背景:神经母细胞瘤(NB)是最常见和最致命的小儿实体瘤。超过50%的高危神经母细胞瘤病例复发,强调了新的药物靶点和治疗策略的必要性。在神经母细胞瘤中,肿瘤相关巨噬细胞(TAMs)的存在与患者预后不良相关.然而,与神经母细胞瘤中TAMs浸润相关的调节基因的临床相关性和预后意义尚不清楚,需要进一步研究。
方法:我们利用与神经母细胞瘤相关的三个主要数据集(GSE45547,GSE49710,TARGET)的转录组表达谱进行了综合分析,以鉴定与神经母细胞瘤中的免疫逃避有关的枢纽基因。随后,我们利用17个临床神经母细胞瘤样本的单细胞RNA测序分析来研究这些hub基因的表达和分布,导致TNFAIP3的鉴定。将上述三个公共数据库合并,以通过GO和KEGG分析验证TNFAIP3的分子功能。此外,我们通过多种算法评估了TNFAIP3与免疫浸润的相关性及其潜在的免疫治疗作用.我们的单细胞转录组数据揭示了TNFAIP3在巨噬细胞极化中的作用。最后,初步实验验证证实TNFAIP3介导的TAMs在NB中的生物学功能。
结果:共筛选了6个与免疫逃避相关的基因,我们发现TNFAIP3在巨噬细胞中的表达明显高于其他免疫细胞类型,基于scRNA测序数据。GO和KEGG分析显示TNFAIP3的低表达与多种致癌途径以及免疫相关途径的激活显著相关。然后验证证实,TNFAIP3高表达队列中的个体可能会从免疫治疗干预措施中获得更大的优势,同时表现出增强的免疫反应性。破译巨噬细胞的伪时间轨迹,我们揭示了TNFAIP3诱导巨噬细胞向M1表型极化的潜力.最后,我们证实TNFAIP3高表达组的患者可能从免疫治疗或化疗中获益更多,如RT-qPCR和免疫荧光检查所证实.此外,验证了TNFAIP3在巨噬细胞极化中的作用.初步实验表明TNFAIP3介导的TAMs抑制细胞增殖,NB细胞的迁移和入侵能力。
结论:我们的结果表明,TNFAIP3首次被确定为有希望的免疫疗法生物标志物和NB的潜在分子靶标。此外,TAMs中TNFAIP3的存在可能为NB提供一种新的治疗策略.
BACKGROUND: Neuroblastoma (NB) is the most prevalent and deadliest pediatric solid tumor. With of over 50% of high-risk neuroblastoma cases relapse, the imperative for novel drug targets and therapeutic strategies is accentuated. In neuroblastoma, the existence of tumor-associated macrophages (TAMs) correlates with an unfavorable patient prognosis. However, the clinical relevance and prognostic implications of regulatory genes linked to TAMs infiltration in neuroblastoma remain unclear, and further study is required.
METHODS: We conducted a comprehensive analysis utilizing transcriptome expression profiles from three primary datasets associated with neuroblastoma (GSE45547, GSE49710, TARGET) to identify hub genes implicated in immune evasion within neuroblastoma. Subsequently, we utilized single-cell RNA sequencing analysis on 17 clinical neuroblastoma samples to investigate the expression and distribution of these hub genes, leading to the identification of TNFAIP3. The above three public databases were merged to allowed for the validation of TNFAIP3\'s molecular functions through GO and KEGG analysis. Furthermore, we assessed TNFAIP3\'s correlation with immune infiltration and its potential immunotherapeutic impact by multiple algorithms. Our single-cell transcriptome data revealed the role of TNFAIP3 in macrophage polarization. Finally, preliminary experimental verifications to confirm the biological functions of TNFAIP3-mediated TAMs in NB.
RESULTS: A total of 6 genes related to immune evasion were screened and we found that TNFAIP3 exhibited notably higher expression in macrophages than other immune cell types, based on the scRNA-sequencing data. GO and KEGG analysis showed that low expression of TNFAIP3 significantly correlated with the activation of multiple oncogenic pathways as well as immune-related pathways. Then validation affirmed that individuals within the TNFAIP3 high-expression cohort could potentially derive greater advantages from immunotherapeutic interventions, alongside exhibiting heightened immune responsiveness. Deciphering the pseudotime trajectory of macrophages, we revealed the potential of TNFAIP3 in inducing the polarization of macrophages towards the M1 phenotype. Finally, we confirmed that patients in the TNFAIP3 high expression group might benefit more from immunotherapy or chemotherapy as substantiated by RT-qPCR and immunofluorescence examinations. Moreover, the role of TNFAIP3 in macrophage polarization was validated. Preliminary experiment showed that TNFAIP3-mediated TAMs inhibit the proliferation, migration and invasion capabilities of NB cells.
CONCLUSIONS: Our results suggest that TNFAIP3 was first identified as a promising biomarker for immunotherapy and potential molecular target in NB. Besides, the presence of TNFAIP3 within TAMs may offer a novel therapeutic strategy for NB.