Molecular profiling of core immune-escape genes highlights TNFAIP3 as an immune-related prognostic biomarker in neuroblastoma

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.

Immune infiltration; Macrophage; Neuroblastoma; TNFAIP3; Tumor immune microenvironment.