骨肉瘤肿瘤免疫微环境相关分析

Analysis of the tumor immune microenvironment of osteosarcoma

目的探讨骨肉瘤的肿瘤微环境,以促进骨肉瘤免疫疗法的进展。方法从癌症基因组图谱(TCGA)数据库中获取了119例骨肉瘤患者的mRNA表达数据,分析骨肉瘤的肿瘤微环境,通过一致性聚类将患者分为4个聚类,并将骨肉瘤组织依据是否为炎性组织分为冷肿瘤与热肿瘤,对比4个聚类免疫相关评分及免疫相关基因的表达水平,对冷、热肿瘤进行生存分析,最后进行聚合酶链反应(PCR)、基因敲除、细胞计数试剂盒(CCK-8)以及细胞凋亡实验进行验证。组间比较采用ANOVA分析。结果聚类4拥有较高的免疫评分与基质评分(P<0.01),其与热肿瘤特点相似,而CD276在聚类4中表达较低(P<0.01),是骨肉瘤治疗的潜在靶点。生存分析曲线显示热肿瘤有相对较好的预后,但其差异无统计学意义(P>0.05)。此外,在冷肿瘤中NPTX2的表达与CD8A、GZMB和IFNG的表达呈负相关,且NPTX2可抑制CCL4的分泌。最后,体外实验发现骨肉瘤细胞中NPTX2的敲减可抑制肿瘤生长并增加肿瘤细胞凋亡。结论本研究探索骨肉瘤中免疫细胞的浸润特征,为改善骨肉瘤患者的免疫治疗现状提供依据。

Objective To understand the tumor microenvironment(TME)of osteosarcoma to promote the development of immunotherapy against this cancer.Methods The mRNA expression data of 119 osteosarcoma patients were obtained from the cancer genome atlas(TCGA)database,and the tumor microenvironment of osteosarcoma was analyzed.The patients were divided into four clusters,and the tissues were divided into cold tumors and hot tumors according to whether they were inflammatory.The immune related scores and the expression levels of immune related genes of the four clusters were compared,and the survival analysis of cold and hot tumors was carried out.Finally,polymerase chain reaction(PCR),gene knockout,cell counting kit-8(CCK-8)and apoptosis were performed.Results Cluster 4 had a higher immune score and stromal score(P<0.01),while CD276 expression was low in the cluster 4(P<0.01),which was a potential target for osteosarcoma treatment.Survival analysis curve showed that hot tumors had a good prognosis,but the difference was insignificant(P>0.05).In addition,the expression of NPTX2 was negatively correlated with the expression of CD8A,GZMB and IFNG in cold tumors,and NPTX2 could inhibit the secretion of CCL4.Finally,knockdown of NPTX2 in osteosarcoma cells was found to inhibit tumor growth and increase tumor cell apoptosis in vitro experiments.Conclusion This study explored the infiltration characteristics of immune cells in osteosarcoma,and provided basis for improving the immunotherapy of osteosarcoma.