METHODS: In this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization.
RESULTS: In the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells.
CONCLUSIONS: CCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.
方法:在这项工作中,我们构建了CCR7基因敲除小鼠和OSCC小鼠模型。采用单细胞RNA测序(scRNA-seq)和生物信息学方法分析3只CCR7基因敲除小鼠(KO)和3只野生型小鼠(WT)OSCC微环境的差异。免疫组织化学,免疫荧光染色,和流式细胞术用于分析KO和WT组之间显着不同细胞类型中关键基因的表达。体外实验验证CCR7对M2巨噬细胞极化的影响。
结果:在小鼠OSCC模型中,KO组肿瘤生长速率显著低于WT组。八种主要细胞类型(包括肿瘤细胞,成纤维细胞,巨噬细胞,粒细胞,T细胞,内皮细胞,单核细胞,和B细胞)通过Seurat分析鉴定。scRNA-seq结果显示肿瘤细胞比例较低,但KO组炎症细胞比例显著高于WT组。CellPhoneDB分析结果表明肿瘤细胞与巨噬细胞之间存在很强的相互作用关系,T细胞,成纤维细胞,和内皮细胞。功能富集结果表明,在各种细胞类型中,KO组Dusp1基因的表达水平普遍高于WT组。巨噬细胞亚聚类结果表明,KO组M2巨噬细胞比例低于WT组。体外实验结果表明,CCR7可以促进M2巨噬细胞的极化,从而促进了扩散,OSCC细胞的侵袭和迁移。
结论:CCR7基因敲除可通过促进M2巨噬细胞的极化而显著抑制小鼠口腔鳞癌的生长。