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

SIMPLE SUMMARY: Oral 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. ABSTRACT: 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 RT(2) 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.