Single-Cell Sequencing Reveals PD-L1-Mediated Immune Escape Signaling in Lung Adenocarcinoma.

Abstract

Background: Lung cancer has the highest mortality rate among all cancers, for which immunotherapy can frequently lead to drug resistance. To understand the molecular mechanisms behind immune escape in patients with lung cancer and develop predictive and therapeutic targets, we carried out analytical experiments using single-cell sequencing. Methods: We collected eight tumor tissue samples from eight patients with lung adenocarcinoma and categorized them based on the positive reactions for programmed cell death ligand 1 (PD-L1) expression levels. Single-cell sequencing analysis was employed to create a comprehensive cellular landscape. Uniform Manifold Approximation and Projection was used to show the proportion of immune and endothelial cells, along with a map depicting the distribution of different cell types. Cells were subdivided according to molecular markers; the subpopulations were grouped based on PD-L1 levels and tumor marker-positive reactions. The correlation between the occurrence of the PD-L1 reaction and the response time of immune cells was explored; differential gene expression between the groups was elucidated. Finally, quantitative polymerase chain reaction (qPCR) was used to examine the relationship between key differentially expressed genes and PD-L1 immune escape checkpoint response. Results: A total of 58,810 single cells were analyzed, identifying seven distinct cell types. In the PD-L1-positive sample group, B cells, astrocytes, endothelial cells, outer skin cells, and tissue stem cells were present in higher proportions, whereas T and dendritic cells were the main cells in the PD-L1-negative sample group. According to the molecular markers, the seven cell types were divided into 17 cell clusters, with one cluster classified as tumor cells, showing PD-L1 positivity. Eleven molecular markers with different expression levels were simultaneously screened (NAPSA, MUC1, WFDC2, MYO6, LYZ, IGHG4, IGLL5, IGHM, IGKC, AQP3, and IGFBP7), and their association with the PD-L1/PD-1 immune escape axis response was confirmed by qPCR. Conclusion: Our study suggests that PD-L1-mediated immune escape may occur at a later stage of tumor progression, involving both PD-L1-positive and negative immune cells. Additionally, we identified 11 differentially expressed genes that could provide insights into the potential mechanisms of immune escape in patients with lung cancer. These findings offer promising molecular targets for the detection and treatment of immune escape in clinical settings.