only in the epithelial compartment (31). It is important to highlight that the study included OL cases with MT occurring within a five-year timeframe. This extended period of transformation does not allow to exclude the possibility of a gradual shift from M1 to M2 polarization, where MΦ may initially display an M1 phenotype to prevent malignant transformation. The presence and grade of OED was associated with macrophage infiltration (31). The presence of CD163+ MΦ in the epithelial compartment has also been reported to precede MT by up to two years (63) and it has been proposed as a red flag in cases where incisional biopsy results appear negative for OSCC, but there is a high clinical suspicion (33). In studies nonspecifically addressing MΦ, the presence of an inflammatory microenvironment has been observed in 57% of non- dysplastic OL that later underwent MT (64). Similarly, the inflammatory microenvironment associated to PL is not a recent discovery. Silverman, shortly after identifying Proliferative Verrucous Leukoplakia, reported an “inflammatory infiltrate in the connective tissue quite variable, ranging from mild and diffuse to dense subepithelial clustering” (65). This finding has been confirmed in subsequent case studies and is more common in the early stages, where multiple white plaque-like lesions may present with chronic sub-epithelial inflammation in the absence of OED (66). Such evidence suggests a potential role for inflammatory microenvironment not limited to OPMDs with a recognized inflammatory pathogenesis (e.g., OLP).

Notably, although most studies report an increase in CD163+ cell infiltration in the transition from high-grade OED to OSCC, a couple of studies do not confirm this finding (23, 49). Kouketsu et al. did not find a significant difference when analyzing CD204+ cells either; conversely, a significant increase was observed between non-dysplastic OL and severe OED (including carcinoma in situ). It could be speculated whether merging severe OED and carcinoma in situ may have affected such results (23). Nevertheless, the absence of significant differences between high-grade OED and OSCC could reflect early M2 polarization occurring in high-grade OED, thus favoring MT rather than acting as an immune effector against malignant progression. Looking at the IHC data from Yuan and Li, the progressive increase in CD163+ cell expression from moderate to severe OED to OSCC results in a statistically significant difference only when comparing severe OED or OSCC to normal mucosa, but not when addressing the transition from OED to OSCC (49).

In OPMDs, chronic inflammation may recruit CD11c+ monocytes that differentiate into macrophages (CD68+) under the influence of cytokines such as M-CSF, GM-CSF, and TGF-β. Subsequent activation of NF-κB in these macrophages can further regulate their inflammatory and immunosuppressive functions. Moreover, NF-κB induces the expression of indoleamine 2,3-dioxygenase (IDO) expression in MΦ (67). Zhang et al. identified Macro-IDO1 and Macro-PLA2G2D as dominant subsets in OL-OSCC and almost not existent in distant normal mucosa, thus conceivably involved in carcinogenesis (13). IDO is an intracellular enzyme that is primarily expressed in antigen presenting cells such as in dendritic cells (CD11c+) and MΦ (CD68+) and represents a mechanism of acquired immune tolerance in cancer (68). The expression of IDO in OPMDs has been investigated in a couple of studies with the same laboratory methods (double/multiple IF). In AC, CD68+ cells did not show IDO expression (36). Similarly, in OL, IDO1+ macrophages were nearly absent, but their infiltration significantly increased in OL associated with OSCC (13). Moreover the same study reported a positive relationship between the proportions of IDO1 + CD68+ cells (IDO1 + macrophages) and the PD-1 + CD3 + CD8+ cells (exhausted CD8+ T cells) in OL associated to OSCC and OSCC (13).

Further assessments in mice revealed that IDO1 inhibitors significantly reduces 4NQO induced oral carcinogenesis. These findings suggest that Macro-IDO1 is a key macrophage sub-cluster potentially associated to the shift of OL to OSCC (13).

In OSCC IDO1 + macrophages were strongly positively correlated with IL6/JAK/STAT3 signaling (69). IL-6/JAK/STAT3 and NF-κB signaling form a feed-forward loop, promoting sustained inflammation and disease progression. IL-6-induced STAT3 activation enhances NF-κB signaling by increasing pro-inflammatory cytokines such as IL-6 itself, TNF-α, and IL-1β while also suppressing NF-κB inhibitors such as SOCS proteins.

