Single-cell RNA sequencing and m6A RNA methylation sequencing and reveal cellular and molecular mechanisms of radiation combined with PD-1 blockade in NSCLC

Abstract

Abstract Background: Lung cancer is the leading cause of cancer-related mortality, and more than 85% of lung cancer cases are non-small cell lung cancer (NSCLC). The heterogeneity and molecular basis of this disease remain incompletely understood. Methods: To address this question, we have performed a single-cell RNA sequencing as well as m6A RNA methylation sequencing of matched untreated and radiation combined with PD-1 blockade NSCLC samples. Results: A total of 21019 cells are categorized into eleven distinct cell types, including fibroblast, macrophages\monocyte, B cell, dendritic cell, endothelial cells, neutrophils, T\Natural killer (NK) cells, NK cells, and cancer stem cells (CSCs). Further analysis of the CSCs showed that radiotherapy combined with immunotherapy effectively reduced the number of CSCs and exhausted CD8+T cells, and increased the naive CD8+ T cells. Dysregulation of multiple signaling pathways, including the Wnt, is associated with lung cancer metastasis through the TCGA tumor dataset analysis. Wnt10b is a member of the Wnt family, which plays an important role in the Wnt signalling pathway. Moreover, N6-methyladenosine (m6A) sequencing has identified obesity-associated protein (FTO) as a direct target of m6A modification whose levels were regulated by Wnt10b, suggesting the FTO as a potential therapeutic target for treatment of lung cancer. Finally, our study reveals the potential pathogenesis of FTO/Wnt10b/b-catenin signaling pathway in NSCLC development. Conclusions: Collectively, this study proves that radiation combined with PD-1 blockade inhibits the stemness of CSCs through the FTO/Wnt10b/β-catenin pathway in NSCLC, which might provide insights for cancer immunotherapies.