In this study, we aimed to identify potential therapeutic targets and develop more effective approaches for the treatment of TSCC. By analyzing mRNA expression profiles and immune cell infiltration in TSCC, a total of 24 DEGs related to TSCC were identified. By constructing a PPI network and performing enrichment analysis, seven key genes were screened, including MMP13, POSTN, MMP9, MMP10, MMP3, SPP1, and MMP1. Among them, SPP1 was found to have prognostic value for TSCC. Secretory phosphoprotein 1 (SPP1), also known as osteopontin (OPN) or early T lymphocyte activation 1 protein, is a multifunctional secretory acidic glycoprotein involved in various physiological and pathological processes [3]. While healthy organisms only secrete small amounts of SPP1 for normal physiological functions, its overexpression has been observed in numerous human tumors, including lung, prostate, breast, colorectal, and liver cancers [30]. High levels of SPP1 expression in tumor tissues have been associated with poorer patient prognosis, indicating its potential as a biomarker for monitoring tumor progression [13, 19, 20, 31, 41].

Previous studies have linked SPP1 expression to various aspects of tumorigenesis and tumor progression, such as angiogenesis [14], distant metastasis [17, 23, 29], maintenance of tumor cell stemness [24], and activation of cell proliferation pathways [12, 28]. In the context of oral squamous cell carcinoma (OSCC), several studies have suggested that SPP1 may play a role in its occurrence and development [9, 10, 42, 46]. In the present study, SPP1 expression was found to be significantly elevated in TSCC tissues compared to normal tissues, consistent with previous findings by other researchers [38]. Moreover, the expression levels of SPP1 were found to significantly correlate with patients’ survival compared to other differentially expressed genes (DEGs). These findings highlight the potential of SPP1 as a molecular therapeutic target in TSCC.

The tumor microenvironment (TME) is composed of various cell populations that play critical roles in tumor pathogenesis. Among the immune cells infiltrating tumors, macrophages are the major cell type involved and serve as a key link between inflammation and cancer. Macrophages can be polarized into two distinct phenotypes: M1 and M2 [36]. M1 macrophages are associated with cytokine production, recruitment of pro-immune stimulated leukocytes, and phagocytosis of tumor cells, while M2 macrophages promote tumor development through basement membrane rupture, leukocyte recruitment, angiogenesis, and immune evasion. Higher levels of M1 macrophages have been associated with better patient prognosis, while higher levels of M2 macrophages have been linked to worse prognosis in various tumor types [21, 40]. Therefore, the balance between M1 and M2 macrophages in the tumor microenvironment can influence clinical outcomes. Understanding the composition and functional characteristics of immune cell infiltration, particularly macrophages, provides valuable insights into tumor biology and potential therapeutic targets. In this study, the increased expression of SPP1 was found to be localized mainly to CD68-expressing macrophages, indicating that macrophages are associated with the production of SPP1 in TSCC. Previous studies have shown that SPP1 can influence macrophage behavior and polarization in different cancer models. For example, in a hepatocellular cancer model, SPP1 produced by tumor cells was found to activate the PI3K-AKT-p65 signaling pathway, leading to the secretion of colony-stimulating factor 1 by macrophages, resulting in macrophage infiltration and M2 polarization [45]. Similarly, in a glioblastoma model, SPP1 was found to promote macrophage migration and preserve the properties and phenotype of M2 macrophages through binding to integrin avβ5 receptors. The expression levels of SPP1 were also found to be associated with the degree of macrophage infiltration and glioma grade in patients, suggesting that the interaction between SPP1 and macrophages affects immunological regulation and tumor progression [37].

In our study, using the “deconvolution algorithm,” we analyzed immune cell infiltration in paraneoplastic tissues and tongue carcinoma tissues and observed significant differences in the immune cell composition between the two. CIBERSORT calculates based on gene expression profile data, which can classify and identify mixed cell types, and can help researchers better understand the gene expression profile data and dig deeper into the background biological information, which is of great significance in future research. The parameter adjustment in CIBERSORT is mainly permutation, which refers to the number of times of alignment on the analysis, and the larger the number, the more accurate the final result will be. Elevated SPP1 expression was associated with a higher prevalence of M2 macrophages during immune cell infiltration analysis. Previous studies have demonstrated that M2 macrophages can play a role in promoting tumor cell proliferation and suppressing lymphatic T cell function through the release of various cytokines. They have been identified as important factors influencing host immunity and promoting immune evasion by tumor cells [27]. While several studies have explored the impact of SPP1 expression on tongue tumor cells, there is relatively less research on the involvement of SPP1-affected macrophages in TSCC.

In the past several years, compelling evidence has demonstrated that the posttranscriptional regulation of mRNA, such as N6-methyladenosine (m6A) RNA methylation, plays a critical role in controlling RNA metabolism and function in tumor immune response [7, 39]. A recent study has revealed that m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment of periodontitis [43]. Understanding the role of posttranscriptional regulation of mRNA in TSCC pathogenesis and immune response could provide a promising therapy for TSCC.

This study, however, is subjected to some limitations that could be addressed in future research. First, the expression of SPP1 in TSCC was found to be correlated with the expression of macrophage marker on our study. To improve further upon these findings, we are now collaborating with other institutions to enroll additional patients to carry out external validation of the expression of SPP1 and other immunological biomarkers in TSCC. Second, the molecular mechanism regulating SPP1 expression and immune microenvironment in TSCC is unclear. In the past several years, compelling evidence has demonstrated that the posttranscriptional regulation of mRNA, such as N6-methyladenosine (m6A) RNA methylation, plays a critical role in controlling RNA metabolism and function in tumor immune response [7, 39]. A recent study has revealed that m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment of periodontitis [43]. Understanding the role of posttranscriptional regulation of mRNA in TSCC pathogenesis and immune response could provide a promising therapy for TSCC. Further study may focus on the potential roles of mRNA modification in TSCC immunity and therapy.

Based on the findings of our analysis, the interaction between SPP1 and macrophages could represent a potential immune-related therapeutic target in TSCC. Further research into the role of SPP1 in modulating macrophage behavior and its impact on the immune response in TSCC may uncover novel therapeutic strategies for the disease.