IFN-γ+ MΦ were predominantly detected in OL rather than in OSCC and their presence was negatively correlated with the progression of oral dysplasia in OL (28). STAT is an interferon-inducible product, and the activation of STAT1 in tumor-associated MΦ (TAMs) can lead to the upregulation of both inducible nitric oxide synthase (iNOS) and Arginase I. These enzymes play significant roles in suppressing T cell function—iNOS by generating nitric oxide, which can directly inhibit T cell activity (70), and Arginase I by depleting L-arginine, an amino acid essential for T cell proliferation and function (71).

Notably, a study comparing OED and normal mucosa reported an increased presence of both CD163+ and iNOS+ cells, though only the rise in iNOS+ cells was statistically significant (24). This finding may indicate a predominant M1 polarization in the early stages of carcinogenesis.

CD163 + STAT1+ MΦ likely represent a plastic or intermediate phenotype, balancing between inflammatory and immunosuppressive functions. They likely represent a potential target for studies investigating OPMDs and particularly conditions characterized by chronic inflammation.

In moderate/severe OED CD163 + STAT1+ were predominantly located beneath the epithelium, but they have also been observed as part of the intraepithelial compartment. In both these locations they seem to account for almost half of immunoreactive cells: 51.5% and 55.1% in the subepithelial and epithelia compartment respectively (5, 35). Both studies found a positive correlation between the presence of CD163+/STAT1+ cells and the distribution of CD4+ cells, supporting the notion that Th1 CD4+ T cell-derived IFN-γ may contribute to their recruitment and activation.

The pro-tumorigenic activity of CD163+ MΦ was shown to be dependent on the presence of nuclear NF-κB (72). Few studies jointly assessed CD163+ cells and the expression of nuclear NF-κB; among them Vered et al. reported an almost complete lack of nuclear expression of NF-κB within the inflammatory cells, indicating a proinflammatory rather than a protumorigenic direction of the CD163+ MΦ in OLP (43).

The PD-1/PD-L1 expression is positively correlated with macrophage infiltration and is often associated with higher concentrations of CD4+ and CD8+ T cells in the immune infiltrate. The PD-1/PD-L1 associated pathway is a key immune checkpoint mechanism that regulates immune tolerance and immune evasion in diseases like cancer (73). MΦ, particularly those polarized toward the M2 phenotype, can actively contribute (up to 14%–32% of PD-L1 expression) to immune suppression through PD-L1 upregulation leading to T cell exhaustion and immune evasion. Several studies consistently suggest that OPMDs may evade the host immune system by PD-L1 expression on not only dysplastic epithelial cells but also the recruited subepithelial microenvironment.

A joint investigation of the expression of CD163 and PD-L1 revealed that more than 90% of CD163+ cells were distributed in the superficial lamina propria (similarly to CD8+ cells). In a mean follow-up of 45.6 months, subepithelial PD-L1+ cell count was found to be a significant risk factor for MT. Though, the characteristics of subepithelial PD-L1+ cells remain to be elucidated as double IF revealed the origin of a limited number of subepithelial cells co-expressing PD-L1, being MΦ (CD163+) 16.6% or CD8+ cells 14.1% (26). Of interest a phase II nonrandomized controlled trial assessing the safety and efficacy of nivolumab in PL patients showed a 2-y cancer-free survival of 73%, consistent with only potential anti-PD-1 activity (74).

Sun et al. observed increasing immunosuppressive macrophage subclusters, specifically Macro_NRG1 and Macro_APOE, when comparing OED-OSCC to OSCC. This suggests that these cells may play a role in MT (15).

The detection of APOE+ MΦ (Macro_APOE) in OPMDs suggests potential involvement in immune suppression and disease progression. These cells were frequently found alongside other immunosuppressive populations as NRG1+ MΦ or regulatory T cells (Tregs) expressing NFRSF4, reinforcing their potential role in immune evasion (15). In other cancers, APOE+ TAMs seem to resemble lipid-associated MΦ (LAMs) and exhibit an M2-like immune profile Braun et al. (75)).

Single-cell analysis also suggests that tissue-resident-like MΦ (Macro_SELENOP) may give rise to Macro_APOE/NRG1, particularly during the transition from OED-OSCC to OSCC (15). This is in keeping with findings from other malignancies, such as renal cell carcinoma, where APOE-enriched M2-like TAMs have been associated with disease progression (75). Finally, high numbers of MΦ co-expressing APOC1 and APOE were found in metastatic lymph nodes of esophageal squamous cell carcinoma, a cancer with a metastatic pattern similar to OSCC (76). The fact that APOE+ macrophages are consistently linked to tumor progression across multiple cancers suggests they may have a conserved, pro-tumorigenic function.

