肿瘤相关巨噬细胞和 PD-L1 在肝癌患者中的表达及预后模型的构建。

BACKGROUND: Hepatocellular carcinoma (HCC) is an inflammation-associated tumor involved in immune tolerance and evasion in the immune microenvironment. Immunotherapy can enhance the immune response of the body, break immune tolerance, and then recognize and kill tumor cells. The polarization homeostasis of M1 and M2 macrophages in tumor microenvironment (TME) is involved in the occurrence and development of tumors and has been considered a hot topic in tumor research. Programmed cell death ligand 1 (PD-L1) plays an important role in the polarity of TAM and affects the prognosis of HCC patients as a target of immunotherapy. To this end, efforts were hereby made to further explore the application value of PD-L1, M1 macrophages (CD86), and M2 macrophages (CD206) in the prognosis assessment of HCC, their correlation with immune cell infiltration in HCC tissues, and their bioenrichment function. METHODS: The gene expression omnibus (GEO) and the Cancer Genome Atlas (TCGA) database were used to analyze the expression of PD-L1, CD86, and CD206 in different tumor tissues. The correlation between the expression of PD-L1, CD86, and CD206 and the infiltration of immune cells was analyzed using the Tumor Immune Estimation Resource (TIMER). The tissue specimens and clinicopathological data of hepatocellular carcinoma patients having undergone surgical treatment in our hospital were collected. Immunohistochemistry was used to verify the expression of PD-L1, CD86, and CD206, and analyze the relationship with clinicopathological features and prognosis of patients. Besides, nomogram was constructed to predict the overall survival (OS) of patients at 3 and 5 years. Finally, the protein-protein interaction network information was analyzed using STRING database, and GO analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis were performed to study the biological functions of PD-L1, CD86, and CD206. RESULT: Bioinformatics analysis found that PD-L1, CD86, and CD206 were underexpressed in various tumor tissues including liver cancer, while the present immunohistochemical detection found that PD-L1, CD86, and CD206 were overexpressed in liver cancer tissues. Expressions of PD-L1, CD86, and CD206 were positively correlated with the infiltration level of immune cells in liver cancer, while the expression of PD-L1 was positively correlated with the degree of tumor differentiation. Meanwhile, the expression level of CD206 was positively correlated with gender and preoperative hepatitis, and patients with high expression of PD-L1 or low expression of CD86 had poor prognosis. AJCC stage, preoperative hepatitis, and the expression levels of PD-L1 and CD86 in cancer tissues were independent risk factors affecting survival of patients after radical hepatoma surgery. KEGG pathway enrichment analysis showed that PD-L1 was significantly enriched in T cell aggregation and lymphocyte aggregation, and might be involved in the formation of T cell antigen receptor CD3 complex and cell membrane. Besides, CD86 was significantly enriched in positive regulation of cell adhesion, regulation of mononuclear cell proliferation, regulation of leukocyte proliferation, and transduction of T cell receptor signaling pathway, while CD206 was significantly enriched in type 2 immune response, cellular response to LPS, cellular response to LPS, and involvement in cellular response to LPS. CONCLUSION: In conclusion, these results suggest that PD-L1, CD86, and CD206 may be involved not only in the occurrence and development of HCC, but also in immune regulation, indicating the potential role of PD-L1 and CD86 as potential biomarkers and new therapeutic targets for prognosis assessment of liver cancer.

背景：肝细胞癌（HCC）是一种与炎症相关的肿瘤，涉及免疫微环境中的免疫耐受和逃逸。免疫疗法可以增强机体的免疫反应，打破免疫耐受，从而识别和杀伤肿瘤细胞。肿瘤微环境（TME）中 M1 和 M2 巨噬细胞的极化平衡与肿瘤的发生发展有关，已被认为是肿瘤研究的热点。程序性死亡配体 1（PD-L1）在 TAM 的极性中起着重要作用，并作为免疫治疗的靶点影响 HCC 患者的预后。为此，我们进一步探讨了 PD-L1、M1 巨噬细胞（CD86）和 M2 巨噬细胞（CD206）在 HCC 预后评估中的应用价值及其与 HCC 组织中免疫细胞浸润的相关性，以及它们的生物富集功能。

方法：利用基因表达综合数据库（GEO）和癌症基因组图谱（TCGA）数据库分析不同肿瘤组织中 PD-L1、CD86 和 CD206 的表达。利用肿瘤免疫估计资源（TIMER）分析 PD-L1、CD86 和 CD206 的表达与免疫细胞浸润的相关性。收集我院行手术治疗的 HCC 患者的组织标本和临床病理资料。采用免疫组织化学法验证 PD-L1、CD86 和 CD206 的表达，并分析其与患者临床病理特征和预后的关系。此外，构建列线图预测患者 3 年和 5 年的总生存率（OS）。最后，利用 STRING 数据库分析蛋白-蛋白相互作用网络信息，并进行 GO 分析和京都基因与基因组百科全书（KEGG）分析，研究 PD-L1、CD86 和 CD206 的生物学功能。

结果：生物信息学分析发现 PD-L1、CD86 和 CD206 在包括肝癌在内的各种肿瘤组织中表达下调，而目前的免疫组织化学检测发现 PD-L1、CD86 和 CD206 在肝癌组织中表达上调。PD-L1、CD86 和 CD206 的表达与肝癌中免疫细胞的浸润水平呈正相关，而 PD-L1 的表达与肿瘤分化程度呈正相关。同时，CD206 的表达水平与性别和术前肝炎呈正相关，PD-L1 高表达或 CD86 低表达的患者预后不良。AJCC 分期、术前肝炎以及肿瘤组织中 PD-L1 和 CD86 的表达水平是影响肝癌根治性手术后患者生存的独立危险因素。KEGG 通路富集分析表明，PD-L1 显著富集于 T 细胞聚集和淋巴细胞聚集，可能参与 T 细胞抗原受体 CD3 复合物和细胞膜的形成。此外，CD86 显著富集于细胞黏附的正调控、单核细胞增殖的调控、白细胞增殖的调控和 T 细胞受体信号通路的转导，而 CD206 显著富集于 2 型免疫反应、细胞对 LPS 的反应、细胞对 LPS 的反应和细胞对 LPS 的反应的参与。

结论：综上所述，这些结果提示 PD-L1、CD86 和 CD206 不仅可能参与 HCC 的发生发展，还可能参与免疫调节，表明 PD-L1 和 CD86 作为潜在的生物标志物和新的治疗靶点，可用于评估肝癌的预后。