Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4+, CD8+, and Foxp3+ Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis.

Abstract

Simple SummaryOral squamous cell carcinoma (OSCC), the most common oral malignancy, severely impacts patient quality of life because of oro-facial destruction. OSCC is preceded by oral premalignant lesions (OPLs). Moreover, lower T cell infiltration in OPLs is associated with OSCC, suggesting that T cell-mediated adaptive immunity protects against malignant transformation. In this study, we used the carcinogen 4NQO, which mimics tobacco-related carcinogenesis, in a mouse model to examine the gene expression kinetics of chemokines/cytokines during OPL and OSCC development. Our results demonstrate that both Th1- and Treg-associated chemokines were simultaneously expressed in 4NQO-induced OPL, with their expression correlating with the infiltration of CD8+ and Foxp3+ cells, respectively. These results indicate that antitumor immune responses and immunosuppression are simultaneously initiated during OLP development.Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.