In OLP activated cytotoxic (CD8+) T-lymphocytes are thought to interact with other inflammatory cells such as helper (CD4+) subpopulations, Langerhans cells (CD1a+), and MΦ (CD68+), as well as basal keratinocytes, leading ultimately to keratinocyte apoptosis (77). The renowned pathogenetic role of chronic inflammation in OLP makes it an intriguing disease to investigate and compare to other OPMDs. In two studies both OLP and OLL were included (21, 38). In the absence of OED, both studies reported a higher CD163+ infiltrate in OLP compared to OLL, but the difference was significant in only one of them. Since STAT is an interferon-inducible product, it would have been interesting to examine the potential co-expression of CD163 and STAT1. When compared to OL a significant increase in CD68+ and IFN-γ+ cells was observed as expected, but no other characterization was performed in the study (28). It has been hypothesized that long-term constant use of steroids may contribute to the possible MT in OLP (77), this appears to be consistent with the described upregulation of CD163+ MΦ by glucocorticoids (78). Considering the potential pro-tumorigenic interaction between NF-κB and CD163+ cells, this hypothesis could suggest that alternative therapeutic agents could modulate the immune response in OLP attenuating the inductive effect of the epithelial NF-κB on the interface inflammatory response and regulating the action of TGF-β (43).

In OLP submucosa, MΦ but not T-lymphocytes were identified by merged fluorescent double staining as TRPA1 immunopositive (79), this led to hypothesize that macrophage-derived TRPA1 may contribute to the transition from early immune responses to a chronic inflammatory condition (80). Li et al. explored cell-cell interactions in OLP finding the ITGB2 pathway enriched in T cells, myeloid cells, and fibroblasts when compared to normal mucosa (14). This pathway influences immune cell adhesion, migration, and activation and is supposed to positively regulates cellular adhesion of tissue MΦ. ITGB2 encodes for integrin LFA-1, which plays a pivotal role in T cell and possibly MΦ chemotaxis and tissue infiltration. The importance of the ITGB2 pathway in MΦ function is emphasized by a positive correlation between ITGB2 expression and CD163+ MΦ infiltration in esophageal carcinoma where scRNA-seq analysis indicated a progressive increase in ITGB2 with the acquisition of a tumor-promoting phenotype (81). Similarly, in ovarian cancer, ITGB2 is upregulated when compared to normal tissue and a high ITGB2 expression correlate positively with the infiltration of immune cells, particularly of M2 macrophages (82).

Current research on MΦ in the OPMD microenvironment is still scanty and presents potential gaps. Only one study retrospectively compared progressing and non-progressing OLs, trying to identify microenvironment features associated with MT (31). The lack of comparative studies assessing different OPMDs while properly accounting for the presence and grade of dysplasia, which should be considered a major confounding factor, represents a significant gap in the current literature. Such an approach might offer valuable insights, as the nature and composition of the inflammatory infiltrate could identify common inflammatory signatures related to carcinogenesis or, conversely, could differentiate OPMDs sharing similar clinical features. Moreover, the anatomical subsite could represent a potential bias related to risk habits (e.g., the mucobuccal fold in the case of tobacco or betel quid chewing, and the gingiva in the presence of plaque-related inflammation).

Looking at methods and data reporting, heterogeneity in study design and scoring methods limits reproducibility and the development of MΦ-based prognostic models, and the lack of quantitative data reporting prevents the possibility of performing meta-analyses. Finally, the assumption of anti-inflammatory or immunomodulating drugs was rarely considered as an exclusion criterion or at least as a confounding factor.

Despite the observed heterogeneity among the included studies, evidence from the present review supports an active role of MΦ in regulating immune suppression, oncogenesis, and tumor progression in OPMDs and during the transition to OSCC. They appear therefore to be the direct precursors of TAM subsets observed in other malignancies. Future research should focus not merely on cell quantification and general M1/M2 polarization but rather on the expression of specific markers potentially linked to immunomodulatory pathways involved in oncogenesis, particularly in light of the capabilities offered by new technologies such as scRNA-seq and CIBERSORTx.