摘要：

目的：探究CD74⁺成纤维细胞在口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）中的表达情况及其与OSCC免疫抑制微环境的相关性。方法：通过免疫组织化学法检测CD74⁺成纤维细胞在OSCC肿瘤组织与癌旁组织中的表达情况，并进一步探讨肿瘤相关成纤维细胞（cancer-associated fibroblasts，CAFs）中CD74表达水平的高低与患者临床病理学参数、预后及免疫细胞浸润的相关性。提取原代CAFs、配对正常成纤维细胞（normal fibroblasts，NFs）及非OSCC患者来源的人外周血单个核细胞（peripheral blood mononuclear cells，PBMCs），比较2种成纤维细胞中CD74表达的差异；随后将2种成纤维细胞与PBMCs共培养，通过酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测其培养液中白细胞介素-10（interleukin-10，IL-10）及IL-35含量。通过Transwell实验及流式细胞术比较不同CD74表达水平的CAFs对PBMCs迁移的影响。结果：OSCC肿瘤组织中CD74⁺成纤维细胞数目明显高于癌旁组织（P<0.001），且CD74高表达的OSCC患者5年总生存率明显更低（P<0.001）。免疫组织化学结果显示，相对于低表达组，在CD74⁺成纤维细胞高表达的肿瘤组织中，FOXP3⁺细胞/CD4⁺细胞的比例显著更高（P<0.001），且CD74⁺成纤维细胞数目与FOXP3⁺细胞数目呈正相关（r=0.439 3）。体外实验结果示，CAFs的CD74表达水平高于对应NFs，与PBMCs共培养12 h后，CAFs CD74高表达组培养液中的IL-10及IL-35的含量显著高于NFs培养液（P<0.05），且CAFs CD74高表达组培养液中IL-35的含量高于CAFs CD74低表达组（P<0.05）；流式细胞术结果显示，CD74高表达的CAFs对于CD4⁺ FOXP3⁺细胞具有更强的趋化能力（P<0.05）。结论：CD74⁺成纤维细胞与OSCC患者不良预后有关，并可能与CD4⁺ FOXP3⁺细胞募集有关。

关键词: 口腔鳞状细胞癌, 成纤维细胞, 免疫抑制微环境, 人外周血单个核细胞

Abstract:

Objective: To investigate the expression of CD74⁺ fibroblasts in oral squamous cell carcinoma (OSCC) and their correlation with the immunosuppressive microenvironment of OSCC. Methods: The expression of CD74 of fibroblasts in OSCC tumor tissues and adjacent non-cancerous tissues will be detected using immunohistochemistry. The correlation between the expression of CD74 in cancer-associated fibroblasts (CAFs) and patients' clinicopathological parameters, prognosis, and immune cell infiltration will be further investigated. Primary CAFs, paired normal fibroblasts (NFs), and human peripheral blood mononuclear cells (PBMCs) from non-OSCC patients will be extracted to compare the differences in the expression of CD74 between the two types of fibroblasts. Subsequently, these two types of fibroblasts will be co-cultured with PBMCs, and the concentrations of interleukin-10 (IL-10) and IL-35 in the culture supernatant will be measured by enzyme-linked immunosorbent assay (ELISA). The effects of CAFs with different CD74 expression levels on the migration of PBMCs were compared by Transwell assay and flow cytometry. Results: The number of CD74⁺ fibroblasts in OSCC tumor tissues was significantly higher than that in adjacent non-cancerous tissues (P<0.001). Moreover, OSCC patients with high CD74 expression had a significantly lower 5-year overall survival rate (P<0.001). Immunohistochemical results showed that in tumor tissues with high CD74⁺ fibroblast expression, the ratio of FOXP3⁺ cells to CD4⁺ cells was significantly higher (P<0.001), and there was a significant positive correlation between the number of CD74⁺ fibroblasts and FOXP3⁺ cells (r=0.439 3). In vitro experiments confirmed that the expression of CD74 in primary CAFs from OSCC tumor tissues was higher than that in the paired NFs. After co-culturing with PBMCs for 12 h, the concentrations of IL-10 and IL-35 in the culture medium of the CAFs CD74 high-expression group were significantly higher than those in the NFs group (P<0.05). Additionally, the concentrations of IL-35 in the culture medium of the CAFs CD74 high-expression group were higher than those in the CAFs CD74 low-expression group (P<0.05). The results of flow cytometry showed that CAFs with high CD74 expression had a stronger chemotactic ability toward CD4⁺ FOXP3⁺ cells (P<0.05). Conclusion: CD74⁺ fibroblasts are associated with poorer prognosis in OSCC patients, and may be involved in the recruitment of CD4⁺ FOXP3⁺ cells.

Key words: oral squamous cell carcinoma, fiborblasts, immunosuppressive microenviroment, peripheral blood mononuclear cells

中图分类号: 

• R782

• R739.8